Extensive lipidation of a Torpedo cysteine string protein.

Abstract

Cysteine string proteins are relatively low mass components of synaptic vesicle membranes. Structurally, their primary sequence is distinguished by a remarkable, cysteine-rich motif. Investigations revealed an unprecedented degree of lipidation of these cysteine residues. At least 11 of the 13 cysteines of the Torpedo protein were modified, principally by palmitoyl moieties. This fatty acylation creates a prominent hydrophobic domain flanked by polar amino and carboxyl termini. An amphipathic structure of this type is uniquely suited to mediate events at membrane interfaces. Thus, cysteine string proteins are candidates to participate in exocytotic membrane fusion.

Full text

Page 1

Communication
Vol. 269, No. 30, Issue of July 29, pp. 19197-19199, 1994
0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
THE JOURNAL
OF BIOLOGICAL
CHEMISTRY
Printed in U.S.A.
Extensive Lipidation of a
Torpedo Cysteine String Protein"
(Received for publication, April 25, 1994)
Cameron B. GundersenSS,
Alessandro Mastrogiacomot., Kym Faulllll**, and
Joy k UmbachS
From the Departments of $Molecular and Medical
Pharmacology, llChemistry and Biochemistry, and
IRsychiatry and Biobehauioral Sciences and the
**Neuropsychiatric Institute, UCLA School of Medicine,
Los Angeles, California 90024
Cysteine string proteins are relatively low mass com-
ponents of synaptic vesicle membranes. Structurally,
their primary sequence is distinguished by a remarka-
ble, cysteine-rich motif. Investigations revealed an un-
precedented degree of lipidation of these cysteine resi-
dues. At least 11 of the 13 cysteines of the ICbrpedo
protein were modified, principally by palmitoyl moi-
eties. This fatty acylation creates a prominent hydro-
phobic domain flanked by polar amino
mini. An amphipathic structure of this type is uniquely
suited to mediate events at membrane interfaces. Thus,
cysteine string proteins are candidates to participate in
exocytotic membrane fusion.
and carboxyl ter-
Cysteine string proteins (Csps) were originally identified as
targets of a nervous system-specific monoclonal antibody
Drosophila (1). Subsequently, a suppression cloning approach
revealed that a Torpedo homolog of the fruit fly Csps is a likely
subunit or modulator of presynaptic calcium channels
When this Torpedo Csp (T-Csp) was found to be an intrinsic
component of the synaptic vesicle membrane (3), it was sug-
gested that Csps mediate a regulatory interaction between
docked synaptic vesicles and presynaptic calcium channels (3).
Studies of temperature-sensitive, paralytic mutants of the Csp
locus in Drosophila confirmed the importance of Csps for pre-
synaptic function (41.' A pervasive question has concerned the
role of the highly conserved cysteine string motif
teins. Our discovery that a large majority of the Cys residues
the Torpedo protein are fatty acylated has novel structural and
functional implications.
in
(2).
of these pro-
of
EXPERIMENTAL PROCEDURES
Post-translational Modification of TCsp in
t35SlCys-labeled T-Csp was produced by in vitro translation (2, 6), di-
luted with 1 m~ dithiothreitol in water, and concentrated (Microcon-30;
Amicon, Beverly, MA) twice to remove [35SlCys. Xenopus oocytes were
injected with 2-4 x lo3 cpm of labeled proteidoocyte and cultured for 2
days in Barth's medium at 17-19 "C (2). For the experiment shown in
Fig. lA,20 oocytes were homogenized in a solution of 100 m~ NaC1,50
m M Tris (pH 7.4 with HCl), 1 m M EGTA with 10 m~ CHAPS.' The
Xenopus Oocytes-
* The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
"aduertisement" in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
5 To whom correspondence should be addressed. Tel.: 310-825-3423;
Fax: 310-206-5052.
J. A. Umbach, K. E. Zinsmaier, K. K. Eberle, E. Buchner, S. Benzer,
' The abbreviation used is: CHAPS, 3-[(3-cholamidopropyl)dimethyl-
ammoniol-1-propanesulfonic acid.
and C. B. Gundersen, submitted for publication.
homogenate was centrifuged (1,000 x g for 3 min) to remove yolk plate-
lets and after 45 min on ice the insoluble material was removed by
centrifugation for 30 min at 100,000 x g. The solubilized material was
concentrated and diluted 1:lOO in phosphate-buffered saline for immu-
noprecipitation (6). The immunoprecipitate that was bound to the in-
soluble protein A immunoadsorbent (Sigma) was dissolved in SDS
sample buffer (without 2-mercaptoethanol) for polyacrylamide gel elec-
trophoresis and autoradiography (2, 6).
For the studies in Fig. U3, oocytes (injected 2 days previously with
labeled T-Csp) were treated to obtain soluble and membrane fractions
essentially as described by Colman (7) except that the homogenization
solution was 10% sucrose in 100 m~ NaCl, 50 m M Tris (pH 7.4), and 1
m M EGTA. Membranes were solubilized, and T-Csp was immunopre-
cipitated. Where indicated, these immunoprecipitates were treated di-
rectly at 21-22 "C with fresh 0.1 M methanolic KOH, 1 M hydroxylamine
(Aldrich; pH 7.0), or 1 M Tris (pH 7.0) to test for the displacement of
protein fatty acyl thioesters (8, 9). Protein was recovered either by
precipitation or evaporation of solvent (after neutralization of pH), and
equivalent countdmin in each sample were subjected to electrophoresis
and autoradiography as above.
To assess the incorporation of L3H1palmitate into T-Csp, oocytes were
injected with unlabeled, in vitro translated T-Csp and cultured in a
siliconized vial with 0.5 mCi ml" of L3H1palmitate (DuPont NEN). After
2 days, T-Csp was immunoprecipitated as above. A sample of the im-
munoprecipitate was pretreated with 0.1 M KOH in methanol before
analysis by electrophoresis and autoradiography (2, 6).
Deacylation Analysis of T-Csp from Electric Organ-T-Csp-contain-
ing membranes were prepared from frozen, pulverized Torpedo califor-
nica electric organ by homogenization (in 10 volumes of 0.1 M NaCl, 50
m M Tris (pH 7.4), 1 m M EDTA, 1 m M EGTA) for 2 min in a blender at
4 "C. After a 30-min centrifugation at 15,000 x g (4 "C), the S, was
centrifuged for 1 h at 4 "C at 100,000 x g to yield membrane samples.
These samples were suspended by hand homogenization and treated
with deacylating reagents before immunoblot analysis using antibodies
specific for the COOH terminus of T-Csp (3, 6). For the time course of
hydroxylamine-mediated hydrolysis, samples were removed at specified
intervals for immunoblot analysis (3, 6).
Quantitation ofthe Number of Free and Fatty Acylated Cys Residues
of TCsp-T-Csp (3-5 pmol) was immunoprecipitated from CHAPS (10
mM) solubilized electric organ membranes (6) and divided into two
equivalent samples. One sample of immunoadsorbent-bound T-Csp was
treated at 22 "C with methanolic KOH (0.1 M) to displace fatty acyl
groups (8,9). The other sample was incubated in methanol only. After 2
h, the KOH was neutralized with HCl(1 N) and the solvent was evapo-
rated. Free thiol groups in each sample were reduced using 2 m M di-
thiothreitol in 10 m~ Tris (pH 7.0) for 2 h at 22 "C. Alkylation of the
reduced protein thiol groups used ['4Cliodoacetamide (5.5 mM, DuPont
NEN) at 22 "C in the dark. After 2 h, these samples were resolved on a
15% SDS-polyacrylamide gel and protein was transferred to nitrocellu-
lose. Regions corresponding to the acylated and deacylated proteins
(judged by immunoblot) were excised, dissolved in dioxane, and counted
by liquid scintillation spectrometry.
Identity ofFatty Acids Esterified to TCsp-Authentic samples of pal-
mitic hydroxamate were prepared as described by Gutnikov and Streng
(10). Samples of oleic and stearic hydroxamate were gifts of G. Gutnikov
(Pomona, CAI. To derivatize biological samples, dried material (corre-
sponding to the hydroxylamine-hydrolyzed fatty acyl groups of about
10-20 pmol of immunoprecipitated T-Csp) and authentic standards were
reacted with
N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide
(MTBSTFA, 5 pl, Pierce Chemical Co.) in ethyl acetate at 75 "C for 1 h.
For the control sample, primary antibody against T-Csp was omitted.
Samples were analyzed by gas chromatography-mass spectrometry in
the selected ion mode (at mlz 442,468, and 470 corresponding to the base
peaks, M - 57 in the electron impact mass spectra of the tert-butyldi-
methylsilyl derivatives of the three fatty acyl hydroxamates).
RESULTS
Xenopus oocytes have been used widely to study the post-
translational modification of proteins (7). Based on our earlier
results, which suggested that T-Csp was subjected to a post-
19197

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19198
-
97.4 .
69
-
46
1 4
91.4
69
46
. I
30
Y
"
22
14
B
Palmitoylation of Cysteine String Proteins
97.4
69
46
14
B
D
kDa
1 2
46
30
22
14
FIG. 1 . Fatty acylation of T-Csp in Xenopus oocytes. A, Xenopus
oocytes converted a portion of the injected 27-kDa in vitro translated
T-Csp (lane 1) to a higher mass (34-kDa) form (lane 2). B, the 27-kDa
form of T-Csp distributed only in the soluble fraction of oocytes, while
the 34-kDa species partitioned with membranes.
agents (1 M hydroxylamine (pH 7) or methanolic KOH) converted the
34-kDa form of T-Csp to a 27Da form. As a control, Tris (pH 7) did not
alter the 34-kDa T-Csp. D, radioactivity from ["Hlpalmitic acid was
incorporated into unlabeled, in vitro translated T-Csp (lane 1). This
T-Csp-associated radioactivity was displaced by pretreatment of the
labeled protein with methanolic KOH (lane 2).
C, deacylating re-
translational modification that increased its hydrophobicity
(61, we examined whether T-Csp was post-translationally al-
tered in these frog oocytes. We observed that, when oocytes
were injected with [35S]cysteine-labeled T-Csp, they converted a
portion of the 27-kDa, in vitro translated T-Csp to a higher
mass (34-kDa) form (Fig. IA). Note also that under the condi-
tions of these experiments, no intermediate mass forms (be-
tween 27 and 34 kDa) were detected. If this initial oocyte ho-
mogenate was partitioned into
fractions, the 34-kDa T-Csp distributed detectably only with
the membranes; the soluble fraction contained only the 27-kDa
T-Csp (Fig. 1B ). Since fatty acylation is a plausible explanation
both for this dramatic change in electrophoretic mobility and
for the subcellular redistribution
reagents that selectively cleave fatty acyl thio-esters of proteins
(8,9) induced the 34-kDa form of T-Csp to revert to the
T-Csp form. Both 1 M hydroxylamine (pH 7.0) and methanolic
KOH had this effect (Fig. 1 0 , but, as a control, 1 M Tris (pH 7.0)
did not. These data imply that T-Csp is post-translationally
fatty acylated in oocytes. Moreover, this observation extends
previous findings that the 34-kDa form of T-Csp is very hydro-
phobic, while the 27-kDa form is hydrophilic (6).
In experiments parallel to those of Fig. 1 (A-C), oocytes were
injected with unlabeled, in vitro translated T-Csp. When these
oocytes were incubated with [3H]palmitic acid, radioactivity
soluble and membrane
of T-Csp, we explored whether
27-kDa
A
8
46 -
30
" -
& "
46
30
72
FIG. 2. T-Csp is fatty acylated in lbrpeah electric organ. A,
deacylating reagents (methanolic KOH or hydroxylamine) converted
the endogenous (34-kDa) T-Csp to a 27-kDa species. B, time course of
hydroxylamine-mediated hydrolysis of T-Csp. The control sample was
untreated and complete deacylation was effected by methanolic KOH.
Samples treated with hydroxylamine (1 M, pH 7.0) were removed at the
times indicated.
was incorporated into a 34-kDa band of immunoprecipitable
T-Csp (Fig. l D 1 . Because methanolic KOH displaces this radio-
activity (Fig. lD), we conclude that ["Hlpalmitate is esterified
to T-Csp in oocytes. Moreover, since the mass of this radioactive
product (about 34 kDa) is essentially the same as T-Csp from
electric organ (34 kDa, Ref. 6), it is likely that T-Csp is palmi-
toylated in vivo.
Evidence that T-Csp is palmitoylated in vivo is shown in
Figs. 2 and 3. Deacylating reagents (hydroxylamine or metha-
nolic KOH) convert T-Csp from the 34-kDa form to the 27-kDa
form. As controls, neither methanol alone (data not shown) nor
Tris (1 M, pH 7) has this effect (Fig. 2A). Given the relative
specificity of these deacylating reagents (8,9), this 7-kDa shift
in mass is consistent with T-Csp being fatty acylated. Again,
because the deacylating agents cause a drop in the mass of
immunoreactive T-Csp to a level that is indistinguishable from
that of in vitro translated T-Csp (Fig. 1; Ref. 11, we conclude
that fatty acylation is predominantly responsible for the post-
translational increase in mass of T-Csp (to 34 kDa).
To confirm and extend the above results, several additional
experiments were performed. First, we assessed the time
course of hydroxylamine treatment of T-Csp. This was done
with the expectation that removal of individual fatty acyl
groups from T-Csp might yield immunoreactive intermediates
between the fully modified (34-kDa) form of T-Csp, and the fully
deacylated (27-kDa) form of this protein. Eleven or 12 immu-
noreactive T-Csp species were detected
stances (Fig. 2B 1. One interpretation of this stepladder pattern
(Fig. 2B) is that each band is a sequentially deacylated variant
of the 34-kDa form of T-Csp. Thus, these data suggest that as
many as 12 of the 13 Cys residues of T-Csp are fatty acylated.
However, another approach was used to estimate more conclu-
under these circum-

Page 3

Palmitoylation of Cysteine Proteins String
19199
Retention Time (min)
FIG. 3. Identity of fatty acyl groups of T-Csp. Figure shows se-
lected ion monitoring of mlz 442, 468, and 470 corresponding, respec-
tively, to the fragmented derivatives of palmitic, oleic, and stearic acid
hydroxamates. The gas chromatographic retention times of authentic
standards are indicated by arrows. The control samples were treated
the same as experimental samples except that primary (anti-T-Csp)
antibody was omitted during the immunoprecipitation.
sively the number of free and fatty acylated Cys residues of
T-Csp. This was accomplished by measuring the incorporation
of ['4Cliodoacetamide into the 34- and 27-kDa forms of this
protein after reduction of -SH groups by 2 II~M dithiothreitol(8).
The proportion of radioactivity incorporated into these two
forms of the protein is an index of the number of "free" (non-
acylated) thiol groups of native T-Csp. The empirical ratio was
10.7 2 0.4 (mean 2 S.D., n = 3). This result is intermediate
between the values expected for one (13:l) or two (6.5:l) free
thiol groups in T-Csp. From this, we conclude that T-Csp in
electric organ has at least 11 fatty acylated Cys residues, and
one or two free -SH groups. Since the Cys residues of T-Csp are
all present in a span of only 2 dozen residues (Cys-113 to Cys-
136, see Ref. 2 1 , this means that fatty acylation occurs in a
highly restricted region of this protein.
To establish the identify of the fatty acyl groups of T-Csp, we
used gas chromatography-mass spectrometry (Fig. 3). The hy-
droxamate of palmitic acid is the predominant species detected
after treating T-Csp with hydroxylamine (Fig. 3). There is al-
most no detectable oleic acid, but small quantities of stearic
acid may be coupled to T-Csp (Fig. 3). This finding is compatible
with previous observations that palmitate is the principal fatty
acid esterified to Cys residues of proteins (11-13).
DISCUSSION
Lipidation of proteins has been documented in organisms as
diverse as viruses and man (8, 9, 11-15). Among the various
forms of protein lipidation, fatty acylation of Cys residues is
relatively common. It occurs in such disparate proteins as my-
elin proteolipid protein (14, 151, members of the major histo-
compatibility complex (81, transferrin receptors (16), rhodopsin
(171, GAP43 (181, SNAP-25 (19), and the
channel (20). In nearly all of these instance, palmitoylation is
confined to 3 or fewer Cys residues of each protein monomer. The
one exception is the proteolipid protein of myelin (14,15). It has
a! subunit of the sodium
six thioester-linked fatty acids at Cys residues 5, 6, 9, 108, and
140 (15). In conjunction with its relatively hydrophobic amino
acid composition, this high degree of lipidation makes myelin
proteolipid protein one of the most hydrophobic proteins iden-
tified (14, 15). Using this proteolipid protein as an index, one is
led to question the functional significance of the 11, closely
spaced fatty acyl groups of T-Csp. Previous studies of fatty acy-
lated proteins have implicated this post-translational modifi-
cation in the membrane attachment
teins (11-13). Additional postulates have
involvement of fatty acylation in signal transduction (9), entry
of viruses into cells (21), and membrane fusion (22). It is in this
lattermost context of membrane fusion that the unique struc-
ture of T-Csp is most suggestive. With its compact, fatty acylated
cysteine string domain flanked by highly polar amino and car-
boxyl termini, T-Csp is distinctively amphipathic. Amphipathic
molecules are well suited to mediate interactions
interfaces. Thus, in addition to the proposed modulation by T-
Csp ofpresynaptic calcium channels (21, it will also be of interest
to explore the interfacial activity of this protein in the context
of membrane fusion. For instance, the fatty
may serve as guides or templates for the lipid rearrangements
that are necessary for fusion of lipid bilayers.
The finding that T-Csp is fatty acylated raises questions
about the mechanisms and dynamics of the acyl transfer proc-
ess. For instance, there is considerable evidence for dynamic
cycles of protein palmitoylation and depalmitoylation in other
systems (5, 18, 23, 24). Thus, it is of some interest that we
recently obtained evidence for a small, non-acylated pool of
T-Csp in Torpedo electric organ3 This raises the
there are similar cycles of acylation and deacylation of T-Csp at
nerve endings in electric organs. Hence, it will be of some
importance to relate these different pools of T-Csp to the mul-
tiple proposed functions of this protein. Moreover, because both
in Torpedo and in Xenopus oocytes we do not detect acylation
intermediates of Csp, there may be mechanisms that produce a
concerted modification of this protein. It will be of considerable
interest to identify the enzyme(s) that regulate this process.
or orientation of these pro-
included an
at membrane
acyl groups of T-Csp
possibility that
REFERENCES
1. Zinsmaier, K. E., Hofbauer,A., Heimbeck, G., Pnugfelder, G. O., Buchner, S. &
Buchner, E. (1990) J. Neurogenet. 7, 1529
2. Gundersen, C. B. & Umbach, J. A. (1992) Neuron 9, 527-537
3. Mastrogiacomo, A., Parsons, S. M., Zampighi, G. A., Jenden, D. J., Umbach, J.
A. & Gundersen, C. B. (1994) Science 263, 981-982
4. Zinsmaier, K. E., Eberle, K. K., Buchner, E., Walter, N. & Benzer, S. (1994)
Science 263, 977-980
5. Magee, A. I., Gutierrez, L., McKay, 1. A., Marshall, C. J. & Hall, A. (1987)
EMBO J. 11,33533357
6. Mastrogiacomo, A,, Evans, C. J. & Gundersen, C. B. (1994) J. Neurochem. 62,
873-880
7. Colman, A. (1984) in Dunscription and Dunslation (Hames, B. D. & Higgins,
S. J., eds) pp. 271-302, IRL Press, Washington, DC
8. Kaufman, J. E, Krangel, M. S. & Strominger, J. L. (1984) J. Biol. Chem. 259,
7230-7238
9. Huang, E. M. (1989) Biochirn. Biophys. Acta 1011, 134-139
10. Gutnikov, G. & Streng, J. R. (1991) J. Chromatogr. 587, 292-301
11. Sefton, B. M. & Buss, J. E. (1987) J. Cell Bid. 104, 1449-1453
12. McIlhinney, R. A. J. (1990) 'IFends Biochem. Sci. 15, 387-391
13. James, G. & Olson, E. M. (1990) Biochemistry 29, 2623-2634
14. Folch-Pi, J. & Lees, M. (1951) J. Bid. Chem. 191, 807-817
15. Weimbs, T. & Stoffel, W. (1992) Biochemistry 31, 12289-12296
16. Bishr-Omary, M. & Trowbridge, I. S. (1981) J. Biol. Chem. 256,47154718
17. OBrien, P. J. & Zatz, M. (1984) J. Bid. Chem. 259, 5054-5057
18. Skene, J. H. P. & Virag, I. (1989) J. Cell Biol. 108, 613424
19. Hess, D. T., Slater, T. M., Wilson, M. C. & Skene, J. H. P. (1992) J. Neurosci. 12,
4634-4641
21. Schmidt, M. E G. & Lambrecht, B. (1985) J. Gen. Virol. 66, 2635-2647
22. Sollner, T., Whiteheart, S. W., Brunner, M., Erdjument-Bromage, H., Geroma-
nos, S., Tempst, P. & Rothman, J. E. (1993) Nature 362, 318-324
20. Schmidt, J. W . & Catterall, W. A. (1987) J. Biol. Chem. 262, 13713-13723
23. Linder, M. E., Middleton, P. Hepler, J. R., Taussig, R., Gilman, A. G. & Mumby,
S. M. (1993) Proc. Nutl. Acad. Sci. U. S. A. 90, 3675-3679
24. Hess, D. T., Patterson, S. I., Smith, D. S. & PateSkene, J. H. (1993)Nuture 366,
562-564
T. Coppolla and C. Gundersen, unpublished data.