Mammalian Gup1, a homolog of Saccharomyces cerevisiae
glycerol uptake/transporter 1, acts as a negative regulator
for N-terminal palmitoylation of Sonic hedgehog
Yoichiro Abe
1
, Yoshiko Kita
1
and Takako Niikura
1,2,
*
1 Department of Pharmacology, Keio University School of Medicine, Tokyo, Japan
2 Department of Neurology, Georgetown University, Washington, DC, USA
Sonic hedgehog (Shh), a member of the vertebrate
Hedgehog (Hh) family [1–4], is an extracellular
secreted signaling molecule that is involved in embry-
onic patterning and organogenesis (for example, in the
dorsal–ventral polarity of the spinal cord and in the
anterior–posterior polarity in the limb bud) in a con-
centration-dependent manner [5].
Shh is initially translated as a precursor protein of
 45 kDa. After excision of the signal sequence, it
undergoes automatic cleavage to release a biologically
Keywords
Gup1; hedgehog acyltransferase;
membrane-bound O-acyltransferase;
palmitoylation; Sonic hedgehog
Correspondence
Y. Abe, Department of Pharmacology,
Keio University School of Medicine,
35 Shinanomachi, Shinjuku-ku,
Tokyo 160-8582, Japan

nize the palmitoylated N-terminal signaling domain of Shh under denatur-
ing conditions. On the other hand, Gup1 interfered with the palmitoylation
of Shh catalyzed by endogenous Skn in COS7 and NSC34. These results
suggest that Gup1 is a negative regulator of N-terminal palmitoylation of
Shh and may contribute to the variety of biological actions of Shh.
Abbreviations
CHO, Chinese Hamster ovary; CM, conditioned medium; EGFP, enhanced green fluorescent protein; ER, endoplasmic reticulum; GRP78,
78-kDa glucose-regulated protein; Gup1, glycerol uptake ⁄ transporter 1; HHAT, hedgehog acyltransferase; HRP, horseradish peroxidase;
IP, immunoprecipitation; IRES, internal ribosome entry site; MBOAT, membrane-bound O-acyltransferase; Shh, sonic hedgehog; Shh-N,
N-terminal signaling domain of Shh without cholesterol modification; Shh-Np, autoprocessed N-terminal signaling domain of Shh;
TRITC, tetramethylrhodamine isothiocyanate.
318 FEBS Journal 275 (2008) 318–331 ª 2007 The Authors Journal compilation ª 2007 FEBS
active N-terminal signaling domain of  19 kDa [6–11],
which is followed by the addition of cholesterol to its
C-terminal Gly residue, a process catalyzed by the
C-terminal catalytic domain [12]. This autoprocessed
N-terminal signaling domain of Shh (Shh-Np [9]) is also
palmitoylated at its N-terminal Cys residue by the
hedgehog acyltransferase (HHAT) called Skn [13,14], a
homolog of the Drosophila skinny hedgehog (also called
sightless, central missing,orrasp) [15–18] gene product,
in an amide-linked manner [14]. These unique lipid
modifications greatly reduce the diffusibility of Shh-Np
and tether it to the cellular membrane. However, they
are necessary to regulate the movement of the protein
to form the proper concentration gradient. The critical
role of cholesterol modification in the movement of
Hh protein has been demonstrated in both vertebrates
and invertebrates [9,19–23]. Palmitoylation is also
involved in the regulation of movement of Shh-Np in

toylated in a mammalian cell line transfected with full-
length human Shh [14]. Thus, it is possible that, in
addition to palmitoylated Shh-Np, nonpalmitoylated
Shh-Np is also produced in vertebrates in vivo,
and that a combination of palmitoylated and non-
palmitoylated Shh-Np contributes to cell fate specifica-
tion during development.
Mammalian glycerol uptake ⁄ transporter 1 (Gup1) is
described in the National Center for Biotechnology
Information gene database as a homolog of Saccharo-
myces cerevisiae Gup1 [29] from its sequence homol-
ogy. It has also been found to have sequence
homology to Drosophila skinny hedgehog gene product
and to mammalian Skn [13,30]. The function of the
mammalian Gup1 is still unclear. However, it has a
motif characteristic of the membrane-bound O-acyl-
transferase (MBOAT) superfamily [31], like Drosoph-
ila skinny hedgehog gene product and mammalian Skn,
as well as yeast Gup1 [32]. One strange thing that has
been observed, however, is that in mammalian Gup1,
the highly conserved His residue in the motif indis-
pensable to the acyltransferase activity of the MBOAT
superfamily has been replaced with a Leu residue.
Therefore, it is possible that Gup1 has some function
related to the post-translational modification of the
mammalian hedgehog family, although it may have no
acyltransferase activity. In this work we examined
whether mammalian Gup1 has a role in regulating the
palmitoylation of Shh, by using a novel technique,
developed in this study, for detecting the palmitoylated

(Fig. 1). In our examination so far, the majority of the
Shh was autoprocessed and 19-kDa Shh-Np was pre-
dominantly detected in the lysate of all lines using
another anti-Shh IgG, H-160, which was raised against
the N-terminal portion (amino acids 41–200) of
human Shh (Fig. 1A–D, lane 2). Consistent with the
previous report, 5E1 failed to recognize Shh-Np in the
lysate of CHO cells (Fig. 1A, lane 2), although it rec-
ognized full-length Shh (Fig. 1A, lanes 2 and 6). This
was also the case with Shh-Np in the lysate of HeLa
cells (Fig. 1B). Remarkably, 5E1 recognized Shh-Np in
the lysate of COS7 and NSC34 cells, even under dena-
turing conditions (Fig. 1C and D, lane 2). The differ-
ence between CHO ⁄ HeLa and COS7 ⁄ NSC34 cell lines
in the reactivity of 5E1 with Shh-Np was attributed to
the existence of endogenous Skn in the latter lines, as
determined by RT-PCR analysis (Fig. 2), suggesting
that Skn affects the reactivity of Shh-Np with 5E1,
regardless of cell type. To confirm this, we transfected
full-length Shh together with FLAG-tagged mouse Skn
into these lines. As expected, 5E1 efficiently recognized
Shh-Np in the lysate of all lines under this experimen-
tal condition without affecting the level of Shh-Np
(Fig. 1A–D, lane 3). Ectopic expression of Skn led to
a reduction in the amount of Shh-Np secreted into the
conditioned media (CM) from all lines (Fig. 1A–D,
lane 3), suggesting increased hydrophobicity of the
protein, probably as a result of palmitoylation
catalyzed by Skn. Similar results were obtained by
using monoclonal anti-Shh N-terminal fragment,

recognized by 5E1 (Fig. 3A,B, lanes 3 and 5), whereas
wild-type Shh-Np clearly was (Fig. 3A,B, lane 1). In
the presence of exogenously transfected Skn, C25A-
Shh was not recognized by 5E1 (Fig. 3A,B, lane 6).
These results indicate a strong correlation between the
N-terminal palmitoylation of Shh-N(p) and the reactiv-
ity of 5E1 with Shh-N(p). Unexpectedly, C25S-Shh-Np
retained the 5E1 epitope when Skn was exogenously
overexpressed (Fig. 3A,B, lane 4). Considering that
Skn is a member of the MBOAT superfamily [32], it is
possible that excess Skn transferred an acyl group onto
the hydroxyl group of the N-terminal Ser of C25S-
Shh-Np, although the efficiency seems much lower
than that for wild-type Shh-Np. To confirm this possi-
bility, we labeled COS7 cells with [
3
H]palmitic acid
and examined whether the radioactivity is incorporated
into C25S-Shh-Np, as observed in wild-type Shh
(Fig. 3C, lanes 1 and 2). As expected, we detected a
band corresponding to C25S-Shh-Np, as well as full-
length C25S-Shh, only when Skn was co-expressed
(Fig. 3C, lane 4).
In COS7 cells, a band migrating more slowly than
Shh-N and strongly recognized by 5E1 was observed
when Shh (1–198) alone was expressed (Fig. 3A,
lane 7, asterisk). This species was not prominently
observed in lysate from NSC34, CHO, or HeLa cells.
Thus, there may be a third post-translational modifica-
tion of the N-terminal signaling domain of Shh specific

and calculation of hydrophobicity using several
programs revealed that both proteins have a similar
structure, with a signal sequence and at least nine
transmembrane domains (Fig. 4). In addition, the open
reading frame of both Skn and Gup1 genes consists
of 11 exons; each corresponding exon of the two
genes is similar in size (Fig. 4), suggesting that
these two genes evolve from the same origin. The
transcript of Gup1 was detectable in E9.5 mouse
embryo (Fig. 2, lane 6), in which Shh transcript is also
detected [2]. These facts prompted us to examine
whether Gup1 is involved in regulating N-terminal
palmitoylation in the mammalian hedgehog family,
including Shh.
We cloned Gup1 cDNA by RT-PCR from adult
mouse lung poly (A)
+
RNA and first examined its sub-
cellular localization by transiently expressing Gup1,
whose C terminus was fused to enhanced green fluores-
cent protein (Gup1-EGFP), as well as EGFP-tag-
ged Skn (Skn-EGFP) in HeLa cells, which express little
endogenous Skn or Gup1 (Fig. 2, lane 5). Consistent
with a previous report [13], Skn–EGFP (Fig. 5A,C)
localized on the endoplasmic reticulum (ER), as deter-
mined by immunofluorescent staining of 78-kDa
glucose-regulated protein (GRP78) (Fig. 5B,C). This
was also the case with Gup1-EGFP (Fig. 5D,F), which
was co-localized with GRP78 (Fig. 5E,F). These obser-
vations imply that these two proteins interact with

ged Gup1 in several cell lines, as detected by western
blotting of unboiled samples using anti-FLAG IgG as
a probe, revealed two major bands with molecular
masses of  45 and 40 kDa (Fig. 6A–C, lane 3). The
expression of Skn-FLAG was undetectable in some
lines (Fig. 6A,C, lane 2) even when the samples
were not boiled. However, in COS7 cells transfected
with Skn-FLAG, a band with a molecular mass
of  40 kDa was detected when probed with anti-
FLAG IgG (Fig. 6B, lane 2). These observations
suggest that Skn without EGFP is more hydrophobic
than Skn with EGFP.
As we observed that Gup1 is localized on the ER,
we examined whether Gup1 can interact with Shh
by immunoprecipitation. We transiently expressed
full-length Shh, together with Gup1-FLAG or Skn-
FLAG, in COS7 cells and immunoprecipitated these
proteins using anti-FLAG IgG. As expected, both full-
length Shh and the N-terminal fragment of Shh were
coprecipitated with Skn-FLAG (Fig. 7A, upper panel,
lane 5), whereas none of the fragment of Shh was
detected in immunoprecipitate from cells transfected
with Shh and empty vector (Fig. 7A, upper panel,
lane 4). Full-length Shh also coprecipitated with Gup1-
FLAG, indicating an interaction between Gup1 and
Shh (Fig. 7A, upper panel, lane 6).
Fig. 2. Expression of Skn and Gup1 transcripts in mammalian cell
lines. Total RNA extracted from CHO (lane 1), NSC34 (lane 2),
COS7 (lane 3), HEK293 (lane 4), HeLa (lane 5) and mouse embry-
onic day 9.5 (E9.5) embryo (lane 6) was subjected to RT-PCR analy-

MBOAT superfamily, the His residue indispensable to
MBOAT activity is replaced by Leu (Fig. 4, asterisk).
To examine whether Gup1 has HHAT activity, we trans-
fected Gup1 cDNA together with full-length Shh cDNA
into CHO cells. As shown in Fig. 6A, Shh-Np in the
lysate of CHO cells was not recognized by 5E1 (lane 3),
demonstrating that Gup1 has no HHAT activity.
Next, we examined whether Gup1 affects the palmi-
toylation of Shh-Np in cells expressing endoge-
nous Skn, such as COS7 and NSC34 cells, by
expressing full-length Shh in the presence of Gup1-
FLAG in these cells. Co-expression of Shh with
Gup1-FLAG resulted in a reduction of the total
amount of Shh-Np, determined using H-160 in the
A
B
C
Fig. 3. Requirement of Cys
25
of Shh for Skn-dependent retention
of the 5E1 epitope on the N-terminal fragment of Shh in western
blotting. COS7 (A) and NSC34 (B) cells were transiently transfected
with pCAG-Shh (lanes 1 and 2), pCAG-C25S-Shh (lanes 3 and 4),
pCAG-C25A-Shh (lanes 5 and 6) or pCAG-Shh (1–198) (lanes 7 and
8) together with either pFLAG-CMV5a (lanes 1, 3, 5 and 7) as a
vector control or pCMV-Skn-FLAG (lanes 2, 4, 6 and 8). Cellular pro-
teins (50 lg) were subjected to western blotting, followed by prob-
ing with anti-Shh N-terminal domain H-160, anti-Shh N-terminal
domain 5E1, or anti-actin IgG. Both full-length Shh and the N-termi-
nal fragment of Shh are indicated by arrows. The asterisk indicates

(Fig. 6B,C, lane 3, and Fig. 6D,E, solid column). The
levels of modified Shh-Np in COS7 and NSC34 cells,
as detected using 5E1, which is expected to recognize
palmitoylated Shh-Np, were further reduced to 6.4%
and 10.7%, respectively, as compared with control
cells, suggesting that the expression of Gup1 inhibits
palmitoylation of Shh-Np catalyzed by endoge-
nous Skn in these cells (Fig. 6B,C, lane 3, and
Fig. 6D,E, open column). It seemed that the overex-
pression of Skn-FLAG in these cells slightly increased
the level of palmitoylated Shh-Np, although the differ-
ence was not statistically significant (Fig. 6D,E).
Taken together, these observations strongly suggest
that mammalian Gup1 acts as a negative regulator of
the N-terminal palmitoylation of Shh.
Discussion
In this report, we found that mammalian Gup1, a mem-
ber of the MBOAT superfamily bearing sequence simi-
larity to HHAT, acts as a negative regulator of
N-terminal palmitoylation of Shh. Several reports have
demonstrated the critical role of N-terminal palmitoyla-
tion of Hh protein for its activity in Drosophila [15–
18,26]. Drosophila Hh protein without palmitoylation
not only loses its activity but also obstructs endogenous
Hh signaling in vivo [26]. By contrast, mammalian Shh
without palmitoylation can act in some tissues
[13,26,28]. Analysis of both Skn knockout and C25S-
Shh knockin mice revealed that the responsiveness to
nonpalmitoylated Shh-Np varied among tissues [13].
Thus, it is possible that while palmitoylated Hh-Np is

-palmi-
toylation of Ga
s
is regulated also remains unclear.
However, we assume that mammalian Gup1 competes
with Skn for Shh to prevent palmitoylation rather
than catalyzing depalmitoylation of Shh because
other known N
a
-acylations, namely N
a
-acetylation
and N
a
-myristylation, are irreversible [37,38]. Further
in vitro analyses are necessary to determine whether
Gup1 can depalmitoylate Shh-Np.
G
ABC
DEF
Fig. 5. Subcellular localization of mouse Skn and Gup1. (A–F) To
visualize the subcellular localization of mouse Skn (A–C) and Gup1
(D–F), EGFP was fused to the C terminus of these proteins and
expressed in HeLa cells. Forty-eight hours after transfection, the cells
were fixed, permeabilized and stained with an ER marker (78-kDa
glucose-regulated protein) followed by TRITC-labeled secondary anti-
body. The fluorescence of EGFP (A, C, D and F, green) and TRITC
(B, C, E and F, red) was observed using a confocal microscope.
Scale bar, 25 lm. (G) COS7 cells were transiently transfected with
pIRES2-EGFP (lanes 1 and 4) as a vector control, pCMV-Skn-EGFP

transfection conditions. Therefore, palmitoylation may
not be a component of the 5E1 epitope but may influ-
ence the structure of the 5E1 epitope under denaturing
conditions. Crystal structure analysis revealed that the
residues Pro
42
, Lys
46
, Arg
154
, Ser
157
, Ser
178
and Lys
179
are located close to each other on the surface of the
mouse Shh-N protein and are essential for Shh-N to
bind both Patched and 5E1 [34,39,40]. Among the resi-
dues, Ser
178
at least is found to be included in the 5E1
epitope [39]. In addition, mouse Shh-N lacking the
N-terminal 25 amino acids [Shh (50–198)] loses the
ability to bind not only Patched but also 5E1 in
immunoprecipitation [34]. These observations indicate
the requirement of the N-terminal region of the Shh-
N, including Pro
42
and Lys

as a vector control, pCMV-Skn-FLAG
(lane 2), or pCMV-Gup1-FLAG (lane 3).
Cellular proteins (50 lg) were subjected to
western blotting, using polyclonal anti-
Shh N-terminal domain H-160, monoclonal
anti-Shh N-terminal domain 5E1, or
monoclonal anti-FLAG IgG. The intensity of
the signals obtained from the western blot
analysis was quantified using
QUANTITY ONE
software (Bio-Rad). The effect of Skn or
Gup1 on the amount of total Shh-Np,
determined with H-160 (solid column), and
on the amount of modified Shh-Np, deter-
mined with 5E1 (open column), in COS7 (D)
and NSC34 (E) cells was expressed as the
ratio of the intensity of the band of Shh-Np
to that from control cells transfected with
Shh and empty vector in the same blot.
Values were the mean ± SD of three
independent experiments. The difference
between total Shh-Np and modified Shh-Np
was determined using a paired t-test.
*, P < 0.01; NS, not significant. The level of
Shh-Np in control cells is shown by the
dotted line.
A negative regulator for palmitoylation of Shh Y. Abe et al.
326 FEBS Journal 275 (2008) 318–331 ª 2007 The Authors Journal compilation ª 2007 FEBS
region to form the 5E1 epitope, even under denaturing
conditions. The other possibility is that the N-terminal

and 5¢-AGCTGGCCCAGCAGCCATACACAGTTAAAG-
3¢. The cDNA was subcloned into the EcoRV site of pBlue-
script SK(+) (Stratagene, La Jolla, CA, USA) and
sequenced using an automated sequencer (ABI-PRISM310
Genetic Analyzer; Perkin-Elmer Applied Biosystems, Foster
AB
Fig. 7. Gup1 interacts with both full-length Shh and Skn. (A) COS7 cells were transiently transfected with pCAG-Shh (lanes 1–6), together
with pFLAG-CMV-5a (vector) (lanes 1 and 4), pCMV-Skn-FLAG (Skn-F) (lanes 2 and 5), or pCMV-Gup1-FLAG (Gup1-F) (lanes 3 and 6). Cells
were lysed with IP buffer, as described in the Experimental procedures, and subjected to immunoprecipitation using anti-FLAG IgG. Then,
the samples were boiled and subjected to western blot analysis using either anti-Shh N-terminal IgG H-160 or HRP-conjugated anti-FLAG IgG
(lanes 4–6). Some of the lysate (1 ⁄ 20 volume) was unboiled and also subjected to western blot analysis as input (lanes 1–3). Both full-length
Shh and the N-terminal fragment of Shh are indicated by arrows. (B) COS7 cells were transiently transfected with pCMV-Gup1-EGFP (Gup1-
G), together with pFLAG-CMV-5a (vector) (lanes 1 and 3), or pCMV-Skn-FLAG (Skn-F) (lanes 2 and 4). Cells were lysed with IP buffer and
subjected to immunoprecipitation using anti-FLAG IgG. Then, samples were boiled and subjected to western blot analysis using anti-GFP IgG
as a probe (lanes 3 and 4). Some of the lysate (1 ⁄ 20 volume) was unboiled and also subjected to western blot analysis as input (lanes 1 and
2). Immunoglobulin G heavy and light chains (IgG-H and IgG-L, respectively) are indicated by arrows. Putative Gup1–EGFP is indicated by the
arrowhead.
Y. Abe et al. A negative regulator for palmitoylation of Shh
FEBS Journal 275 (2008) 318–331 ª 2007 The Authors Journal compilation ª 2007 FEBS 327
City, CA, USA). A BamHI site was introduced immediately
before the termination codon for addition of the FLAG-tag
to the C terminus of Skn by PCR using primers 5¢-AA
GCTTCCGGAGGCTGCTAGAGAC-3¢ and 5¢-GGATC
CAAGAACTGTGTATGTCTG-3¢. The 1.6-kbp full-
length Skn cDNA, whose termination codon was changed
to a BamHI site, was inserted between the SalI and the
BamHI sites of pFLAG-CMV5a (Sigma, St Louis, MO,
USA) (pCMV-Skn-FLAG, SF) or between the XhoI and
the Bam HI sites of pEGFP-N3 (pCMV-Skn-EGFP). The
cDNA encoding FLAG-tagged Skn was then excised by

AGGTTATATAG-3¢ and 5¢-GGGCCCA
GAGGCCAGG
CCGGGGCACACCAG-3¢, and primers 5¢-GGCATGC
TGGCTCGCCTGGCTGTGGAAGCA-3¢ and 5¢-GGAT
CCTGGGAAA
GCGCCGCCGGATTTGGC-3¢, respec-
tively. Shh (1–198) (Shh lacking the C-terminal catalytic
domain) was constructed by PCR, which changed the
codon TG
T corresponding to the Cys
199
to TGA (Stop)
and added an EcoRV site immediately after the stop
codon using a sense primer 5¢-GGCATGCTGGCT
CGCCTGGCTGTGGAAGCA-3¢ and an antisense pri-
mer 5¢-AAGCTTGATATC
TCAGCCGCCGG ATTTGGC- 3¢.
C25A mutation of Shh was introduced by a QuikChange
site-directed mutagenesis kit (Stratagene) using primers
5¢-CCGGGCTGGCCGCTGGCCCCGGCAGGGG-3¢ and
5¢-CCCCTGCCGGGGCCAGCGGCCAGCCCGG-3¢.
Cell culture and transient transfection
CHO cells were cultured in Ham’s F12 supplemented with
10% fetal bovine serum, 50 unitsÆmL
)1
of penicillin and
50 lgÆmL
)1
of streptomycin. COS7, HeLa, HEK293 and
NSC34 [41] cells were maintained in Dulbecco’s modified

and 5¢-GGATCCCTCCAGCTTCTCTCTGTCCTGC-3¢.
These primer sets were designed based on the mouse seque-
nce and were compatible with human species. As an inter-
nal control, glyceraldehyde-3-phosphate dehydrogenase was
amplified using the primers 5¢-TCCACCACCCTGTTGCT
GTA-3¢ and 5¢-ACCACAGTCCATGCCATCAC-3¢ (25 cycles
at 94 °Cfor1min,65°C for 1 min and 72 °C for 1 min).
Western blot analysis
Cells were washed twice with NaCl ⁄ P
i
(PBS) and lysed with
lysis buffer containing 20 mm Tris-HCl (pH 7.5), 1 mm
EDTA, 1% Triton X-100 and Complete
TM
protease inhibi-
tor cocktail tablets (Roche Diagnostics, Indianapolis, IN,
USA). Each sample was analyzed using a bicinchoninic acid
protein assay kit (Pierce, Rockford, IL, USA), and 30–50 lg
of cellular protein was subjected to SDS-PAGE, followed by
transfer to polyvinylidene difluoride membrane (Pall Life
Sciences, East Hills, NY, USA) and blocking with 10% skim
milk (Becton-Dickinson, Franklin Lakes, NJ, USA) in
NaCl ⁄ P
i
containing 0.1% Tween 20 (Wako, Osaka, Japan).
Signals were detected with enhanced chemiluminescence
reagents (GE Healthcare Bio-Sciences, Piscataway, NJ). As
for the analysis of the secreted N-terminal signaling domain
of Shh, 20 lL of CM was diluted with an equivalent volume
of 2· SDS-PAGE sample buffer and subjected to SDS-

hours later, the immunocomplexes were washed four times
with the IP buffer. Then, the beads were boiled in 20 lLof
2 · SDS-PAGE sample buffer, and the eluted samples were
subjected to western blot analysis as described above.
Isotope labeling with [
3
H]palmitic acid
COS7 cells seeded onto 60-mm dishes at a density of
2 · 10
5
cells ⁄ dish were transiently transfected with pCAG-
Shh ⁄ CMV-IRES-EGFP, pCAG-Shh ⁄ CMV-Skn-FLAG-
IRES-EGFP, pCAG-C25S-Shh ⁄ CMV-IRES-EGFP, or
pCAG-C25S-Shh ⁄ CMV-Skn-FLAG-IRES-EGFP. Twenty-
four hours after transfection, cells were incubated in
Dulbecco’s modified Eagle’s medium with 10% fetal bovine
serum containing 200 lCiÆmL
)1
of [9,10-
3
H]palmitic acid
(Perkin Elmer) for 24 h and lysed with IP buffer. Shh was
immunoprecipitated from the lysate as described above
using 5E1 and subjected to SDS-PAGE. The gel was fixed
with isopropanol ⁄ water ⁄ acetic acid (25 : 65 : 10, v ⁄ v ⁄ v) for
30 min and treated with Amplify Fluorographic Reagent
(GE Healthcare Bio-Sciences) for 30 min. Then, the gel was
dried and exposed to an X-ray film at )80 °C for 14 days.
Confocal microscopy
HeLa cells were seeded onto six-well plates at a density of

maintained by the University of Iowa Department of
Biological Sciences (Iowa City, IA 52242, USA). This
work was supported, in part, by grants from Japan
Society for the Promotion of Science Grant-in-Aid for
Scientific Research (C) 16590845 (YA), 17590893 (YK),
17590894 (TN), Keio Gijuku Academic Development
funds (YA and TN), National Grant-in-Aid for the
Establishment of High-Tech Research Center in a
Private University (YA), and the Nakabayashi Trust
for ALS Research (TN). The authors gratefully dedicate
this article to late Professor Ikuo Nishimoto.
References
1 Riddle RD, Johnson RL, Laufer E & Tabin C (1993)
Sonic hedgehog mediates the polarizing activity of the
ZPA. Cell 75, 1401–1416.
2 Echelard Y, Epstein DJ, St-Jacques B, Shen L, Mohler
J, McMahon JA & McMahon AP (1993) Sonic hedge-
hog, a member of a family of putative signaling mole-
cules, is implicated in the regulation of CNS polarity.
Cell 75, 1417–1430.
3 Krauss S, Concordet J-P & Ingham PW (1993) A func-
tionally conserved homolog of the Drosophila segment
Y. Abe et al. A negative regulator for palmitoylation of Shh
FEBS Journal 275 (2008) 318–331 ª 2007 The Authors Journal compilation ª 2007 FEBS 329
polarity gene hh is expressed in tissues with polarizing
activity in zebrafish embryos. Cell 75 , 1431–1444.
4 Roelink H, Augsburger A, Heemskerk J, Korzh V,
Norlin S, Ruiz i Altaba A, Tanabe Y, Placzek M,
Edlund T, Jessell TM et al. (1994) Floor plate and
motor neuron induction by vhh-1, a vertebrate homo-

the amino-terminal cleavage product of Sonic hedgehog
autoproteolysis. Cell 81, 445–455.
12 Porter JA, Young KE & Beachy PA (1996) Cholesterol
modification of Hedgehog signaling proteins in animal
development. Science 274, 255–259.
13 Chen M-H, Li Y-J, Kawakami T, Xu S-M & Chuang
P-T (2004) Palmitoylation is required for the production
of a soluble multimeric Hedgehog protein complex and
long-range signaling in vertebrates. Genes Dev 18, 641–
659.
14 Pepinsky RB, Zeng C, Wen D, Rayhorn P, Baker DP,
Williams KP, Bixler SA, Ambrose CM, Garber EA,
Miatkowski K et al. (1998) Identification of a palmitic
acid-modified form of human Sonic hedgehog. J Biol
Chem 273, 14037–14045.
15 Amanai K & Jiang J (2001) Distinct roles of Central
missing and Dispatched in sending the Hedgehog signal.
Development 128, 5119–5127.
16 Chamoun Z, Mann RK, Nellen D, von Kessler DP,
Bellotto M, Beachy PA & Basler K (2001) Skinny
hedgehog, an acyltransferase required for palmitoyla-
tion and activity of the Hedgehog signal. Science 293
,
2080–2084.
17 Lee JD & Treisman JE (2001) Sightless has homology
to transmembrane acyltransferases and is required to
generate active Hedgehog protein. Curr Biol 11, 1147–
1152.
18 Micchelli CA, The I, Selva E, Mogila V & Perrimon N
(2002) rasp, a putative transmembrane acyltransferase,

freely diffusible multimeric form. J Biol Chem 281,
4087–4093.
26 Lee JD, Kraus P, Gaiano N, Nery S, Kohtz J, Fishell
G, Loomis CA & Treisman JE (2001) An acylatable
residue of Hedgehog is differentially required in Dro-
sophila and mouse limb development. Dev Biol 233,
122–136.
27 Kohtz JD, Lee HY, Gaiano N, Segal J, Ng E, Larson
T, Baker DP, Garber EA, Williams KP & Fishell G
(2001) N-terminal fatty-acylation of sonic hedgehog
enhances the induction of rodent ventral forebrain neu-
rons. Development 128, 2351–2363.
28 Williams KP, Rayhorn P, Chi-Rosso G, Garber EA,
Strauch KL, Horan GSB, Reilly JO, Baker DP, Taylor
FR, Koteliansky V et al. (1999) Functional antagonists
of sonic hedgehog reveal the importance of the N termi-
nus for activity. J Cell Sci 112, 4405–4414.
A negative regulator for palmitoylation of Shh Y. Abe et al.
330 FEBS Journal 275 (2008) 318–331 ª 2007 The Authors Journal compilation ª 2007 FEBS
29 Holst B, Lunde C, Lages F, Oliveira R, Lucas C &
Kielland-Brandt MC (2000) GUP1 and its close homo-
logue GUP2, encoding mutimembrane-spanning pro-
teins involved in active glycerol uptake in
Saccharomyces cerevisiae. Mol Microbiol 37, 108–124.
30 Feng J, White B, Tyurina OV, Guner B, Larson T &
Lee HY (2004) Synergistic and antagonistic roles of the
Sonic hedgehog N- and C-terminal lipids. Development
131, 4357–4370.
31 Hofmann K (2000) A superfamily of membrane-bound
O-acyltransferases with implications for Wnt signaling.

10995–11001.
40 Tanaka Hall TM, Porter JA, Beachy PA & Leahy DJ
(1995) A potential catalytic site revealed by the 1.7-
Angstrom crystal structure of the amino-terminal sig-
nalling domain of Sonic hedgehog. Nature 378, 212–
216.
41 Cashman NR, Durham HD, Blusztajn JK, Oda K, Ta-
bira T, Shaw IT, Dahrouge S & Antel JP (1992) Neuro-
blastoma · spinal cord (NSC) hybrid cell lines resemble
developing motor neurons. Dev Dyn 194 , 209–221.
42 Bendtsen JD, Nielsen H, von Heijne G & Brunak S
(2004) Improved prediction of signal peptides: Sig-
nalP3.0. J Mol Biol 340, 783–795.
43 Sonnhammer EL, von Heijne G & Krogh A (1998) A
hidden Markov model for predicting transmembrane
helices in protein sequences. Proc Int Conf Intell Syst
Mol Biol 6, 175–182.
44 Hofmann K & Stoffel W (1993) TMbase-A database
of membrane spanning protein segments. Biol Chem
Hoppe-Seyler 374, 166.
45 Tusna
´
dy GE & Simon I (2001) The HMMTOP trans-
membrane topology prediction server. Bioinformatics
17, 849–850.
46 Horton P & Nakai K (1997) Better prediction of pro-
tein cellular localization sites with the k nearest neigh-
bors classifier. Proc Int Conf Intell Syst Mol Biol 5,
147–152.
47 Hirokawa T, Boon-Chieng S & Mitaku S (1998)