Electrical properties of plasma membrane modulate
subcellular distribution of K-Ras
Guillermo A. Gomez and Jose L. Daniotti
Centro de Investigaciones en Quı
´
mica Biolo
´
gica de Co
´
rdoba (CIQUIBIC, UNC-CONICET), Departamento de Quı
´
mica Biolo
´
gica, Universidad
Nacional de Co
´
rdoba, Argentina
Ras proteins are small GTPases localized mainly on
the cytoplasmic leaflet of cellular membranes, where
they operate as binary molecular switches between a
GDP-bound inactive and GTP-bound active state,
regulated by the concerted action of guanine nucleo-
tide exchange factors (GEFs) and GTPase-activating
proteins [1,2]. There are three ubiquitous isoforms
of Ras: K-Ras4B (referred to hereafter as K-Ras),
H-Ras, and N-Ras. These isoforms, encoded by differ-
ent genes, are more than 90% homologous, and their
functions are not redundant [3]. Ras proteins share a
conserved G-domain which contains a GTP-binding
cassette and an effector sequence involved in inter-
actions between Ras proteins and their prominent

E-mail:
(Received 28 November 2006, revised 16
February 2007, accepted 27 February 2007)
doi:10.1111/j.1742-4658.2007.05758.x
K-Ras is a small G-protein, localized mainly at the inner leaflet of the
plasma membrane. The membrane targeting signal of this protein consists
of a polybasic C-terminal sequence of six contiguous lysines and a farnesyl-
ated cysteine. Results from biophysical studies in model systems suggest
that hydrophobic and electrostatic interactions are responsible for the
membrane binding properties of K-Ras. To test this hypothesis in a cellular
system, we first evaluated in vitro the effect of electrolytes on K-Ras mem-
brane binding properties. Results demonstrated the electrical and reversible
nature of K-Ras binding to anionic lipids in membranes. We next investi-
gated membrane binding and subcellular distribution of K-Ras after dis-
ruption of the electrical properties of the outer and inner leaflets of plasma
membrane and ionic gradients through it. Removal of sialic acid from the
outer plasma membrane caused a redistribution of K-Ras to recycling
endosomes. Inhibition of polyphosphoinositide synthesis at the plasma
membrane, by depletion of cellular ATP, resulted in a similar subcellular
redistribution of K-Ras. Treatment of cells with ionophores that modify
transmembrane potential caused a redistribution of K-Ras to cytoplasm
and endomembranes. Ca
2+
ionophores, compared to K
+
ionophores,
caused a much broader redistribution of K-Ras to endomembranes. Taken
together, these results reveal the dynamic nature of interactions between
K-Ras and cellular membranes, and indicate that subcellular distribution
of K-Ras is driven by electrostatic interaction of the polybasic region of

toylation sites; instead, it contains a polybasic stretch
of six contiguous lysines which is critical for targeting
K-Ras to plasma membrane [8]. Together, the CAAX
motif and the second signal constitute the minimal
plasma membrane targeting signal of these proteins
[9,10]. Recent studies have demonstrated that protein
kinase C (PKC)-dependent phosphorylation on S181
at the hvr of K-Ras promotes translocation of this
protein to mitochondria, where it induces cell death
[11].
Ras isoforms, by regulating different effectors as
above, affect different signaling pathways. Recent
experimental evidence indicates that Ras signaling is
restricted to particular plasma membrane micro-
domains (e.g., caveolae and cholesterol-dependent or
-independent membrane domains) and to particular
intracellular compartments (including Golgi complex,
ER, mitochondria, and membranes from early and
recycling endosomes) [11–18]. Although recent studies
have shown that subcellular distribution and ⁄ or mem-
brane association dynamics of Ras isoforms are
important for their proper function, underlying mecha-
nisms of intracellular transport and distribution of
these proteins is not completely understood. Palmitoyl-
ation of H-Ras and N-Ras causes membrane trapping
early in the classical secretory pathway, and subse-
quent transport to plasma membrane through
association with exocytic vesicles [9,10]. Unlike farn-
esylation, which is a stable lipid modification of
proteins, depalmitoylation of H-Ras was shown to be

and cellular membranes through electrostatic and
hydrophobic interactions.
In the present study, we combined biochemical tech-
niques and fluorescence confocal microscopy analysis
to clarify the role of electrical properties of the plasma
membrane in the subcellular distribution of K-Ras. In
particular, we investigated (a) the role of surface
charge on inner and outer leaflet of plasma membrane
and (b) effect of ionic gradients through plasma mem-
brane on membrane binding and subcellular distribu-
tion of K-Ras in Chinese hamster ovary (CHO)-K1
cells. At steady state, K-Ras is associated with plasma
membrane, cytosol, and endosomal compartments, but
not with ER or Golgi membranes. Results from our
in vitro experiments demonstrate the electrical and
reversible nature of K-Ras binding to cellular mem-
branes, consistent with a proposed model of K-Ras
membrane association based on electrostatic interac-
tion [33]. Confocal microscopy analysis, in combina-
tion with live cell imaging, demonstrated that
enzymatic removal of sialic acid from the outer leaflet
caused a significant accumulation of K-Ras, but not
H-Ras, in recycling endosome membranes. Inhibition
of synthesis of polyphosphoinositides (poly PIs) in live
cells, by depletion of cellular ATP, resulted in signifi-
cant accumulation of K-Ras in a perinuclear region,
G. A. Gomez and J. L. Daniotti Membrane targeting of K-Ras
FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS 2211
colocalizing with recycling endosome and Golgi com-
plex markers. Finally, the dependence of ionic strength

(YFP), were described and partially characterized in
our previous study [16]. In order to evaluate expression
and subcellular distribution of these proteins, CHO-K1
cells were transiently transfected with corresponding
DNA constructs, and expression was monitored by
western blot analysis with an antibody directed to the
fluorescent protein. The antibody detected YFP and
YFP-K-Ras
C14
as bands of 27 kDa and 27.5 kDa,
respectively, and YFP-K-Ras
full
as a band of  55 kDa
according to the expected molecular mass (Fig. 1A).
Membrane association of the expressed fusion proteins
was investigated by ultracentrifugation of extracts
from mechanically lysed cells. YFP-K-Ras
C14
and
YFP-K-Ras
full
were associated mainly with the particu-
late fraction (65% and 63%, respectively) (Fig. 1B).
To analyze the degree of post-translational modifica-
tion, and to rule out possible association of these pro-
teins with insoluble components such as cytoskeleton,
nuclear remnants, or extracellular matrix, we per-
formed Triton X-114 partitioning assay on particulate
fractions of cells transiently expressing the fusion
proteins [34,35] (Fig. 1C). Fifty percent and 44% of

and YFP-K-
Ras
full
were extensively colocalized in cells that expres-
sed K-Ras mostly in plasma membrane (Fig. 1E),
as well as in the other phenotypes (data not shown).
These findings indicate that the C-terminal domain
of K-Ras operates as a membrane targeting motif when
fused to a soluble protein, and that the polybasic region
and post-translational modifications on this domain
could be relevant for proper function of K-Ras.
At steady state, K-Ras is associated with
plasma membrane, cytosol, and endosomal
compartments
In order to characterize subcellular distribution of
K-Ras in CHO-K1 cells at steady state, we performed
extensive colocalization analyses with markers of
organelles (Fig. 2 and Fig. S1). No colocalization was
observed between YFP-K-Ras
C14
and major histocom-
patability complex class II invariant chain isoform
lip33 fused to cyan fluorescent protein (lip33-CFP) and
calnexin, two ER markers, suggesting that the diffuse
pattern in the cytosol probably represents a soluble
fraction of the expressed protein. There was also no
colocalization between K-Ras
C14
and mannosidase II
(Man II), a medial Golgi marker or mitochondria

membrane and cytosol, and to a minor degree with
membranes from recycling endosomes.
Membrane binding properties of K-Ras
Results from model system experiments and theoretical
analyses suggest that membrane association and
plasma membrane targeting of K-Ras are a conse-
quence of the electronegative sensing function of the
C-terminal domain of this protein, and that membrane
association depends on both electrostatic and hydro-
phobic interactions between this domain and the
plasma membrane [25,26,37]. The models predict that
electrostatic interactions and plasma membrane associ-
ation are reduced when ionic strength of the medium
increases or when negative surface charge density of
membranes or net charge of the C-terminal domain
decreases. Mutagenesis experiments to reduce net
charge of the polybasic region of K-Ras gave results
consistent with the models [8,9,27,31,38].
To better characterize the membrane binding prop-
erties of K-Ras to biological membranes we per-
formed extensive biochemical experiments to evaluate
effects of various electrolytes (including poly l-lysine,
NaCl, and CaCl
2
) on membrane association of
K-Ras. We also investigated effects of these factors
on membrane binding properties of CFP-H-Ras
C20
[16], which is dually palmitoylated and does not
A

or YFP-K-Ras
full
were fixed with paraformaldehyde and visualized by confocal microscopy. Left, representative cell phenotypes show-
ing YFP-KRas
C14
subcellular distribution. Right, frequency of phenotypes (%) showing YFP-K-Ras
C14
and YFP-K-Ras
full
subcellular distribution.
Values are mean ± SEM for three or more experiments (300 cells analyzed for each condition). (E) CHO-K1 cells expressing both YFP-K-Ras
full
(pseudocolored red) and CFP-K-Ras
C14
(pseudocolored green). Right panel is a merged image from YFP-K-Ras
full
and CFP-K-Ras
C14
. Scale
bars ¼ 20 lm.
G. A. Gomez and J. L. Daniotti Membrane targeting of K-Ras
FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS 2213
contain a polybasic domain, and of GPI-YFP, a
fluorescent protein containing a glycosylphosphatidy-
linositol (GPI) attachment signal. When membrane
fractions from cells expressing YFP-K-Ras
C14
or
YFP-K-Ras
full

2+
is a central second messenger having a higher
affinity for anionic than zwitterionic and neutral
phospholipids [39]. Ca
2+
also promotes the formation
of lateral domains of phosphatidylserine (PS) in bila-
yers of mixed phosphatidylcholine and PS because of
the different affinities of these lipids [40–42]. It was
recently reported that the polybasic-prenyl motif of
K-Ras acts as a Ca
2+
⁄ calmodulin-regulated molecular
switch that controls plasma membrane concentration
of K-Ras, and redistributes its activity to internal sites
[43]. In view of these previous findings, we studied the
Fig. 2. At steady state, most of K-Ras is associated with plasma membrane, cytosol and to a minor extent to endosomal compartments. CHO-
K1 cells transiently expressing YFP-K-Ras
C14
were fixed and immunostained with antibodies for Man II, a medial Golgi marker; or fixed and
examined for the intrinsic fluorescence of CFP from lip33-CFP, an ER marker;
N27
GalNAc-T-CFP (
N27
GalNAc-T), a TGN marker or incubated with
MitoTracker or Alexa
647
-Tf (Tf) and then fixed. The expression of YFP-K-Ras
C14
was analyzed by the intrinsic fluorescence of YFP (pseudocolored

cantly dissociated under the same conditions.
Having demonstrated that K-Ras membrane associ-
ation depends on electrostatic interaction, we analyzed
in vitro the reversibility of such interaction. Cytosolic
A
i
ii
iii
BC
Fig. 3. Membrane binding properties of K-Ras. (A) Membrane fractions of CHO-K1 cells expressing YFP-K-Ras
C14
or YFP-K-Ras
full
or CFP-H-
Ras
C20
were obtained as described in Fig. 1B and then incubated for 1 h in solutions containing 0, 0.012 and 0.12 mgÆmL
)1
poly L-lysine (i)
or 0, 3 · 10
)6
, 1.5 · 10
)4
,3· 10
)2
,15· 10
)2
and 1.5 M NaCl (ii) or 0.1 · 10
)6
,5· 10

input. Proteinase K activity was monitored by measuring the degradation of a-tubulin present in total CHO-K1
extracts (lower panel).
G. A. Gomez and J. L. Daniotti Membrane targeting of K-Ras
FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS 2215
and particulate fractions were prepared from cells
expressing YFP, YFP-K-Ras
C14
, and YFP-K-Ras
full
,
and from nontransfected cells. Soluble fractions from
transfected cells were incubated with membranes from
nontransfected cells; conversely, membrane fractions
from transfected cells were incubated with cytosol
from nontransfected cells. Samples were incubated for
1 h at 4 °C and then ultracentrifuged to separate sol-
uble and particulate fractions. Presence of fluorescent
proteins in the fractions was evaluated by western blot
analysis. Results (Fig. 3B) showed that cytosol from
nontransfected cells caused 30% dissociation of mem-
brane associated K-Ras. Cytosolic YFP (a soluble pro-
tein) was recovered mostly in the soluble fraction,
indicating that it was not associated with membranes
from nontransfected cells. In contrast,  50% of sol-
uble K-Ras
C14
and K-Ras
full
was associated with mem-
branes from nontransfected cells. The K-Ras fraction

leaflet, and may also be involved in molecular rear-
rangement at the inner leaflet, and in cytosolic events
[44,45]. To evaluate the role of sialic acid in subcellular
distribution of K-Ras, CHO-K1 cells were treated with
neuraminidase (NANase). Neuraminidase activity was
assayed by conversion of GD1a (disialoganglioside) to
GM1 (monosialoganglioside) in a CHO-K1 clone stably
expressing UDP-GalNAc:LacCer ⁄ G3 ⁄ GD3 N-acetyl-
galactosaminyltransferase (GalNAc-T) and UDP-Gal:-
GA2 ⁄ G2 ⁄ GD2 ⁄ GT2 galactosyltransferase (Gal-T2)
glycosyltransferases [46] (Fig. 4A). Live cell imaging
analysis showed that neuraminidase treatment incre-
ased K-Ras
C14
, but not H-Ras
C20
, expression in a peri-
nuclear compartment (Fig. 4B), and that K-Ras
colocalized with recycling endosome markers but not
with cis ⁄ medial Golgi and TGN markers (Fig. 4C).
These changes were not due to modifications in shape
of neuraminidase-treated cells (results not shown).
Quantification of neuraminidase effect on subcellular
distribution of K-Ras (Fig. 4B) suggested that the
increase in number of cells showing K-Ras at the peri-
nuclear compartment is a consequence of a reduction
in number of cells showing cytosolic K-Ras expression.
Taken together, these results suggest a dynamic
interplay between the cytosolic, recycling endosome
and plasma membrane fractions of K-Ras. Independ-

ments with a TGN marker (
N27
GalNAc-T) and endo-
cytosed human Alexa
647
-Tf, a recycling endosome
marker [50]. We observed colocalization of YFP-K-
Ras
C14
with endocytosed Tf, and with TGN marker,
in ATP-depleted cells (Fig. 5B). No colocalization
was observed between K-Ras and
N52
Gal-T2-CFP
Membrane targeting of K-Ras G. A. Gomez and J. L. Daniotti
2216 FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS
(
N52
Gal-T2), a medial Golgi marker. These results sug-
gest that surface charges from poly PIs at the inner
leaflet are necessary for proper membrane binding and
subcellular distribution of K-Ras.
Calcium ionophore redistributes K-Ras
to endomembrane
In vitro experiments in this study and others have dem-
onstrated that binding of lipid modified cationic pep-
tides, YFP-K-Ras
C14
and YFP-K-Ras
full

Calcium affects membrane surface potential shielding
negative charges of plasma membrane, stimulating PI
hydrolysis and PS ‘flipping out’ in a Ca
2+
-scramblase
dependent fashion (Fig. S4) [25,40,42,54,55]. Following
treatment of YFP-K-Ras
C14
-expressing CHO-K1 cells
with A23187, live cell confocal microscopy showed a
clear dissociation of this protein from plasma membrane
(Fig. 6A and Video S1). Perinuclear and scattered struc-
tures were also decorated with K-Ras. Similar redistri-
bution was observed for full-length K-Ras fused to YFP
A
B
C
Fig. 4. Enzymatic release of sialic acid redis-
tributes K-Ras to recycling endosomes. (A)
CHO-K1 cells or a parental clone 4 stably
expressing GalNAc-T and Gal-T2-HA were
treated or not with 1.5 UÆmL
)1
NANase for
2 h at 37 °C. Then, cells were shifted to
4 °C and incubated with cholera toxin for
30 min. Homogenates were analyzed by
western blot using antibodies to reveal the
A subunit of cholera toxin (CTx-A) and
Gal-T2-HA (left). Densitometric analysis of

N27
GalNAc-T-CFP (
N27
Gal-
NAc-T, red) or cells expressing YFP-K-Ras
C14
(K-Ras
C14
, green) and labeled with Alex-
a
647
-Tf (Tf; red) were treated (NANase) or
not (control) with 1.5 UÆmL
)1
NANase for
2 h, fixed and visualized by confocal micros-
copy. Panels are merged images from
YFP-K-Ras
C14
and the corresponding organ-
elle marker. The insets show details of the
boxed area at higher magnification. Scale
bars ¼ 10 lm for (B) and 5 lm for (C).
G. A. Gomez and J. L. Daniotti Membrane targeting of K-Ras
FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS 2217
(data not shown). In contrast, YFP-H-Ras
C20
and
GPI-CFP showed no redistribution under the same con-
ditions (Fig. 6A and Video S1). A23187 function was

distribution of YFP-K-Ras
C14
.
Increase in cytosolic Ca
2+
can cause PKC activa-
tion and consequent K-Ras phosphorylation [11]
and ⁄ or Ca
+2
⁄ calmodulin binding to K-Ras [43]. We
evaluated membrane affinity of K-Ras
C14
under the
conditions described in Fig. 6B. Membrane affinity of
K-Ras was not changed by any of the experimen-
tal conditions (Fig. 6C). These results suggest that
redistribution of K-Ras from plasma membrane to
endomembranes is not a consequence of further post-
translational modifications or association with cytoso-
lic protein; rather, K-Ras responds to local changes in
membrane properties which are lost during subcellular
fractionation.
To further characterize the subcellular distribution
of YFP-K-Ras
C14
under the different conditions shown
in Fig. 6B, we performed extensive colocalization
experiments using organelle markers (Fig. 6D). Chan-
ges in calcium level caused alterations in morphology
of Golgi complex and ER. This phenomenon was evi-

; green) and
N52
Gal-T2-CFP (
N52
Gal-T2; red) or
N27
GalNAc-
T-CFP (
N27
GalNAc-T; red) or cells expressing
YFP-K-Ras
C14
(green) and labeled with
Alexa
647
-Tf (Tf; red) were treated as des-
cribed above, fixed and visualized by confo-
cal microscopy. Panels are merged images
from YFP-K-Ras
C14
and the corresponding
organelle marker. The insets show details of
the boxed area at higher magnification.
Membrane targeting of K-Ras G. A. Gomez and J. L. Daniotti
2218 FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS
endogenous resident proteins (data not shown). K-Ras
was colocalized to a minor extent with lip33-YFP,
an ER marker, in Ca
2+
-depleted cells and Ca

membrane to the endomembrane system, according
probably to their physical and chemical properties.
Change in intracellular pH does not affect K-Ras
subcellular distribution
Because ionophore A23187 operates as a Ca
2+
⁄ H
+
exchanger (see above), its observed effect on K-Ras
distribution could conceivably result from modification
of not only calcium homeostasis but also intracellular
pH. To test this possibility, we abolished pH gradients
across the endomembrane system using the polyether
ionophore monensin (a Na
+
⁄ H
+
exchanger), and
AB
C
D
-Ca
2+
+Ca
2+
+A23187
Fig. 6. Ca
2+
influx causes K-Ras to redistribute from plasma membrane to the endomembrane system. (A) CHO-K1 cells expressing
YFP-K-Ras

PM > Cyt phenotypes for YFP-K-Ras
C14
expression (%). (C) Homogenates from cells expressing K-Ras
C14
were treated as described in
(B), lysed and ultracentrifugated. The supernatant (S) was recovered and the particulate fraction (P) resuspended in lysis buffer.
YFP-K-Ras
C14
expression was investigated by western blot. The percentage of YFP-K-Ras
C14
associated to P fraction is indicated. (D)
CHO-K1 cells coexpressing CFP-K-Ras
C14
(K-Ras
C14
) and lip33-YFP (lip33) or YFP-K-Ras
C14
and
N52
Gal-T2-CFP (
N52
Gal-T2) or
N27
GalNAc-T-
CFP (
N27
GalNAc-T) or cells expressing YFP-K-Ras
C14
and labeled with MitoTracker or endocyted Alexa
647

of the monovalent cation K
+
on K-Ras subcellular
distribution in CHO-K1 cells, we used the K
+
iono-
phore valinomycin, which forms K
+
-selective pores
through which K
+
can flux across the cell membrane
[57]. K-Ras
C14
showed a rapid and significant accumu-
lation (10% increment) in a perinuclear compartment
defined as recycling endosome by colocalization with
endocytosed Alexa
647
-human Tf (Fig. 8A,B). This
effect was enhanced (15% increment) when extracellu-
lar K
+
was increased to 55 mm. The results for valino-
mycin and for A23187 suggest that cytosolic ionic
composition and transmembrane potential are relevant
for plasma membrane targeting of K-Ras.
Discussion
Membrane potential in biological membranes is deter-
mined by three main components: (a) transmembrane

B
Fig. 7. pH gradients does not affect subcellular distribution of
K-Ras. (A) CHO-K1 cells transiently expressing YFP-K-Ras
C14
(upper
panels) and YFP-H-Ras
C20
(middle panels) were incubated with
10 l
M monensin (Monensin) or vehicle (Control) for 30 min at
37 °C and visualized alive by confocal microscopy. Cells treated as
described above and labeled with Lysotracker are shown at the
bottom. Images from control and monensin treated cells were
acquired with identical acquisition settings. (B) Cells were treated
as described above, fixed and visualized by confocal microscopy.
Graphic shows the frequency of phenotypes (%) showing
YFP-K-Ras
C14
subcellular distribution both in control and monensin
treated cells. Scale bars ¼ 20 lm.
Membrane targeting of K-Ras G. A. Gomez and J. L. Daniotti
2220 FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS
potential regulates lateral distribution of K-Ras after it
binds to membrane.
The third component of membrane potential, elec-
trostatic membrane surface potential, is a consequence
of incomplete quenching of the net excess of surface
charge found in membrane surfaces [64]. Strength of
this potential depends on surface charge density, ionic
strength, and the dielectric constant of the membrane

M KCl + DMSO) or DMEM and 10 lM valinomicyn (5 mM KCl + valinomicyn) or incubated in
Locke’s, high K
+
, and dimethylsulfoxide (55 mM KCl + DMSO) or Locke’s, high K
+
, containing 10 l M valinomicyn (55 mM KCl + valynomicin)
and visualized alive by confocal microscopy. Images are representative from PM > Cyt phenotype (media plus dimethylsulfoxide) and perinu-
clear phenotype (media plus valinomicyn) of YFP-K-Ras
C14
subcellular distribution (left). Cells were treated as described above, fixed and visu-
alized by confocal microscopy. The graphic (right) shows the frequency of phenotypes (%) showing YFP-K-Ras
C14
subcellular distribution in
cells incubated in 5 m
M KCl or 5 mM KCl ⁄ 10 lM valinomicyn or 55 mM KCl ⁄ 10 lM valinomicyn. (B) CHO-K1 cells coexpressing YFP-K-Ras
C14
(K-Ras
C14
; green) and
N27
GalNAc-T-CFP (
N27
Gal-NAc-T; red) or cells expressing YFP-K-Ras
C14
and labeled with Alexa
647
-Tf (Tf; red) were incu-
bated for 20 min at 20 °C in Locke’s media and dimethylsulfoxide (55 m
M KCl) or Locke’s, high K
+

of calmodulin with not significant differences from
results from NaCl experiments, suggesting an unspecific
effect of Ca
2+
on membrane affinity of K-Ras.
A significant proportion of ectopically expressed
YFP-K-Ras
C14
and YFP-K-Ras
full
was found in the
soluble fraction (35% and 37%, respectively) after
ultracentrifugation. This could be a consequence of (a)
equilibrium between the soluble and particulate pools;
(b) association with cytosolic escort proteins; and⁄ or
(c) post-translational modification that affect mem-
brane binding of K-Ras. Regarding possibility (c),
recent studies showed that PKC-dependent phosphory-
lation of S181 within the hvr of oncogenic K-Ras leads
to dissociation of K-Ras from plasma membrane [11].
Our present results indicate that a significant propor-
tion of soluble K-Ras
C14
and K-Ras
full
was reversibly
associated with membranes from nontransfected CHO-
K1 cells. Thus, the soluble pool of K-Ras appears to
undergo a dynamic exchange with the particulate pool
under our experimental conditions.

observe significant externalization of PS in ATP-
depleted cells (Fig. S3). The normal subcellular distri-
bution of PIs is unclear [71], but it appears that PIP2
is located at the plasma membrane, while PI(3)P and
PI(4)P are associated with membranes from endo-
somes and Golgi complex. When synthesis of poly PIs
is inhibited, PIP2 is first degraded to phosphatidy-
linositolphosphate by specific phosphatases, resulting
in accumulation of these lipids in the cell. This cata-
bolism can shift to some extent the negative surface
charge density gradient between plasma membrane
and endosomal and Golgi membranes, causing K-Ras
to localize in intracellular compartments. Depletion of
ATP led to cessation of kinase activity, and we spe-
culate that phosphorylation on hvr (S181) of K-Ras
is not operating under this experimental condition.
Because ATP is necessary for intracellular vesicular
transport [78–80], K-Ras may translocate to endo-
membranes via a nonvesicular pathway following its
dissociation from plasma membrane [15,25–28]. This
translocation could result from diffusion down an
electronegative gradient, because negative charge den-
sity in normal cells is greater at the plasma mem-
brane than in intracellular membranes (plasma
membrane > recycling endosomes > Golgi com-
plex > ER) [71,81].
The dependence of ionic strength on plasma mem-
brane targeting of K-Ras was evaluated using a bat-
tery of ionophores. Changes in subcellular distribution
of K-Ras were observed only for ionophores that mod-

the cell. The dynamic interaction of K-Ras with mem-
branes, and the fact that knockout of K-Ras, but not
H-Ras or N-Ras is lethal in mice, suggest that the plei-
otropic subcellular distribution of K-Ras is essential
for its proper activity.
Experimental procedures
Plasmids
Expression plasmids for yellow fluorescent protein (YFP)-
K-Ras
C14
and YFP-H-Ras
C20
,
N27
GalNAc-T-CFP and
N52
Gal-T2-CFP have been described previously [16,82].
Plasmid encoding YFP-K-Ras
full
was kindly supplied by
M. Philips (New York University School of Medicine,
New York, NY). GPI-YFP fusion construct was kindly
supplied by P. Keller (Max-Plank Institute, Dresden,
Germany). Plasmid encoding GFP-PH-PLCd1 was kindly
supplied by M. Lemmon (University of Pennsylvania
School of Medicine, Philadelphia, PA).
Cells lines, cell culture and DNA transfections
The following CHO-K1 cell clones were used: wild-type
CHO-K1 cells (ATCC, Manassas, VA) and clone 4, a
stable double transfectant expressing GalNAc-T and Gal-

and harvested by scra-
ping in 5 mm Tris ⁄ HCl (pH 7.0) (buffer T). Extracts were
centrifuged at 4 °C for 5 min at 13 000 g using a F-45-24-11
rotor in a 5415R centrifuge (Eppendorf, Hamburg, Ger-
many) and resuspended in 400 lL of buffer T in the presence
of 5 lgÆmL
)1
aprotinin, 0.5 lgÆmL
)1
leupeptin and
0.7 lgÆmL
)1
pepstatin (buffer T ⁄ protease inhibitor mixture;
T-PIM). Pellets were dispersed by vortex and passed 60 times
through a 25-gauge needle. Nuclear fractions and unbroken
cells were removed by centrifuging twice at 4 °C for 5 min at
600 g using a F-45-24-11 rotor in a 5415R centrifuge (Eppen-
dorf). Supernatants were then ultracentrifuged at 4 °C for
1 h at 400 000 g using a TLA 100.3 rotor (Beckman Coulter,
Inc., Fullerton, CA). The supernatant (S fraction) was
removed, and the pellet (P fraction) was resuspended in
400 lL of T-PIM for subsequent western blot analysis.
Triton X-114 partition assay
P fractions were solubilized for 1 h at 4 °C in 1% Triton
X-114 in NaCl ⁄ P
i
-PIM. Then, samples were incubated at
37 °C for 3 min and centrifuged at 13 000 g using a F-45-
24-11 rotor in a 5415R centrifuge (Eppendorf). The aque-
ous upper phase (A) and the detergent-enriched lower

FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS 2223
(Beckman Coulter). Membrane binding of soluble K-Ras
protein was assayed by incubation of S fractions obtained
from transfected cells with P fractions obtained from un-
transfected cells during 1 h at 4 °C. The mix was then cen-
trifuged at 400 000 g using a TLA 100.3 rotor in an
Optima TLX ultracentrifuge (Beckman Coulter), for 1 h at
4 °C. K-Ras distribution in S and P fractions was analyzed
by western blot. Membrane dissociation of K-Ras was
assayed in the same condition but using P fractions from
K-Ras transfected CHO-K1 cells and S fractions from un-
transfected CHO-K1 cells.
Topology assays
Membrane fractions from cells expressing YFP-K-Ras
C14
and membrane fractions obtained after incubation of par-
ticulate fractions from unstransfected cells and cytosol from
K-Ras transfected cells were resuspended in 200 lL of buf-
fer T containing 200 lgÆmL
)1
BSA or 200 lgÆmL
)1
trypsin
(Try) and further incubated at 37 °C for 1 h. Reactions
were stopped by addition of 10% (w ⁄ v, final concentration)
trichroloacetic acid. Proteins were then recovered by cen-
trifugation at 13 000 g for 30 min at 4 °C using a F-45-24-
11 rotor in a 5415R centrifuge (Eppendorf), resuspended in
sample buffer and analyzed by western blot.
Electrophoresis and western blot

i
and fixed in 3% (v ⁄ v) paraformaldehyde (30 min
at 4 °C).
ATP depletion treatment
ATP depletion in CHO-K1 cells was performed as des-
cribed by [49]. Briefly, 24 h after transfection cells were
washed twice with DMEM without glucose (Gibco, Invitro-
gen, Carlsbad, CA) and incubated in the same media
containing 5 mm NaN
3
and 50 mm 2-deoxi-d-glucose
(ATP-depleted cells) or water (vehicle) and d-(+)-glucose
(control cells) for 1 h. Then, cells were directly visualized or
washed with NaCl ⁄ P
i
and fixed in 3% (v ⁄ v) paraformalde-
hyde (30 min at 4 °C).
Calcium depletion and A23187 treatment
Twenty-four hours after transfection, cells were washed three
times with extracellular solution [140 mm NaCl, 5 mm KCl,
1mm MgCl
2
,10mm glucose, 0.1% BSA, 15 mm Hepes
pH 7.4, extracellular solution (ECS)] without calcium and
then incubated for 1 h in the same media containing 10 lm
BAPTA-AM (Molecular Probes) and 10 mm EGTA. Then,
cells were washed in the absence of chelators (Chel) and incu-
bated for 1 h with ECS without calcium (–Ca
2+
treatment)

meric proteins were washed twice with DMEM and incu-
bated for 15 min with 10 lm valinomycin or 25 lm
monensin or vehicle for control cells and then visualized
alive or fixed for fluorescence microscopy. For high K
+
and valinomycin incubations, cells were washed with 1·
buffer Lockes, high K
+
(55 mm KCl, 85 mm NaCl,
2.4 mm NaHCO
3
, 1.8 mm CaCl
2
,5mm Hepes pH 7.2) and
then incubated for 20 min in the same media containing
10 lm valinomycin.
Membrane targeting of K-Ras G. A. Gomez and J. L. Daniotti
2224 FEBS Journal 274 (2007) 2210–2228 ª 2007 The Authors Journal compilation ª 2007 FEBS
Live cell imaging
Live cells experiments were performed at 20 °C on a Carl
Zeiss LSM5 Pascal laser scanning confocal microscope
(Carl Zeiss AG, Go
¨
ttingen, Germany) or an Olympus
FluoView FV1000 confocal microscope (Olympus Latin
America, Miami, FL) equipped with a argon laser and a
63· Plan-Apochromat objective using a pinhole appropriate
to obtain 0.8 lm optical slices. Images for each experiment
were taken during 30 min.
Endocytosis of Alexa

lengths and filter set for CFP, GFP, YFP and Alexa
647
were described previously [16]. MitoTracker fluorescence
was detected using the filters for rhodamine or Cy5. Ima-
ges of fixed cells for quantitative purposes were acquired
using a 63· Plan-Apochromat oil immersion objective
using a pinhole appropriate to obtain an optical slice of
0.8 lm. For colocalization experiments, images were taken
using a 100· Plan-Apochromat NA 1.4 oil immersion
objective.
Phenotypic analysis was performed using the meta-
morph
Ò
4.5 (Molecular Devices, Sunnyvale, CA) software
using the threshold function. Statistical significance (P)
(t-student test) was performed from at least three independ-
ent experiments (600 cells per experiment). Asterisks repre-
sent P < 0.1 versus control conditions.
Acknowledgements
This work was supported in part by grants from Secre-
tarı
´
a de Ciencia y Tecnologı
´
a, Universidad Nacional de
Co
´
rdoba (162 ⁄ 06); Consejo Nacional de Investigaci-
ones Cientı
´

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Supplementary material
The following supplementary material is available
online:
Fig. S1. Subcellular distribution of K-Ras.
Fig. S2. Topological distribution of K-Ras in mem-
branes from CHO-K1 cells.
Fig. S3. Effect of ATP depletion on PIP2 content and
PS externalization in CHO-K1 cells.
Fig. S4. Subcellular distribution of A23187 and its
effect on H
+
homeostasis, PS externalization and PIP2
content in CHO-K1 cells.
Video S1. Ca
2+
influx causes K-Ras, but not GPI