Transport Machineries
B1-1
Osteopontin expression in polarized MDCK cells
N. Tascene
1
and Z. S. Isguder
2
1
Ankara University Faculty of Veterinary Medicine, Ankara,
TURKEY,
2
University of Veterinary Medicine, Department of
Physiological Chemistry, Hannover, GERMANY
Osteopontin(OPN) is an arginine-glycine-aspartate (RGD)-contain-
ing adhesive glycoprotein that was first identified as a major sialo-
protein in bone and subsequently found to be expressed in kidney,
brain, macrophages, vascular smooth muscle cells and many cells
of epithelial linings. In this study we examined the expression of
OPN in MDCK cells in different cellular confluences. The cells
were examined at very low density, half confluence or complete
confluence. OPN levels were investigated by western blotting and
RT-PCR. An increase in OPN expression was observed due to the
increasing confluency and subsequent initiation of polarization.
Expression profiles of flotillin-2 in the same cells was used as a
control, since this protein is ubiquitously produced in MDCK cells
and its expression rates are independent of confluence and/or
polarization. Intracellular distribution of OPN was also monitored
by confocal microscopy on preparations immunolabeled with anti-
OPN antibodies. Staining patterns have also confirmed increased
OPN expression during confluency in MDCK cells.
We conclude that the expression of OPN in polarized MDCK cells

that the interaction of VGF to GM1 rafts is relevant for VGF
aggregation since it is not affected by the inhibition of glyco-
sphyngolipid synthesis. We have then looked for proteins capable
to interact with VGF by gel filtration and by SDS-PAGE analysis
of proteins that co-aggregated with VGF. We found a 40 kDa pro-
tein that is co-eluted with VGF and we demonstrated that it is not
flotillin 2, which also forms aggregates but has an intracellular dis-
tribution distinct from that of VGF. FRT-VGF cells were treated
with bafilomycin A1 that alters the proton gradient of the secre-
tory compartment. A dramatic reduction in VGF secretion and
granule formation, and lack of response to PMA were observed.
The secretion of thyroglobulin, that we have stably expressed in
FRT cells and that is also secreted through the apical cell domain
but by a constitutive pathway, was unaffected.
Overall our data support the hypothesis that VGF aggregation
does not depend on rafts association and possibly occurs in the
presence of a 40 kDa protein. Proton gradient perturbation affects
granule formation and VGF secretion.
B1-3
Cellular trafficking of cell penetrating peptides
and protein transport over plasma membrane
A. Lorents
1
, K. Padari
2
, M. Hansen
3
,U
¨
. Langel

cence microscopy. The inhibitors of macropinocytosis and cellular
metabolism decreased the uptake of complexes by K562 cells in a
concentration dependent manner, indicating that CPP-avidin con-
structs are taken up mostly by energy-dependent mechanisms, invol-
ving different types of endocytosis. The cellular trafficking of CPP-s
in HeLa cells was mapped on ultra-structural level by transmission
electron microscopy by using nanogold-labelled peptides.
B1-4
Cellular uptake and subcellular distribution of
Ap
n
A family of signaling molecules
R. Pe˛cherzewska, A. D. Krakowiak, J. Kaz´ mierczak,
M. Maszewska and W. J. Stec
CMMS PAS, Lodz, POLAND
Diadenosine polyphosphates are recognized as intra- and extracel-
lular signaling molecules and considered as the class of new second
messengers. Biochemical function of dinucleoside tri- and tetra-
phosphates has not been fully elucidated yet [1]. It is known that
Ap
3
A is cleaved by specific enzymes of hydrolases family. One of
them is the tumor suppressor Fhit protein. It is assumed that for-
mation of the FhitAp
3
A complex, recognized as a signaling mole-
cule induces cell apoptosis [2].
The aim of presented studies is to determine the cellular uptake of the
Fhit protein substrates. For our experiments we used fluorescent ana-
logs of Ap

space. ECP and EDN belong to the ribonuclease A superfamily.
Both have been suggested as factors in allergic respiratory diseases,
and thus used as clinical biomarkers for detecting the severity of
asthma. Interestingly, ECP and EDN showed cytotoxicity toward
several cell lines, including HL60 and A431 cells. The cytotoxicity
was positively correlated with the internalization properties of the
RNases, but the exact route by which eosinophil RNases enter
cells has not been clarified. Recently, we hypothesized that ECP
and EDN may internalize target cells through binding to the cell
surface heparan sulfate (HS). In this study, we have investigated
the interaction between ECP/EDN and HS using surface plasmon
resonance instrument (SPR) technology. On the other hand, amino
acid sequence analysis reveals that EDN contains one putative
heparin-binding motif of the type XBBXBX, whose effects on HS
binding has been investigated by site-directed mutagenesis and
binding assays. Our results indicate that the heparin-binding region
plays a critical role in recognition and internalization of eosinophil
RNases.
B1-6
A heparan sulfate-facilitated and
raft-dependent macropinocytosis of eosinophil
cationic protein
T. Fan, S. Lin and M. D. T. Chang
National Tsing Hua University, Hsin-chu, TAIWAN
Eosinophils secrete several basic proteins, among which the eosino-
phil cationic protein (ECP) has been used as bio-markers for the
severity of asthma. ECP, which belongs to the human RNaseA su-
perfamily, is released from activated eosinophils during the inflam-
mation process and contributes to eosinophils’ helminthotoxic,
bactericidal and antiviral activities. The cytotoxicity of ECP is clo-

minal kinase (JNK), and that this resulted in a significant increase
in the ligase activity. It is known that JNK can also be activated
by EGF treatment. Likely, we have found that Itch phosphoryla-
tion is increased after treatment with EGF and that inhibition of
JNK blocks this effect. Remarkably, Itch substrate ubiquitination
after EGF treatment is also reduced after JNK inhibition. As Itch
phosphorylation influences its capacity to interact with its sub-
strates, we examined the interactions of Itch with endophilin, cbl,
and FAM after EGF treatment. We show that Itch interaction
with Cbl and endophilin increases after treatment with EGF, but
interaction with FAM decreases. These results explain the transient
ubiquitination of Itch and the sustained ubiquitination of its sub-
strates after treatment with EGF. We conclude that Itch activity
and stability is regulated by EGFR via JNK and that this regula-
tion affects the ubiquitination and the degradation of Itch sub-
strates, especially cbl and endophilin, which could in turn regulate
receptor internalisation.
B1-8
Identification of a novel Musk binding protein
with a potential role in intracellular trafficking
processes
B. Woller and R. Herbst
Medical University Vienna, Vienna, AUSTRIA
The muscle-specific kinase MuSK plays an essential role during the
formation of the neuromuscular junction. MuSK is activated by
the nerve-derived protein agrin, a process that is essential for all
known aspects of postsynaptic and presynaptic differentiation. The
steps that follow MuSK activation and lead to acetylcholine recep-
tor clustering are not fully understood. Therefore we have a high
interest in the identification and characterization of new MuSK

3
and
N. Santama
1
1
University of Cyprus, Nicosia, CYPRUS,
2
EMBL, Heidelberg,
GERMANY,
3
Centre of Genomic Regulation, Barcelona, SPAIN
Inhibition of motor protein activity is linked with defects in the for-
mation of poles in the mitotic spindle but the molecular mecha-
nisms underlying the functional relationship between motor activity
and centrosome dynamics are unclear. We characterised KIFC5A,
a minus-end-directed mouse kinesin-like protein, highly expressed
in dividing cells. It is nuclear in interphase, localises to the centre of
spindle asters at the beginning of mitosis, and to spindle MTs in
later phases. Overexpression of KIFC5A in mouse cells causes the
formation of non-separated MT asters and mitotic arrest in a pro-
metaphase-like state. KIFC5A knockdown partly rescues the phe-
notype caused by monastrol inhibition of plus-end-directed motor
Eg5, indicating that it is involved in the balance of forces determin-
ing bipolar spindle assembly and integrity. Silencing of KIFC5A
results in centrosome amplification detectable throughout the cell
cycle. Supernumerary centrosomes arise primarily by reduplication
and partly from cytokinesis defects. They contain duplicated centri-
oles and have the ability to organise MT asters, resulting in the for-
mation of multipolar spindles. KIFC5A interacts with nucleotide-
binding proteins 1 and 2 (Nubp1, Nubp2), which have extensive

constructed a chimeric motor with the motor domain of Kinesin-1
and a neck/stalk of a non-processive Kinesin-3. We found that this
novel kinesin behaves qualitatively as conventional Kinesin-1. It
moves processively in single molecule fluorescence assays and
exerts up to 3 pN force in optical trapping experiments. However,
reduced velocities and run lengths at a variety of loads indicate
that the unconventional neck domain hinders the diffusive search
for the next binding site. In the reverse chimera (motor domain of
non-processive Kinesin-3 and neck/stalk of Kinesin-1) the Kinesin-
1 neck allows sequential ADP release from the partner heads upon
microtubule binding. But this motor was unable to make successive
steps.
Our observations suggest that kinesin processivity requires two
independent elements. One, located in the neck/stalk region,
ensures coordinated ADP release from the motor domains. The
other, located in the motor domain itself, allows successive ‘hand-
over-hand’ stepping by coordinating the timing of events between
the two motor domains.
B1-11
Effects of kinesin motor domain structure on
kinesin activity
N. Kalchishkova, E. Unger and K. J. Bo
¨
hm
Leibniz Institute for Age Research - Fritz Lipmann Institute, Jena,
GERMANY
Kinesin-microtubule interaction results in generation of motility,
essential for organelle transport and cell division. Each kinesin
molecule reveals a distinct globular motor domain, a neck-linker
and a neck, a stalk and a cargo-binding tail domain. The force-

,T.Du
2
, R. Jansen
2
and D. Niessing
1
1
GSF & Gene Center Munich University, Munich, GERMANY,
2
Gene Center Munich University, Munich, GERMANY
Motor protein-dependent transport of cargo is a basic cellular
function. To exert their motile activity, the molecular motors kine-
sin and myosin must dimerize. Surprisingly little is known about
how such molecular motors assemble with other proteins into func-
tional translocation complexes. Recent studies on a minus-end
directed myosin, i.e. type VI myosin, show that this motor only di-
merizes upon complex assembly. However, for plus-end directed
myosins, which constitute the vast majority of myosin motors, we
know very little about the mechanisms controlling the assembly of
their translocation complexes. Available data suggest for these my-
osins a constitutive and unregulated interaction of their C-terminal
tails with cargo complexes.
We report a study involving biochemical, biophysical, and in vivo
experiments on the assembly of a yeast translocation complex with
plus-end directed myosin type V. Our results establish that myosin
dimerization significantly increases its affinity for its interaction
partner of the cargo complex and stabilizes the resulting complex.
Thus, myosin dimerization is required for efficient binding to the
core complex. Since only dimeric myosins move processively along
actin filaments, these observations reflect a quality control step for

The ESCRT-III subunit CHMP2B/Vps2b was recently found to be
mutated in patients with frontotemporal dementia (FTD) and
amyotrophic lateral sclerosis (ALS). These diseases are characterized
by abnormal intracellular ubiquitin positive protein deposits in
affected neurons. The ESCRTs, first identified in yeast as vps class E
mutants, have proven important for recognition of ubiquitinated
endocytosed integral membrane proteins and their sorting into the
intralumenal vesicles of the multi-vesicular body (MVB) and subse-
quent degradation in the lysosome/vacuole. We here present evi-
dence that siRNA-mediated depletion of the ESCRT III subunit
Vps24 leads to accumulation of large ubiquitin-positive cytoplasmic
protein aggregates, which also contain p62/Sequestosome-1 and Alfy
(autophagy-linked FYVE protein), but are devoid of the early endo-
some marker EEA1. We show that p62-containing amphisomes
(positive for GFP-LC3 and CD63) do form in Vps24 depleted cells,
but that lysosomal degradation of p62 and LC3 is inhibited/delayed.
Vps24 is also required for efficient clearance of HttQ103 aggregates
in a cell-based model of Huntingtons disease. Taken together, our
data indicate that efficient autophagic clearance of ubiquitinated
proteins requires functional MVBs and provide a possible explan-
ation to the observed neurodegenerative phenotype seen in patients
with CHMP2B mutations.
B1-14
Screen for new factors affecting function of
ubiquitin ligases, human Nedd4 and yeast Rsp5,
in yeast S. cerevisiae
P. Kaliszewski
1
, M. Stawiecka-Mirota
1

situated in its C-terminal tail
M. Boonen, G. Cuvelier, R. Castro, I. Hamer and M. Jadot
URPHYM, FUNDP, Namur, BELGIUM
Transport of newly synthesized lysosomal membrane proteins from
the TGN to the lysosomes is due to the presence in their cytoplas-
mic domains of specific signals which are recognized by clathrin-
associated adaptor complexes. p40, a predicted multispanning pro-
tein of 372 amino acids localized to the lysosomal membrane, con-
tains four putative lysosomal sorting motifs in its sequence: three
of the YXXF-type (Y
6
QLF, Y
106
VAL, Y
333
NGL) and one of the
dileucine-type (EQERL
360
L
361
). Mutations of the critical residues
of these motifs, tyrosine or leucine, revealed that the EQERLL
motif was implicated in the sorting of p40. Confocal microscopy
analyses, performed in transfected HeLa cells, showed that p40-
GFP was mis-targeted to the plasma membrane when its dileucine
motif was disrupted. This result was confirmed by cell surface bio-
tinylation studies which allowed us to estimate that approximately
50% of the p40 dileucine mutants (without GFP) were associated
with the plasma membrane. We also observed that, in self-forming
Percoll density gradients, the dileucine mutants were excluded from

SRP causes an arrest in the elongation (EA) of nascent chains pre-
senting signal sequence and efficiency of their translocation is
dependent on this arrest. We have identified a five amino acid-long
motif in SRP14 required to confer EA activity to SRP. Particles
comprising the defective SRP14 protein (A5), no longer arrest elon-
gation of the nascent chain and have reduced translocation effi-
ciency. To examine the significance of this function for protein
secretion in mammalian cells, we depleted endogenous SRP14 using
RNAi and replaced it with the expression of wild type or mutant
GFP-14. Although the expression of both, GFP-14 and GFP-14A5,
restored cellular SRP levels to normal, the translocation efficiency
of reporter proteins was specifically diminished in cells expressing
GFP-14A5. The defect could be rescued by the addition of aniso-
mycin, which slows down nascent chain elongation. These results
demonstrate the physiological relevance of EA for efficient target-
ing in mammalian cells. Protein translocation into the ER is ineffi-
cient at normal cellular translation elongation rates presumably,
because nascent chains become too long during the time span
required for SRP to target nascent chains successfully to the trans-
locon. The selective slow down of translation elongation by SRP is
therefore required to optimize protein translocation efficiency in
mammalian cells without hampering continued rapid translation of
other cellular proteins.
*equal contribution.
Abstracts Transport Machineries
100 ª 2007 The Authors Journal compilation ª 2007 FEBS
B1-17
Regulation of vesicle trafficking by protein
S-nitrosylation
Y. Daaka

the ER and the Golgi, the first two compartments of the secretory
pathway. Movement of these transport carriers occurs in a stop-
start fashion with frequent bidirectional oscillatory, as well as lon-
ger range, movements. This behaviour is due to regulation of the
binding to, and/or regulated activity of, opposing motor proteins-
in general terms, dynein (along with dynactin) moves cargo
towards the Golgi, while kinesin moves cargo away from it. The
function of these motors is also controlled by protein phosphoryla-
tion, acting at least in part through a protein kinase A-dependent
pathway. We are studying the association of dynactin with carriers
in transit to the Golgi and the regulation of bidirectional motility
by protein phosphorylation by imaging GFP-tagged forms of var-
ious ER-Golgi markers. We have established an experimental sys-
tem that allows us to image the movement of ER-to-Golgi
transport carriers with extremely high spatial and temporal resolu-
tion. Application of 2D-Gaussian fitting models to the data allows
precise determination of object position. This should ultimately
allow us to determine motor step size, which will give key informa-
tion regarding the type of motors associated with these structures.
It will also lead to the detailed analysis of the dynamics of trans-
port carriers operating between early secretory pathway and help
in deciphering the mechanism of their regulation.
B1-19
Molecular analysis of YIP1 family protein
function
C. Chen and R. N. Collins
Cornell University, Ithaca, NY, USA
The YIP1 family is a group of integral membrane proteins with
the capability of binding to dual prenylated Rab GTPases. These
proteins are functionally conserved from yeast to humans, and, in

1
and H. Farhan
1
1
Institute of Pharmacology, Medical University, Vienna, AUSTRIA,
2
Department of Pharmacology, Vanderbilt University, Nashville,
TN, USA,
3
Departments of Neuroscience and Neurology, Johns
Hopkins University, Baltimore, MD, USA
GTRAP3-18 is an ER localized protein belonging to the prenylat-
ed rab-acceptor-family interacting with small Rab GTPases. Its
impact on secretory trafficking has not been investigated. We
report here that GTRAP3-18 inhibits Rab1, which is involved in
ER-to-Golgi trafficking. The effects on the early secretory pathway
were: reduction of the rate of ER-to-Golgi transport in HEK293
cells, inhibition of cargo concentration of the EAAC1 glutamate
transporter into transport complexes in HEK293 cells, an effect
that could be completely reversed in the presence of an excess of
Rab1. In accordance with the role of Rab1 in neurite formation,
overexpression of GTRAP3-18 significantly inhibited neurite out-
growth in CAD cells. Finally, we hypothesized that expression of
GTRAP3-18 in the brain should be lower at stages of active syn-
aptogenesis compared to early developmental stages. This was the
case as expression of GTRAP3-18 was lower in P0 rat brains com-
pared to E17 rat brains.
Transport Machineries Abstracts
ª 2007 The Authors Journal compilation ª 2007 FEBS 101
B1-21

ding a myristoylation site and two hydrophobic (h) regions. After
cleavage by signal peptidase SP
GP-C
was found to accumulate in
cells and virus particles.
In the present study, we identified the LCMV SP
GP-C
as an
essential component of the viral GP complex. By using several
deletion and point mutants we investigated signal sequence require-
ments for GP-C biosynthesis, transport to the cell surface and viral
infectivity. Our results show, that only one SP
GP-C
h-region is nee-
ded for membrane insertion of pGP-C, whereas both h-regions are
required for GP-C processing and cell surface expression. Further-
more, the n-region of the SP
GP-C
and the myristoylation are found
to be essential for viral infectivity, most likely for pH-dependent
fusion with the host cell membrane. Thus different regions of
LCMV SP
GP-C
fulfil essential functions in GP-C maturation and
virus infection.
B1-22
Different domains of GOPC regulate its Golgi
localization
I. Todros
1,2

devoid of the C-terminal PDZ domain suggested that Golgi distri-
bution of GOPC is negatively regulated by the PDZ-mediated
interactions. This was further supported by the fact that overex-
pression of CFTR, a protein interacting with GOPC through its
PDZ domain, substantially reduced Golgi localization of GOPC.
Thus, different domains of GOPC seem to contribute to its subcel-
lular distribution. While the coiled-coil-mediated interactions
ensure efficient targeting to the Golgi, the PDZ-based mechanism,
being responsible for binding a cargo protein, contributes to the
subsequent detachment of the whole complex from the trans-Golgi
membrane.
Acknowledgement: Supported by grant PBZ/KBN/122/P05/01-
06.
B1-23
Role of the multimolecular conserved
oligomeric Golgi complex in intra-Golgi
trafficking and glycosylation disorders
V. Lupashin, A. Shestakova, E. Suvorova, R. D. Smith and
O. Pavliv
University of Arkansas for Medical Sciences, Little Rock, AR, USA
Large oligomeric multiprotein complexes direct membrane trafficking
in eukaryotic cell. One class of traffic regulators, vesicle tethering fac-
tors, mediate the initial loose tethering of transport vesicles to their
target membranes. The peripheral membrane
conserved oligomeric
Golgi (COG) complex directs retrograde intra-Golgi membrane traf-
ficking and ensures proper Golgi functions. Mutations in COG sub-
units cause congenital glycosylation disorders in humans, suggesting
that COG is necessary for the correct sorting of glycosyltransferases,
which create a variety of developmentally important glycans. The

1
, A. Spaar
1
,
D. Di Giandomenico
1
, A. Luini
1
and A. A. Mironov
1
1
Consorzio ‘‘Mario Negri Sud’’, Santa Maria Imbaro, ITALY,
2
Uni-
versity of Ferrara, Ferrara, ITALY
Recent evidence has highlighted the functional importance of Ca
2+
in
intracellular trafficking, and Ca
2+
increases could act downstream of
the SNAREs, proteins that are involved in the fission/fusion processes
that occur during cargo progression through the Golgi apparatus.
Indeed, C a
2+
increases the rate of SNARE-mediated membrane fusion
via C a
2+
ion channel association with the SNARE complex. Our work-
ing hypothesis is based on the fact that the fusion events, that occur

Abstracts Transport Machineries
102 ª 2007 The Authors Journal compilation ª 2007 FEBS
B1-25
Direct interaction of the C-terminus of TRPC6
with Rab9 targets TRPC6 to the trans-Golgi
network
S. Cayouette and G. Boulay
Universite
´
de Sherbrooke, Sherbrooke, PQ, CANADA
TRPC proteins are implicated in Ca
2+
entry following activation of
Gq-protein coupled receptors. We previously showed that the inser-
tion of TRPC6 into the plasma membrane (PM) is regulated by hor-
monal stimulation. Since small G proteins of the Rab family are
involved in vesicle trafficking and fusion, we assessed the intracellu-
lar trafficking of TRPC6 with different Rab dominant negative
(DN) mutants. TRPC6 activation was measured in HEK293 cells
transiently co-expressing either Rab11DN (blocks transport from
the TGN and recycling endosome to the PM) or Rab9DN (blocks
fusion of late endosomes with the TGN). Rab9DN significantly
enhanced carbachol-induced Ca
2+
entry through TRPC6 activation,
whereas Rab11DN completely abolished its activity. Rab11 and
Rab9 have no significant effect on TRPC6 activity. GST-pulldown
assay showed that Rab9 and Rab9DN interact directly with the C-
terminus of TRPC6. These interactions occur also in vivo, since
Rab9 and Rab9DN co-immunoprecipitate with TRPC6. However,

that Casein Kinase 2 (CK2) binds wt-CFTR near the F508 resi-
due and phosphorylates serine residue at position 511. Deletion
of F508 abrogates this residue-dependent interaction [1]. Our
aim here was to identify whether CK2 interaction affects CFTR
biogenesis, turnover and processing. BHK cells were stably
transfected with wt- or F508del-CFTR in which S511 was sub-
stituted by alanine (S511A) or aspartate (S511D). Pulse-chase
experiments followed by CFTR immunoprecipitation were per-
formed in these lines, showing that substitution of S511 does
not affect the turnover or processing of either wt- or F508del-
CFTR. The effect of CK2 inhibition on the turnover and pro-
cessing of CFTR showed that: 1) steady-state levels of CFTR
are reduced. 2) turnover of wt-CFTR (but not F508del-CFTR)
is increased and 3) processing of wt-CFTR is decreased. Our
data suggest a putative stabilizing role for CK2 upon wt-CFTR,
not dependent on the charge that is added by the kinase, so it
is probably indirect.
Acknowledgement: Work supported by CIGMH and POCTI/
SAU/MMO/58425/2004 grant (FCT, Portugal).
Reference
1. Treharne et al. (2007) J Biol Chem (epub).
B1-27
Checkpoints in the traffic pathways of
Tyrosinase Related Proteins in melanoma cells
G. Negroiu
Institute of Biochemistry, Bucharest, ROMANIA
Tyrosinase Related Proteins-(TRPs) are the main regulators of
melanin synthesis in normal and pathological pigmentation and
immune targets in melanoma, glioma and autoimmune depigmen-
tation. TRP-2 has a significant role in intrinsic drug resistance of

NF-jB. We proposed to investigate the effects of progesterone (P)
pretreatment on cytokine-stimulated activation of NF-jB.
Study design: HeLa cells were pretreated with 10
-7
M progester-
one for 24 h and exposed to 1 ng/ml IL-1b for 1 hour. Nuclear
and cytosolic extracts were subjected to Western analysis using
anti-p65 and anti-IjBa antibodies. Densitometric data (n =5)
were compared using Kruskal–Wallis test.
Results: Pretreatment with P interfered with IL-1b-induced inhibi-
tory protein-jBa (IjBa) degradation. However, P pretreatment
resulted in a significant decrease in p65 in the cytoplasm. Pretreat-
ment with P did not reduce the amount of nuclear p65 and did not
interfere with nuclear translocation of p65.
Conclusion: Our observations suggest that any possible role
played by P in preterm labor prevention is not exerted through
anti-inflammatory mechanisms of NF-jB downregulation.
Transport Machineries Abstracts
ª 2007 The Authors Journal compilation ª 2007 FEBS 103
B1-29
Loss of alpha-tubulin polyglutamylation in
ROSA22 mice is associated with abnormal
targeting of KIF1A and modulated synaptic
function
M. Setou
NIPS and MITILS, Machida, JAPAN
Microtubules function as molecular tracks along which motor pro-
teins transport a variety of cargo to discrete destinations within the
cell. The carboxyl termini of alpha- and beta-tubulin can undergo
different posttranslational modifications, including polyglutamyla-

, D. Carrel
1,2
, C. Borg-Capra
3
,
J. Laine
´
2,4
, M. Hamon
1,2
, M. B. Emerit
1,2
and M. Darmon
1,2
1
INSERM U677, Paris, FRANCE,
2
Universite
´
Pierre et Marie
Curie, Paris, FRANCE,
3
Hybrigenics SA, Paris, FRANCE,
4
Dept Physiologie, UPMC, Paris, FRANCE
The 5-HT1A receptor is a dendritic receptor implicated in the con-
trol of brain (5-HT) neurotransmission. Evidence has been reported
that the short C-terminal domain of the rat 5-HT1AR plays a crucial
role in receptor targeting from the endoplasmic reticulum to the
plasma membrane. We used this 17 amino acid region as a bait in a

´
nez, F. Portillo and T. Iglesias
Insituto de Investigaciones Biomedicas, Madrid, SPAIN
Protein kinase D1 (PKD1) belongs to a novel family of diacylglyc-
erol (DAG)-stimulated Ser/Thr kinases, constituted by two more
members, PKD2 and PKD3. We have previously identified the C
terminus of the different PKDs that constitutes a PSD-95, Dlg,
ZO-1 (PDZ)-binding motif in PKD1 and PKD2, but not in PKD3,
to be responsible for the differential control of Kinase D-interact-
ing substrate of 220-kDa (Kidins220) surface localization. Ki-
dins220 is a neural membrane protein identified as the first
substrate of PKD1. PKD1 controls Kidins220 transport by the
interaction with a PDZ protein. In an effort to identify that PDZ
protein we performed yeast two hybrid screenings using as baits
the PDZ-binding motifs of Kidins220 and PKD1. We found a pos-
itive clone containing the complete coding sequence of the PDZ
protein a-syntrophin that strongly interacted with both baits. a-
syntrophin has been described as a Kidins220 binding partner that
plays an important role in the regulation of Kidins220 localization
during neuromuscular junction differentiation. Our results show
that PKD1 phosphorylation of its PDZ-binding motif regulates the
interaction with a-syntrophin in yeast and that Kidins220, PKD1
and a-syntrophin form ternary complexes in neural cells. These
data suggest that the interaction of a-syntrophin with Kidins220
and PKD1 could be relevant for the control the PKD1 exerts on
Kidins220 transport.
B1-32
Cellular control of trafficking and activity of
perforin, a key regulator of immune
homeostasis

B1-33
Regulation of intracellular trafficking plays a
pivotal role in tumor pathogenesis
J. Go
¨
ttert
Max-Delbru
¨
ck-Centrum fu
¨
r Molekulare Medizin, Berlin,
GERMANY
Intracellular trafficking as a fundamental cellular process needs
to be tightly regulated, otherwise disturbances can lead to a
variety of disorders and diseases. Glycans play a pivotal role in
protein sorting, there synthesis proceeds in a hierarchical manner
along the secretory pathway. Mechanisms that alter the intracel-
lular localization of glycosyltransferases and glycosidases may
lead to an alternative glycan repertoire of cells. In addition, gly-
can linkage defects are associated with pathogenetic events in
hereditary and acquired human diseases. Here, we refer to
EBAG9 that has been identified as a primary estrogen respon-
sive gene. High expression levels of the gene product have been
recorded in several carcinomas, suggesting a tumor-promoting
role of the protein. EBAG9 is a ubiquitously expressed Golgi
protein with a peripheral membrane attachment. It was shown
to induce expression of the truncated O-linked-glycans Tn and
TF at the plasma membrane of non-secretory epithelial cell lines.
In general, Tn- and TF are extensively expressed on the cell sur-
faces of diverse tumor tissues and they are thought to be

alone. In contrast, allicin did not enhance the cytotoxic activity of
AmB against cells of human promyelocytic leukemia (HL-60), a
vacuole-less organism.
B1-35
Hereditary Spastic Paraplegia caused by
Kinesin-1 mutants
B. Ebbing
1
, K. Mann
1
, R. Schu
¨
le
2
and G. Woehlke
1
1
Institute for Cell Biology LMU, Munich, GERMANY,
2
Hertie-Institute for Clinical Brain Research, Tu
¨
bingen, GERMANY
Hereditary Spastic Paraplegia is a neurodegenerative motor neuron
disease characterized by lower limb spasticity and weakness. This
autosomal dominant disease is linked to mutation of at least 20
genes, among them several point mutations in the neuronal Kinesin-
1 (KIF5A) gene. Here, for the first time, we investigate the in vitro
properties of the wild-type KIF5A and three of the point mutations,
which are located in the motor domain. The N256S mutant had a
lower gliding velocity, whereas the R280S mutant exhibited low

, M. Maslov
2
and G. Serebrennikova
2
1
Institute of Chemical Biology and Fundamental Medicine SB RAS,
Novosibirsk, RUSSIAN FEDERATION,
2
Lomonosov Moscow State
Academy of Fine Chemical Technology, Moskow, RUSSIAN
FEDERATION
Cationic lipids are extensively used as non-viral vectors for the
gene transfer but a number of the drawbacks limit their use in vitro
and in vivo. One of the ways to solve these problems is creating the
new biodegradable non-toxic cationic lipids based on the natural
compounds and studying their properties. We synthesized and tes-
ted the new biodegradable cationic lipids based on the cholesterol
and glycerol derivatives with the heterocyclic cationic head groups
and various linkers and studied them as gene delivery carriers. It
was found that the new cationic lipids are low-toxic at the concen-
trations below 10 lM for the various cell lines (293, HeLa, BHK,
BHK IR-780). By fluorescent microscopy and FACS we showed
that the presence of the cationic lipids enhanced the delivery of
various nucleic acids (oligonucleotides, plasmid DNA and siRNA)
into cells. The transfection efficiency and toxicity of the new cati-
onic lipids were compared with the commercially available cationic
agent Lipofectamine. Hence, the obtained results suggest that the
new cationic lipids based on the natural compounds could be the
promising agents for the transfection of human cells.
Acknowledgements: This work was supported by RAS pro-

and L. Nyitray
1
1
Eo
¨
tvo
¨
s Lora
´
nd University, Budapest, HUNGARY,
2
Semmelweis
University, Budapest, HUNGARY,
3
Boston Biomedical Research
Institute, Boston, MA, USA
DYNLL1 and DYNLL2 are two mammalian paralogs of the con-
served LC8 family of dynein light chains that were described as tail
subunits of dynein and myosin Va motor proteins. Moreover, they
were shown to interact with a wide variety of other polypeptides.
They may serve as adaptors to bind cargos to the transport motors
however, they also have other functions in the cell unrelated to their
role in motor complexes. We have localized the binding site of DYN-
LL2 on myosin Va tail within an uncoiled region, between two
coiled-coil domains. DYNLL2 binds to this site with a Kd of 40 nM
and stabilizes both flanking coiled-coils. By studying DYNLL bind-
ing to their targets, we have found that PAK1, an important regula-
tor of cell motility, regulates activity of DYNLL1, but not
DYNLL2, by phosphorylating Ser88 and dissociating the homo-
dimer protein into two monomers. The monomeric DYNLL (mim-

cellular distribution and biological activity of the complexes formed
by delivered oligonucleotides and cholesterol-modified carrier-oligo-
nucleotides were studied. We show that incorporation of the desired
antisense oligonucleotide into the self-assembling concatemeric sys-
tem significantly promotes its delivery into cells without the addi-
tion of any supplementary transfection agents and allows to achieve
specific inhibition of the target gene. In contrast to the use of con-
ventional delivery agents, concatemeric oligonucleotide system was
shown to posses no cytotoxic and non-specific intercellular activit-
ies. In conclusion, delivery of pharmacologically active oligonucleo-
tides into the target cells in concatemeric form provides perspective
alternative to the use of conventional transfection methods.
Acknowledgements: This work was supported by RAS programs
‘‘Molecular and cellular biology’’ and ‘‘Science to Medicine’’.
B1-39
The role of cytoplasmic networks in plasmid
DNA intracellular trafficking and intranuclear
movement of plasmid DNA
V. Ondrej, E. Lukasova, S. Kozubek and M. Falk
Institute of Biophysics Academy of Sciences of the Czech Republic,
v.v.i., Brno, CZECH REPUBLIC
The role of microtubule and actin networks in transport and pro-
cessing of transfected plasmid DNA in human cells was studied.
We observed strong binding activity of plasmid DNA to both
networks, their immobilization at actin filaments and directional
motion along the microtubules with average velocity
v = 1.12 lm/min. In addition, actin filaments play an important
role in dissociation of plasmid DNA aggregates at the cell periph-
ery. Disruption of one of these networks led to the abortion of
plasmid transport and accumulation of plasmid DNA in huge

pendently of the cellular Cu content. Co-immunolocalization with
intracellular compartment markers (endoplasmic reticulum, ER;
Golgi apparatus, GA; early endosomes, EE; late endosomes/lyso-
somes, LE/Ly and plasma membrane, PM) showed that 200 lM
CuSO
4
stimulated trafficking of COMMD1 from ER, GA and
EE to LE/Ly, as well as trafficking of ATP7B from ER and EE
to the LE/Ly compartment. No GA and PM localization was
observed. These results were sustained by those obtained after
differential centrifugation and separation on sucrose gradient.
Taken together these results suggested that the mechanism of bil-
iary Cu excretion involves the intracellular trafficking of ATP7B/
COMMD1 and their interaction in the endosomes.
Abstracts Transport Machineries
106 ª 2007 The Authors Journal compilation ª 2007 FEBS
B1-41
Conformational changes in actin-binding
proteins, revealed by single particle electron
microscopy
O. Sokolova
1,2
, S. Maiti
2
, N. Grigorieff
3
, P. Lappalainen
4
and
B. L. Goode

closed conformation the number of open molecules increases dra-
matically after activation. Next, we studied the structure of actin
binding proteins MIM and IPRs53 during their activation. The
obtained results explained ligand-induced conformational changes
inside the MIM/IRSp53 proteins.
B1-42
Fluorescence Correlation Spectroscopy (FCS)
study of l-opioid receptor dynamics in live cells
V. Vukojevic, Y. Ming, C. D’Addario, B. Johansson, R. Rigler
and L. Terenius
Karolinska Institute, Stockholm, SWEDEN
Cellular dynamics of G protein-coupled receptors (GPCRs) is
rather complex and not yet fully elucidated, but it is widely accep-
ted that it is an integral part of their function - the trafficking steps
are regarded to be dynamical regulators of GPCRs density at the
cellular surface. Hence, the surface density of GPCRs can be con-
trolled without modifying the overall number of receptors in the
cell through receptor synthesis (up- or down-regulation) [1, 2].
Recent advances in molecular imaging offer the possibility to study
protein interactions and intracellular trafficking in real time and
with single-molecule sensitivity in living cells. We present results on
nondestructive observation of l-opioid receptor (MOR) inter-
actions with selected ligands using Fluorescence Correlation Spectr-
oscopy (FCS) integrated with Confocal Laser Scanning Microscopy
(CLSM) [3, 4]. With this approach we were able to monitor the
number of MOR exposed at the plasma membrane, determine their
lateral motility at the plasma membrane and evaluate how these
parameters are affected by interaction with selected ligands.
References
1. Gainetdinov, R.R., Premont, R.T., Bohn, L.M., Lefkowitz,

cellular response to hypoxia. Its regulated alpha subunit (HIF-
1alpha) can be induced, in addition to hypoxia, by transition
metals, iron chelators and various growth factors, thus responding
to the activation of several signaling pathways. Regulation of
expression and activity of HIF-1alpha also involves several post-
translational modifications. We have recently shown that MAPK-
dependent phosphorylation of HIF-1alpha blocks its nuclear
export by CRM1 and facilitates its accumulation inside the nucleus
(1). To further understand the mechanism regulating the intracellu-
lar distribution of HIF-1alpha, we have investigated its nuclear
import using recombinant GFP-HIF-1alpha in digitonin-permeabi-
lized HeLa cells. We show that nuclear entry of HIF-1alpha is not
mediated by the classical NLS receptor importin alpha/beta but by
two other monomeric receptors of the importin-beta family. Bind-
ing assays confirm that these two importins can physically interact
with recombinant GST-HIF-1alpha. Taken together, our results
show that shuttling of HIF-1alpha between cytoplasm and nucleus
is a complex and regulated process involving several members of
the nuclear transport receptor family.
Reference
1. Mylonis I. et al., (2006), J. Biol. Chem. 281(44):33095.
B1-44
Searching for NLS and NES signals in the
Methoprene-tolerant protein
B. Greb-Markiewicz
1
, J. Dutko-Gwo
´
z´ dz´
1

enhanced yellow fluorescence protein for cloning full length and
some fragments of Met cDNA. In the next step, vectors were used
for COS-7 cells transfection and finally localization of expressed
fusion proteins was visualized with Confocal Microscopy System.
We will present results suggesting presence of more than one single
NLS and NES signal in the Methopren-tolerant, existing in differ-
ent fragments of protein.
Transport Machineries Abstracts
ª 2007 The Authors Journal compilation ª 2007 FEBS 107
B1-45
Characterisation of NLS in human PHD1 and
PHD3
A. Hilz
1,2
, T. Schillinger
1
, S. G. Schindler
1
, M. Koehler
2
and
R. Depping
1
1
University of Luebeck, Luebeck, GERMANY,
2
Reha Clinic Damp
GmbH, Damp, GERMANY
HIF (hypoxia inducible factor) family members act as transcrip-
tional master regulators of genes involved in cellular and systemic

coding for the Pir family proteins (ccw5 ccw6 ccw7 ccw8) was per-
formed in order to investigate their potential role. After disruption
of the genes coding for all Pir family members, 67 kDa protein
band still remained in the NaOH extract. Disruption of SCW4
resulted in apparent disappearance of the 67 kDa band, indicating
that Scw4p could also be covalently linked to the cell wall. In order
to investigate the role of the Scw4p in the construction of the cell
wall, wilde type yeast was transformed with a high copy number
plasmid containing SCW4 gene. Phenotypes of this strain as well as
scw4, ccw5ccw6ccw7ccw8 and ccw5ccw6ccw7ccw8scw4 were exam-
ined. In order to get further insight in the binding mechanism a
novel, simple binding assay for Pir family proteins, was developed.
It has been shown that pir, as well as scw4 and scw10 mutant cells,
can bind externally added Ccw5p to their cell wall. A study of
appropriate binding conditions revealed the requirement of the
native conformation of Ccw5p. The presence of EDTA blocked the
binding of Ccw5p, indicating the cation dependence of the reaction.
Both wild type and mutant cells showed enhanced binding in 0.6 M
KCl. Further experiments should provide answers to question
regarding exact conditions required, as well as reveal if the binding
occurs autocatalytically or require a particular enzyme in the wall.
B2-2
The haemolysin A system of E.coli: from single
subunits to the whole transport machinery
S. Jenewein
1
, B. Holland
2
and L. Schmitt
1

lytic cycle of the NBD in detail. However, in order to understand the
process of protein secretion in detail we overexpressed other compo-
nents such as the N-terminal C39 domain, full-length HlyB and dif-
ferent domains of HlyD. Furthermore using the haemolysin system
as a tool for heterologous protein overexpression we designed a vec-
tor being able to secrete large amounts of proteins into the media.
B2-3
Interaction analysis of HrpE with other TTSS
components of Pseudomonas syringae pv.
phaseolicola injectisome
M. N. Bastaki
1
, N. J. Panopoulos
1,2
and A. P. Tampakaki
3
1
Department of Biology, University of Crete, Heraklion-Crete,
GREECE,
2
Institute of Molecular Biology and Biotechnology
(IMBB), Heraklion-Crete, GREECE,
3
Department of Agricultural
Biotechnology, Agricultural University of Athens, Athens, GREECE
Pseudomonas syringae pathovars utilize a specialized protein secre-
tion pathway called «type III secretion system» (TTSS) to inject viru-
lence-related proteins into host cells. TTSSs are also used by
mammalian pathogens such as Yersinia, Salmonella and Shigella as
well as by plant and insect symbiotic bacteria. The TTSS injectisome

, H. Robinson
3
, L. L. Burrows
4
and
P. L. Howell
1,2
1
Hospital for Sick Children, Toronto, ON, CANADA,
2
University of
Toronto, Toronto, ON, CANADA,
3
Brookhaven National Laborat-
ories, Upton, NY, USA,
4
McMaster University, Hamilton, ON,
CANADA
The exopolysaccharide alginate is the major component of P. aerugi-
nosa biofilms infecting the lungs of cystic fibrosis patients. Ten pro-
teins, located on the algD operon, are required for alginate
polymerization and export. Since polymerization requires protein
components in both the inner and outer membranes, these proteins
are believed to form a large multi-protein complex that spans the cell
envelope and facilitates export of the polymer. To date, our struc-
tural and functional characterization of the alginate secretion com-
plex has focused on the outer membrane lipoprotein, AlgK. AlgK is
essential for alginate production and is hypothesized to interact with
the outer membrane secretin, AlgE. Recombinant AlgK from P. flu-
orescens has been expressed, purified and crystallized, and native

system, we have undertaken a multi-scale/multi-resolution
approach to reconstructing mammalian (beta) cells in toto in 3D
at the EM level. By providing complete sets of 3D spatio-tem-
poral coordinates for cells at a range of resolutions that will
uniquely inform advanced in silico studies of 3D cell and
molecular organization, we are working to develop the world’s
first navigable ’Visible Cell atlas’. Such an interactive high-reso-
lution map of the 3D landscape of an entire mammalian cell
imaged and reconstructed by ET at £5 nm resolution will serve
as a unique international resource for protein and organelle
annotation, database integration and 3D visualization, and as a
framework for 4D animations of cells at pseudo-molecular reso-
lution.
B2-6
Direct evidence for the involvemen t of
VAMP-8/endobrevin in constitutive exocytosis
T. Takuma, M. Okayama, T. Arakawa and I. Mizoguchi
Health Sciences University of Hokkaido, Hokkaido, JAPAN
Studies with gene knockout mice have established that VAMP-8 is a
v-SNARE for the regulated exocytosis of various exocrine glands.
Although VAMP-8 is also believed to play an important role in con-
stitutive exocytosis and endosomal vesicle fusion, these processes
were normal in the mutant mice, probably because other v-SNAREs
can compensate the role of VAMP-8. In this study we have directly
examined the role of VAMP-2, -7, -8 in constitutive exocytosis by
TIRF microscopy. The plasmids of hGH and VAMPs with C-ter-
minal GFP-tags, prepared as described previously (Histochem Cell
Biol 125: 273–281, 2006), were transfected into HeLa cells and PC12
cells. Cells were incubated at 37 °C in a portable CO
2

2+
in micromolar range concentra-
tions. This fusion step was separated from the docked state in our
model. The specific action of synaptosomal cytosolic proteins on
synaptic vesicles was demonstrated using cytosolic proteins from
different rat tissues and bovine serum albumin. Our results indicate
that incubation synaptic vesicles with liver cytosol proteins or in
the bovine serum albumin solution do not lead to the enlargement
of the vesicles size. Studies of exocytosis in vitro have shown that
this process can be divided into Ca
2+
- independent step, termed
docking followed by rapid fusion step that is triggered by Ca
2+
.
Soluble proteins of synaptosomes have critical roles in the exocy-
totic pathway, perhaps involving modulation of docking site at the
targeting membrane.
Transport Machineries Abstracts
ª 2007 The Authors Journal compilation ª 2007 FEBS 109
B2-8
The proteins participate in binding of
cell-surface extracellular nucleic acids
V. S. Mal’shakova, P. P. Laktionov and V. V. Vlassov
Institute of Chemical Biology and Fundamental Medicine,
Novosibirsk, RUSSIAN FEDERATION
Extracellular nucleic acids are always presented in biological fluids
and at the cell surface. It has been demonstrated that not only apop-
tosis and necrosis, but active secretion by cells input into their gen-
eration. Although the data on mechanisms of secretion of nucleic

, S. Obermueller
1
, I. Lundquist
1
,
N. Brose
2
, E. Renstro
¨
m
1
and P. Rorsman
1,3
1
Lund University, Malmo
¨
, SWEDEN,
2
Max-Planck Institute for
Experimental Medicine, Go
¨
ttingen, GERMANY,
3
Oxford Center for
Diabetes, Endocrinology and Metabolism, Oxford, UK
CAPS1 is thought to play an essential role in mediating exocytosis
from transmitter storing large dense-core vesicles in neuroendocrine
cells. Whereas antibody inhibition studies suggested a role for
CAPS1 in late stages of secretion, studies on CAPS1-knockout mice
pointed to a role in vesicular storage of transmitters. The function of

Dept. of Medical Anatomy, Panum Institute,
University of Copenhagen, Copenhagen, DENMARK,
3
The
Norwegian Radium Hospital, Dept. of Biochemistry, Oslo,
NORWAY
Prostasomes are vesicles secreted by epithelial cells of the prostate
gland. The prostasome membrane is enriched in cholesterol and
sphingomyelin, and a recent proteomic study revealed that prosta-
somes contain at least 139 proteins. We have investigated whether
cholesterol plays a role in prostasome release using the human pros-
tate cancer cell line PC-3 as a model. Prostasomes released to the sup-
ernatants of PC-3 cells treated with cholesterol-depleting drugs were
isolated and analyzed by western blot. Caveolin-1 and LAMP-1, two
proteins found in PC-3 cells prostasomes, were used as markers of
prostasome release. Interestingly, methyl-beta-cyclodextrin (MBCD),
that in our experimental conditions reduced the cholesterol levels of
PC-3 cells by 40%, increased the amounts of both caveolin-1 and
LAMP-1 in prostasomes. Furthermore, the release of prostasomes
from PC-3 cells was also increased when the cellular cholesterol levels
were metabolically reduced by lovastatin and mevalonate. Moreover,
to investigate whether the total protein composition of PC-3 cells
and prostasomes was altered by MBCD, PC-3 cells were labelled
with [35S]methionine. The protein profiles of both cell lysates and
prostasomes were similar in control cells and in MBCD-treated cells,
but as expected, more radioactivity was associated with prostasomes
isolated from MBCD-treated cells. Finally, electron microscopy stud-
ies revealed that the morphology of prostasomes was not changed by
MBCD. In conclusion, these results suggest that cholesterol plays an
important role in the release of prostasomes.

uration? We provided here an example showing that the presence
of the TMD is compulsory for the productive folding and ER
secretion of a membrane glycoprotein. Tyrosinase is a type I mem-
brane protein regulating the pigmentation process in humans. We
have shown that soluble tyrosinase lacking its TMD is retained in
the ER becoming an ERAD substrate (1). We further used con-
structs of tyrosinase ectodomain fused with chimeric TMDs or
GPI anchor and found that all chimeras have been secreted (2).
The TMD controls tyrosinase folding by regulating the chaperone
recruitment at the translocon. This is a novel role for the TMD in
the folding and secretion of membrane bound proteins that adds to
our understanding of the multiple roles of TMDs and of the post-
translational regulation of protein expression.
References
1. Popescu CI, Mares A, Zdrentu L, Zitzmann N, Dwek RA, Pet-
rescu SM. J Biol Chem. 281, 21682–9, 2006.
2. Popescu CI, Paduraru C, Dwek RA, Petrescu SM, J Biol Chem.
280, 13833–40, 2005.
Abstracts Transport Machineries
110 ª 2007 The Authors Journal compilation ª 2007 FEBS
B2-12
Characterization of processing and secretion of
the N-terminal domain of a-dystroglycan
K. Matsumura, Y. Arai, F. Saito and T. Shimizu
Teikyo University School of Medicine, Tokyo, JAPAN
Purpose: Dystroglycan (DG) complex, composed of aDG and
bDG, provides a tight link between the extracellular matrix (ECM)
and intracellular cytoskeleton. aDG is an extracellular heavily gly-
cosylated mucin-type glycoprotein which is involved in the binding
of ECM proteins laminin and agrin. During the glycosylation of

¨
-Repo
University of Oulu, Oulu, FINLAND
The endoplasmic reticulum (ER) is the major intracellular storage
for calcium (Ca
2+
) in animal cells. The high [Ca
2+
]ER /
[Ca
2+
]cytoplasm ratio is established by the Sarco(endo)plasmic ret-
iculum Ca
2+
ATPase 2b (SERCA2b). High [Ca
2+
]ER enables effi-
cient Ca
2+
signaling but is also required for synthesis of several
secreted and membrane proteins, because several ER chaperones
and folding catalysts require Ca
2+
. G protein-coupled receptors
(GPCRs) are polytopic membrane proteins whose function requires
highly dynamic structure - the fact that makes them excellent but
challenging models for studying biogenesis and quality control of
membrane proteins. We found that SERCA2b interacts physically
with newly synthesized incompletely folded precursors of the
human delta opioid receptor (hDOR) as well as other GPCRs in

chondria, and showed that the necessary and sufficient targeting
information was contained in the respective N-termini. We have
now systematically analyzed the subcellular sorting of eleven
acyl-CoA synthetases. GFP reporter proteins were compared by
confocal microscopy to organelle specific markers. While many
fusion proteins were restricted to the ER, we also observed local-
ization to the Golgi apparatus, lipid droplets and the cytosol.
We believe that this remarkable diverse subcellular localization
within one enzyme family has functional implications. From
expression data it is obvious that multiple different acyl-CoA
synthetases are present at the same time in the same cell type.
Substrate specificities likely account for some of this apparent
redundancy. Our tentative hypothesis implies that the spatial
organization of acyl-CoA synthetase activity is also a key factor
in channeling fatty acids towards a particular metabolic fate.
B2-15
Thermodynamic studies on SNARE complex
assembly
K. Wiederhold and D. Fasshauer
Research Group Structural Biochemistry, Department of
Neurobiology, Max Planck Institute of Biophysical Chemistry,
Go
¨
ttingen, GERMANY
The proteins syntaxin 1A, SNAP-25 and synaptobrevin play an
essential role during Ca
2+
-dependent neurotransmitter release.
Their stepwise assembly into a stable SNARE complex between
synaptic vesicle and plasma membrane brings the two membranes

The formation of ternary SNARE-complexes is known to draw
apposing biological membranes close to each other and eventually
enable them to fuse. After fusion these extremely stable complexes
are dissociated into their single SNARE-components to allow fur-
ther rounds of fusion to occur. The disassembly reaction is endo-
thermic and therefore needs to be fuelled by the AAA-ATPase
NSF (N-ethylmaleimide sensitive factor) and its cofactors a-, b-
and c-SNAP. However, only little is known about how the disas-
sembly enzymes attack four-helix bundle SNARE complexes. In
order to investigate the reaction in molecular detail we established
several in vitro FRET and fluorescence anisotropy readouts for the
neuronal SNARE complex consisting of syntaxin1a, synaptobr-
evin2, and SNAP25. For all three neuronal SNAREs several single
cysteine variants exist to which fluorescent dyes can be specifically
attached. The fluorescence intensities of the dyes change upon
interaction of the respective proteins and allow for recording of
the speed of the disassembly process. By comparison of disassem-
bly kinetics of SNARE complexes in solution to that of complexes
incorporated into liposomes we attempt to determine possible
influences of membrane-anchorage on the efficacy of SNARE dis-
assembly. To characterize possible differences between the mammal
machinery to that of other organisms we have also performed
measurements using the yeast homologues of NSF/aSNAP called
sec18 and sec17 respectively.
B2-17
Studies on the molecular mechanism of the
acceleration of SNARE-mediated membrane
fusion by synaptotagmin 1
A. Stein, A. Radhakrishnan, D. Fasshauer and R. Jahn
MPI for Biophysical Chemistry, Go

P. Burkhardt and D. Fasshauer
Research Group Structural Biochemistry, Department of
Neurobiology, Max Planck Institute for Biophysical Chemistry, Go-
ettingen, GERMANY
The SNARE proteins syntaxin1, SNAP-25, and synaptobrevin play
a central role during Ca
2+
-dependent neurosecretion. Syntaxin1
and SNAP-25 are located in the plasma membrane, whereas syna-
ptobrevin resides on the synaptic vesicles. Their assembly into a
membrane-bridging ternary SNARE complex is believed to drive
membrane fusion. Sec1/Munc18-like (SM) proteins functionally
interact with SNARE proteins in vesicular fusion. Yet, structurally
and functionally dissimilar binding modes for SM proteins with
their cognate syntaxins have been uncovered: only a short N-ter-
minal peptide-finger of the syntaxin Sed5 binds to an outer surface
of the SM protein Sly1, whereas Munc18 tightly clasps around a
closed conformation of syntaxin 1, blocking its SNARE complex
association. We performed a detailed kinetic and thermodynamic
analysis of the Munc18/syntaxin1-interaction using a combination
of isothermal titration calorimetry (ITC) and fluorescence spectros-
copy. Furthermore, we investigated the influence of Munc18 on
the assembly pathway of the synaptic SNAREs. We now show that
the homologous peptide-finger of syntaxin1 and the closed confor-
mation of syntaxin1 simultaneously participate in binding to
Munc18. Consequently, the peptide-finger
´
s binding status deter-
mines the accessibility of Munc18-bound syntaxin for its partner
SNAREs, suggesting a common mechanism of how SM proteins

. The peripheral
association of toposome with the bilayer has been confirmed in 2
H-NMR experiments using liposomes composed of perdeuterated
dimyristoyl phosphatidyl serine. Chain orientational order was lar-
gely uneffected by toposome while quadropole echo decay times
were sensitive to toposome in the liquid crystalline but not the gel
phase. Collectively, our data support a model of toposome func-
tion in which Ca
2+
regulates both the association of toposome
with the membrane and subsequent toposome-driven, membrane-
membrane juxtaposition, perhaps as a prelude to a fusion event.
Acknowledgements: This work was supported by grants from
the Natural Sciences and Engineering Research Council of Canada
to JJR and MMR.
Abstracts Transport Machineries
112 ª 2007 The Authors Journal compilation ª 2007 FEBS
B3-1
The yeast proteins Ccz1p, Mon1p and Ypt7p are
involved in the movement and positioning of
vacuoles and nuclei
M. Hoffman-Sommer and J. Rytka
Institute of Biochemistry and Biophysics, Warsaw, POLAND
The Ccz1p protein of Saccharomyces cerevisiae is known to func-
tion in intracellular vesicular transport. It is necessary for efficient
trafficking of proteins to the vacuole and for proper vacuolar bio-
genesis. In its N-terminal part, Ccz1p shows homology to the
human protein HPS4, which is mutated in type 4 Hermansky-Pud-
lak syndrome. HPS4 is involved in intracellular transport and in
the biogenesis of lysosomes (the mammalian counterpart of yeast

P. Bayer
1
1
University of Duisburg-Essen, Essen, GERMANY,
2
Ruhr-Universita
¨
t Bochum, Bochum, GERMANY
The Parvulin Par14 is highly conserved in all metazoans and is
assumed to play a role in cell cycle and chromatin remodeling.
Recently, we confirmed the existence of a longer Parvulin mRNA
species expressed in all human tissues examined so far. This elon-
gated mRNA encodes a novel N-terminal domain whose expres-
sion was shown in human cell lysates. This novel Parvulin was
denoted as Par17 (Mueller et al., BMC Mol. Biol. 2006;7:9).
In contrast to Par14, endogenous Par17 was found in mitochond-
rial and membrane fractions of human cell lysates. Fluorescence of
EGFP fusions of Par17, but not Par14, co-localized with mitoch-
ondrial staining and the Par17 N-terminal domain was a prerequis-
ite for mitochondrial targeting. In vitro translated Par14 and Par17
associated with isolated human, rat and yeast mitochondria at low
salt concentrations, but only the Par17 mitochondrial association
was resistant to higher salt concentrations. Par17 was imported
into yeast mitochondria in a time and membrane potential-depend-
ent manner, where it reached the mitochondrial matrix. Moreover,
Par17 was shown to bind to double-stranded DNA under physio-
logical salt conditions. Based on the observation that the human
genome encodes Par17, but bovine and rodent genomes do not,
Par17 exon sequences from ten different primate species were
cloned and sequenced. Par17 is only encoded in the genomes of

Carrier proteins of the inner mitochondrial membrane are required
for transport of metabolites, nucleotides and cofactors across the
inner membrane. Mitochondrial carrier deficiency in humans leads
to lactic acidosis, cardiomyopathy, muscular hypotonia and causes
death in the first months of life. Like most mitochondrial proteins,
carrier proteins are synthesized as precursors in the cytosol and
imported into mitochondria in a post-translational manner. On the
way to their functional destination, carrier preproteins are first
translocated through the translocase of the outer membrane (TOM
complex). They are transferred across the intermembrane space
with the help of the soluble Tim9/Tim10 chaperone complex to the
inner membrane machinery, the carrier translocase (TIM22 com-
plex). The TIM22 complex consists of the integral components
Tim18, Tim22 and Tim54 and the peripheral components Tim12,
Tim9 and Tim10. Little is known about the function of Tim12 and
its role on the TIM22 complex. We analyzed the import and
assembly of Tim12. We show that newly imported Tim12 requires
endogenous IMS proteins to be assembled into the mature TIM22
complex. Tim12 import assembly is affected in Tim9 and Tim10
mutants, showing the requirement of these proteins for the Tim12
assembly. We purified the TIM22 complex and observed specific
interactions between components of the carrier translocase. Using
novel mutants of Tim12, we show that this protein play a dual role
in maintaining the stability of the TIM22 complex and in the bio-
genesis of mitochondrial carrier proteins.
B3-4
Assembly of TOM core proteins into the
mitochondrial outer membrane
D. Stojanovski, N. Pfanner and C. Meisinger
University of Freiburg, Freiburg, GERMANY

lyse the role of the SAM complex in the import and assembly of
alternative outer membrane proteins. This has revealed a role for
SAM components in the biogenesis of alternative Tom components
pointing to a novel and currently undefined function for the SAM
complex.
Transport Machineries Abstracts
ª 2007 The Authors Journal compilation ª 2007 FEBS 113
B3-5
Biogenesis of mitochondrial intermembrane
space proteins – the MIA pathway
D. Milenkovic
1
, K. Gabriel
1
, B. Guiard
2
, N. Pfanner
1
and
A. Chacinska
1
1
Institute for Biochemistry and Molecularbiology, Freiburg,
GERMANY,
2
Gentre de Ge
´
ne
´
tique Mole

M. Bohnert
1
, P. Rehling
1
, B. Guiard
2
, N. Pfanner
1
and
M. van der Laan
1
1
University of Freiburg, Freiburg, GERMANY,
2
Centre de
Ge
´
ne
´
tique Mole
´
culaire, CNRS, Gif-sur-Yvette, FRANCE
The majority of mitochondrial proteins are synthesized on cytoso-
lic ribosomes. A sophisticated import machinery catalyzes the
transport of these proteins to one of the four mitochondrial com-
partments, the outer membrane, the inner membrane, the inter-
membrane space and the matrix. Import of one subgroup of
mitochondrial proteins which is characterized by a positively
charged, cleavable, N-terminal signal sequence, is mediated by the
presequence translocase (TIM23 complex). The presequence tran-

Institute of Molecular Biology, Bratislava, SLOVAKIA,
2
Department of Biochemistry, Faculty of Natural Sciences,
Komenius University, Bratislava, SLOVAKIA,
3
Institute of
Microbiology, Academy of Sciences of Czech Republic, Prague,
CZECH REPUBLIC,
4
Department of Biochemistry and Molecular
Biology, UMDNJ-New Jersey Medical School, Newark, NJ, USA
Lon protease is important for degradation of misfolded, unassem-
bled, oxidatively damaged and short-lived proteins. In yeast and
mammalian cells the Lon protease is located exclusively in mito-
chondria, were the vast majority of mitochondrial matrix proteins
function as multi-subunit complexes. We founded that human as
well as yeast Saccharomyces cerevisiae Lon protease recognizes and
initially cleaves of folded subunits of mitochondrial processing
peptidase based on their surface determinants. No degradation was
observed in case of the functional complex of these subunits.
Moreover human mitochondrial Lon protease binds the mtDNA
and is responsible for degradation of some mtDNA binding pro-
teins. At present, Lon protease from C. parapsilosis has been
cloned and characterized with the aim to look for the possible role
in mtDNA nucleoid function.
B3-8
Reversible phosphorylation of the
mitochondrial fission/fusion machinery as a
determinant for neuronal survival
J. T. Cribbs

maintaining the integrity of the mitochondrial network. In a search
for relevant substrates, we identified critical phosphorylation sites
in two of the large GTPases that mediate mitochondrial morpho-
genesis. PKA-phosphorylation of dynamin-related protein 1 (Drp1)
sequestered Drp1 in the cytosol and inhibited its mitochondria-
fragmenting activity. PC12 cells in which endogenous Drp1 was
replaced by the constitutively active (Ala) mutant displayed
increased sensitivity to several apoptotic stimuli, while the hypoac-
tive, phospho-mimetic (Asp) Drp1 mutant interfered with caspase
activation and cell death. Phosphorylation at a second site in the
Charcot-Marie-Tooth disease protein mitofusin-2 (Mfn2) promoted
mitochondrial fusion. Thus, reversible phosphorylation of Drp1
and/or Mfn2 by PP2A/Bb2 and PKA/AKAP1 controls the mitoch-
ondrial fission/fusion equilibrium as the pivot point for neuronal
life and death decisions.
Abstracts Transport Machineries
114 ª 2007 The Authors Journal compilation ª 2007 FEBS
B3-9
The peroxisomal serine protease Deg15 from
Arabidopsis thaliana processes the PTS2 signal
H. Schuhmann
1
, P. F. Huesgen
1
, C. Gietl
2
and I. Adamska
1
1
University of Konstanz, Konstanz, GERMANY,

The matrix protein import into peroxisomes uses either one of the
two well-characterized peroxisomal targeting signals, PTS1 or
PTS2 that are recognized and bound by the receptor proteins
Pex5p and Pex7p. This recognition event is supposed to take place
in the cytosol, which however, has not been confirmed. Whereas
Pex5p is capable to perform its role in PTS1 protein targeting on
its own, the PTS2-receptor Pex7p needs the auxiliary proteins
Pex18p and Pex21p. After its formation, the receptor-cargo com-
plex docks to distinct proteins at the peroxisomal membrane, prob-
ably Pex13p and Pex14p. These two proteins together with Pex17p
form the receptor docking complex, responsible for the initial bind-
ing of the receptor-cargo complex at the membrane. We focussed
on the PTS2-dependent protein import into peroxisomes with
Fox3p as target protein. In order to isolate and characterize
Fox3p-complexes, we constructed strains expressing Fox3p fused
to protein A. This fusion-protein, which turned out to be fully bio-
logical active, was used to isolate protein complexes from either a
soluble cell fraction or from Digitonin-solubilized membranes by
affinity chromatography. Soluble Fox3p was associated with the
PTS2-receptor Pex7p and the auxiliary protein Pex18p. Membrane-
bound Fox3p in addition contains components of the docking
complex. Furthermore, we analyzed the size of the protein-com-
plexes by BN-PAGE and gel filtration. From these experiments,
we estimated an approximate size of 200 and 300 kD for the sol-
uble and membrane associated Fox3p-complex. Our data demon-
strate that the receptor-cargo recognition in PTS2-dependent
protein import takes place in the cytosol and that it does not
depend on components of the docking complex.
B3-11
Functional characterization of the co-receptor

are essential for its dislocation from the peroxisomal membrane to
the cytosol as well as for its subsequent proteasomal degradation.
B3-12
Ubiquitination is a prerequisite for the receptor
cycle of the peroxisomal import receptor Pex5p
H. W. Platta, F. El Magraoui, S. Grunau, D. Schlee,
W. Girzalsky and R. Erdmann
Institut fu
¨
r Physiologische Chemie, Ruhr Universa
¨
t Bochum,
GERMANY
The peroxisomal import receptor Pex5p binds its cargo proteins in
the cytosol, targets them to the peroxisomal membrane and shut-
tles back to the cytosol after cargo release. Dislocation of Pex5p
from the peroxisomal membrane back to the cytosol is performed
by the AAA-proteins Pex1p and Pex6p. Pex5p can be modified by
mono- or polyubiquitination at the peroxisomal membrane but the
functional role of this modification was not known. We demon-
strate that Pex5p is polyubiquitinated by Ubc4p when its export is
blocked. In contrast, monoubiquitination is present under wild-
type conditions, most likely as a physiological modification of
Pex5p. We demonstrate by in vivo and in vitro ubiquitination
experiments that Pex4p/Ubc10p facilitates Pex5p monoubiquitina-
tion. Pex4p is essential for the biogenesis of peroxisomes but its
molecular target has been a mystery for 13 years. The functional
role of ubiquitination was tested by in vitro export assays. When
both mono- and polyubiquitination of Pex5p were blocked, release
of the receptor was completely inhibited. From this we conclude

myces cerevisiae was used as a model organism to apply an organ-
ellar proteomic approach. We established a novel isolation
procedure for peroxisomes based on a combination of density gra-
dient centrifugation and affinity purification. Proteins from highly
purified and intact organelles were fractionated and analyzed by
mass spectrometry. This procedure enabled us to identify a number
of proteins previously not known to be associated with peroxi-
somes. Here we present potential candidates, their involvement in
oleate utilization and their localization.
B3-14
Molecular recognition of dysferlin-containing
proteins at the pero xisome membrane
S. Merich
1
, A. Korneli
2
, C. David
2
, R. Erdmann
2
and
C. Brocard
1
1
University of Vienna, Vienna, AUSTRIA,
2
Ruhr-Universita
¨
t
Bochum, Bochum, GERMANY

ing on the cell and tissue type and on the environmental conditions
of the cells. For example, yeast cells possess one or two peroxisomes
when grown on glucose as carbon source, but they contain up to
twenty when grown on oleate. Therefore, a molecular mechanism for
proliferation of peroxisomes must exist. Among the proteins known
to be directly involved in peroxisome proliferation are members of
the PEX11 protein family, small and basic proteins containing at
least one predicted transmembrane domain. In Saccharomyces cere-
visiae this family consists of PEX11, PEX25 and PEX27, the human
counterparts are PEX11A, PEX11B and PEX11G. Most likely, they
exert their function not alone but in a protein complex, and our aim
is the elucidation of the composition and function of these mem-
brane protein complexes.
We complemented pPex11-deleted yeast cells with plasmids expres-
sing either HsPEX11A or HsPEX11B proteins fused to GFP under
the control of the yeast PEX11 promotor, and followed the co-
localisation with the marker protein DsRed-SKL in the fluores-
cence microscope. In addition we generated constructs to express
yeast/human hybrid proteins. We aim to analyse and compare the
composition of the various protein complexes and the function of
their members in peroxisome proliferation.
B3-16
The role of EGY1 in plastid biogenesis
N. Li
The Hong Kong University of Science and Technology, Hong Kong,
CHINA
Egy1 was isolated as an ethylene-dependent gravitropism-deficient
and yellow green mutant. Molecular studies reveal that EGY1 gene
encodes a 59-kDa plastid-targeted metalloprotease and is up-regu-
lated by ethylene and light mainly at post-translational level in

According to the recently released data on Arabidopsis thaliana
genome there are at least three Athsp70 genes which could be pre-
dicted to be localized at the plastids. To identify the suborganeller
localization of the putative AtHsp70s, we have constructed cDNAs
of these three Athsp70 genes into expression vectors. Afterwards,
the respective recombinant precursor proteins were overexpressed
in Escherichia coli and were in vitro imported into intact chloro-
plasts. Currently, positive sub localizations of two of the putative
AtHsp70s have been identified.
We are further experimenting to characterize and to investigate the
possible functions of these proteins at the specific subcompart-
ments and/or their relation to the components of the protein trans-
location pathways of the chloroplasts.
B3-18
Regulation of protein translocation across the
chloroplast envelope
E. Schleiff
LMU Mu
¨
nchen, Mu
¨
nchen, GERMANY
Protein translocation across and into membranes is a fundamental
process as up to 50% of all proteins synthesized in the cytosol need
to traverse at least one membrane to reach their place of function.
The basic principles of protein translocation have been established,
although the extent to which the molecular mechanism of each sys-
tem has been determined varies. Proteins destined for chloroplasts
are synthesised in the cytosol as precursors containing a targeting
signal. They are subsequently delivered to the surface of the organ-

1
, I. Hapala
4
and G. Daum
1
1
Institute of Biochemistry, Graz University of Technology, Graz,
AUSTRIA,
2
Institute for Biophysics and Nanosystem Research,
Academy of Sciences, Graz, AUSTRIA,
3
Research Institute for
Electron Microscopy; TU Graz, Graz, AUSTRIA,
4
Institute of Ani-
mal Biochemistry and Genetics, Slovak Academy of Sciences, Ivanka
pri Dunaji, SLOVAKIA
In Saccharomyces cerevisiae the neutral lipids TAG (triacylglyce-
rols) and SE (steryl esters) are stored in so-called lipid particles.
Major yeast enzymes catalyzing synthesis of these neutral lipids,
namely the TAG synthases Dga1p and Lro1p, and the SE synthas-
es Are1p and Are2p, have been identified and partially character-
ized. To study the coordinate process of neutral lipid synthesis and
the specific contribution of each of the four acyltransferases to
lipid particle biogenesis we made use of triple mutants with only
one of the gene products active. All triple mutants form lipid parti-
cles, although at different rate, lipid composition, size and struc-
ture. These experiments also showed that TAG or SE alone are
sufficient to form lipid particles. Moreover, we studied yeast lipid

the presence of amino acids and activating signal transduction
pathways that induce many intracellular changes. In C. albicans,a
family of Gap1 homologues exists. In our work, we focused on
investigation the role of CaGAP genes. We would like to elucidate
the role of individual CaGap1 permeases in the virulence and path-
ogenecity of this species, together with the characterization of their
transport properties. For this we employ deletion of GAP genes in
the C. albicans SC5314 strain, using the URA3-based deletion cas-
sette, and heterologous expression of CaGAP genes in a S. cerevisi-
ae gap1D mutant to characterize substrate speficity and kinetic
parameters of individual CaGap permeases. This work was suppor-
ted by the GA CR 204/03/H066, MSMT LC531, and Czech-Fla-
mish bilateral project.
Transport Machineries Abstracts
ª 2007 The Authors Journal compilation ª 2007 FEBS 117
B4-2
Topological analysis of a haloacid permease in
a Burkholderia species
Y. Tse, M. Yu and J. Tsang
University of Hong Kong, Pokfulam Road, HONG KONG
There are many computer programs and databases used for the
prediction of protein structures and functions. Despite their useful-
ness, predictions are often incorrect and require experimental veri-
fication. Such limitation is exemplified by the study of a haloacid
permease in the bacterium Burkholderia sp. MBA4.
The haloacid permease Deh4p is a membrane protein of 552 resi-
dues. Analysis with Pfam, ScanProsite and BLAST against TCDB
suggested that Deh4p belongs to the major facilitator superfamily
(MFS). Proteins in the MFS have been proposed to contain 12 or
14 transmembrane regions. Prediction of Deh4p using programs

Deh4p dependent. Deh4p has an apparent K
m
of 5.5 and 8.9 M
and a V
max
of 9.1 and 23.1 nmol/mg/min for acetate and MCA,
respectively. A mutant with a transposon inactivated haloacid
operon failed to grow on MCA even when deh4a was provided
in trans.
B4-4
Influence of epigallocatechin gallate on artificial
lipid membranes
M. Dima, A. Iftime, A. Popescu and C. Ganea
Faculty of Medicine, Bucharest, ROMANIA
Epigallocatechin gallate (EGCG) is a natural flavonoid from the
tea plant (C. sinensis), with reported antioxidant and other health
beneficial effects. The Black Lipid Membranes (BLM) method has
been used to examine the interaction between EGCG and artificial
lipid bilayers, as reflected by recorded changes of the membrane
electrical parameters (capacitance and conductance). Experiments
were performed at constant EGCG concentrations in the BLM cu-
vettes and at increasingly higher concentrations, applied on both
sides of the membrane. At constant EGCG concentrations, a sta-
tistically significant difference (P<0.05) has been noticed between
insertion slopes at 5 lM and 20 lM, but not between the 20 lM
and 50 lM EGCG concentrations. This might suggests saturation
of insertion at higher EGCG concentrations.
In the presence of increasing EGCG concentrations the conduct-
ance showed non-significant statistical fluctuations, a possible clue
to a non-disruptive insertion of EGCG in the lipid bilayer (differ-

ratio of the iso-anteiso fatty acids.
Acknowledgements: This project is co-funded by the European
Union-European Social Fund (ESF) and National Sources, in the
framework of the program ‘Pythagoras II’ of the ‘Operational Pro-
gram for Education and Initial Vocational Training’ of the 3rd
Community Support Framework of the Hellenic Ministry of Edu-
cation
Abstracts Transport Machineries
118 ª 2007 The Authors Journal compilation ª 2007 FEBS
B4-6
The role of multidrug resistance gene 1 (MDR1)
in breast carcinoma resistance
R. Vaclavikova
1
, G. Alnaes
2
, M. Hubackova
1
, E. Kubala
3
,
R. Kodet
3
, M. Mrhalova
3
, J. Novotny
4
, I. Gut
1
,

affect function of P-gp and modify the clinical outcome.
Acknowledgements: Supported by GACR 305/07/P347, NIPH
Young Scientist Program and the National Program FUGE no.:
151924/150 and 15204/150, funded by The Research Council in
Norway.
B4-7
Crystal structures of a bacterial multidrug
transporter reveal a functionally rotating
mechanism
S. Murakami
Osaka University, Osaka, JAPAN
The emergence of bacterial multidrug resistance is an increasing
problem in the treatment of infectious diseases. AcrB and its
homologues are the major multidrug efflux transporter in gram-
negative bacteria, which confer intrinsic drug tolerance and multi-
drug resistance when they are overproduced. AcrB exports a wide
variety of toxic compounds including anionic, cationic, zwitterion-
ic, and neutral compounds directly out of the cells bypassing the
periplasm driven by proton motive force. It cooperates with mem-
brane fusion protein AcrA and outer membrane channel TolC.
The X-ray crystal structure of AcrB was first solved by Murakami
et al.[1] in 2002.
Now we solve the crystal structures of AcrB with and without sub-
strates in the new crystal form [2]. The new crystal structure solved
with new crystal form is asymmetric. The AcrB-drug complex con-
sists of asymmetric three protomers, each of which has different
conformation corresponding to one of the three functional states
of the transport cycle. Bound substrate was found in the periplas-
mic domain of one of the three protomers. The voluminous bind-
ing pocket is aromatic and allows multi-site binding. The

yeast is similar to MDR occurring in mammalian tumor cells.
Little is known about physiological substrates for ABC transporters
or their molecular mechanisms of function, mainly due to limited
structural information. Human ABC proteins have been implicated
in the transport of lipids and sterols. Likewise, Yor1p and Pdr5p in
the yeast S. cerevisiae may also be involved in maintaining or modu-
lating membrane lipid homeostasis, which is also true for their main
transcription factors Pdr1p and Pdr3p.
Our goal is to overexpress and purify the major ABC transporters
Pdr5p, Pdr10p, Pdr15p, Pdr12p and Snq2p from S. cerevisiae.We
use an approach as used successfully for Pdr5p, by genomically
tagging the genes with the 14-HIS tag at the N-terminus. This sys-
tem also allows for overexpression, as the PDR5 promoter drives
all genes, which is highly active in a genetic background harboring
the PDR1–3 gain-of-function alleles encoding a hyperactive tran-
scription factor. Purified ABC transporters will be subjected to
reconstitution experiments as well as structural studies to unravel
their molecular mechanism of function, which could perhaps lead
to a better understanding of clinically relevant ABC transporters.
B4-9
Localization and function of ABCC/MRP efflux
pumps and organic anion uptake transporters
in human brain
A. T. Nies
German Cancer Research Center, Heidelberg, GERMANY
Integral plasma membrane proteins decisively control cellular sub-
stance uptake and efflux. We analyzed the localization of multidrug
resistance proteins (MRPs) and of organic anion uptake transporters
(OATPs) in human brain. MRPs belong to branch C of the ATP-
binding cassette (ABC) transporters and mediate the ATP-driven

IBCP, Lyon, FRANCE
Multidrug resistance protein 1 (MRP1) is a member of the ATP-
binding cassette (ABC) superfamily of membrane transporters, some
of which, like MRP1, are involved in cancer cell Multidrug Resist-
ance (MDR) phenotype. It has been previously shown for
P-glycoprotein (Pgp) and MRP1 that verapamil could modulate
their function. Verapamil acts on Pgp as an inhibitor, whereas it acts
on MRP1 as a modulator. Verapamil activates glutathione transport
by MRP1, leading to cell apoptosis induced by a strong cellular
glutathione efflux. Verapamil exists under two enantiomeric forms,
and we therefore investigated the potential effects of verapamil e-
nantiomers on the MRP1 transport function. The results show that
S-verapamil is responsible for the strong cellular glutathione efflux,
while R-verapamil is not cytotoxic. However, S-verapamil alone has
a stronger effect than the racemic mixture and R-verapamil was
demonstrated to antagonize the activity of S-verapamil on MRP1.
Indeed, we showed that the R-enantiomer, as the S enantiomer,
inhibited calcein transport by MRP1. Furthermore, R-verapamil can
revert the cell growth resistance to vincristine. A molecular study on
purified MRP1 shows that, in correlation with cellular effects, both
enantiomers bind to MRP1 and the conformational changes of
MRP1 induced by the two enantiomers are quite different, mediating
two distinct and specific effects of the isomers on the transporter.
These results could be of great potential interest in therapy.
They show that verapamil enantiomers display opposite effects on
MRP1 function, S-verapamil acting as a modulator inducing gluta-
thione efflux, and R-verapamil acting as an inhibitor blocking the
transport activity.
B4-11
Contribution of non-apoAI:ABCA1 pathways for

(62.58 ± 24.8, 79.8 ± 7.44) and in LCAT(-)/ABCA1(-) (80.3 ±
15.4, 197.3 ± 16.34). CE content in steroidogenic tissue was signifi-
cantly decreased in LCAT(-)/ABCA1(-). FC level of female spleen
and male brain were also significantly decreased in ABCA1(-) back
ground mice. No significant differences of TC contents were
observed in kidney, thymus, heart, and lungs. These results demon-
strate that ABCA1 null mice showed significantly higher cholesterol
accumulation in the liver. The result corresponds to our previous
finding with the ABCA1 specific inhibitor, probucol, which demon-
strated the liver is the major organ for apoAI:ABCA1 mediated cel-
lular cholesterol release, production of nascent HDL particles.
Interestingly, the cholesterol deposition was accelerated in ABC/
LCAT double deficient mice. This indicates that one of cellular non-
apoAI:ABCA1 pathways must also highly contribute to cholesterol
homeostasis in liver. Involvement of ABCG1 transporter in this phe-
nomenon will also be examined in future studies.
B4-12
Lateral compartmentation of proteins and lipids
in the plasma membrane: involvement of the
membrane potential
M. Opekarova, Sr.
1
, G. Grossmann, Jr
2
, J. Malinsky
3
and
W. Tanner
4
1

M, Malinsky J, Weig-Meckl I and
Tanner W. Membrane potential governs lateral segregation of
plasma membrane proteins and lipids in yeast. EMBO J 2007;
26: 1–8.
B4-13
The change of taurine transport by oxidative
stress in the retinal capillary endothelial cell
and syncytiotrophoblast cells
Y. Kang
Sookmyung Women’s University, Seoul, REPUBLIC OF KOREA
Taurine transport to fetus occurs through syncytiotrophoblast, the
layer of placenta villi between maternal blood and fetal blood. The
retina is formed by retinal capillary endothelial cells (inner BRB,
iBRB) and retinal pigment epithelial cells (outer BRB). The inner
BRB plays important role in supplying nutrients to the retina from
the circulating blood.
We examined taurine transport by measuring [
3
H]taurine under
various conditions inducing cytokines, bacterial endotoxin, a com-
pound inducing a depletion of antioxidant, hydrogen peroxide and
nitric oxide donor at the iBRB (TR-iBRB cells) and in placenta
(TR-TBT cells). Taurine uptakes through taurine transporter
(TauT) showed a time, sodium ion and chloride ion-dependency,
and taurine uptake was decreased by PKC activator in TR-TBT
cells. Also, taurine uptake was decreased in calcium free condition
in those cells. Taurine transport in TR-iBRB cells was inhibited by
calcium channel blockers. The taurine uptake was increased by
TNF-a, LPS and DEM stimulation but decreased by H
2

aliphatic side chain with the positively charged functional groups.
A guanidino-containing cholesterol derivative, GEC-Chol, was syn-
thesized to improve the transfection efficiency and reduce cytotox-
icity. The physical parameters of these gene complexes including
morphology, size, surface charge of were evaluated. The GEC-
Chol formed cationic liposomes or micelles with diameters less
than 100 nm, and with the positive surface charges of 20–35 mV.
The transfection efficiency of GEC-Chol micelles was two order of
magnitude higher than other cationic cholesterols such as DC-
Chol. In addition, the micellar forms of lipid nanoparticles after
further functionalization displayed the structural stability and func-
tional selectivity in the cell culture system. Finally, the cationic
lipid-based nanostructures that contain inorganic nanoparticles
such as magnetic iron oxide nanoparticles or quantum dots have
been formulated and employed for live cell imaging and in vivo cell
tracking analysis.
B4-15
Dissecting a purine transporter: intrinsic
topogenetic signals, substrate binding and a
selectivity filter
A. Vlanti, A. Pantazopoulou, S. Amillis, I. Papageorgiou,
N. Lemuh, C. Gournas and G. Diallinas
University of Athens, Athens, GREECE
UapA, a member of the NAT family, is a uric acid-xanthine/H
+
symporter in Aspergillus nidulans. Mutant, chimaeric and truncated
versions of UapA were analysed by physiological tests, fluorescent
microscopy and kinetic assays using a plethora of purine ana-
logues. Specific internal regions were found to be critical for
expression in the plasma membrane, substrate binding and sub-

+
symport
E. Georgopoulou, G. Mermelekas, E. Karena, P. Karatza,
P. Panos and S. Frillingos
Laboratory of Biological Chemistry, University of Ioannina Medical
School, Ioannina, GREECE
To study structure-function relationships of the nucleobases-ascor-
bate transporter (NAT) family, we employ Cys-scanning mutagen-
esis of YgfO, the xanthine permease of E. coli. In this context, we
analyzed
324
QN
325
, a sequence motif conserved in all purine-trans-
porting NATs, and the flanking
304
DGLVSVIASAVG-
SLPLTTFA
323
and
326
NGVIQMTGVASRYVGR
341
. We found
that: (a) Q324 is essential for high-affinity xanthine transport;
(b) N325 is irreplaceable for active transport; (c) D304 is also
essential, as mutants D304E, D304N and D304C display strikingly
low uptake activity; (d) the environment of
324
QN

1
, M. A. Castro
1
, R. Maldonado
1
, D. Segretain
2
,
A. J. Yan
˜
ez
1
, J. C. Slebe
1
, J. C. Vera
3
and I. I. Concha
1
1
Universidad Austral de Chile, Valdivia, CHILE,
2
Universite
´
Paris
V, Paris, FRANCE,
3
Universidad de Concepcio
´
n, Concepcio
´