Adipocytes and muscle cells play a major role in blood glucose homeostasis. This is dependent upon the expression of Glut4, an insulin-responsive facilitative glucose transporter. Glut4 is localised to specialised intracellular vesicles that fuse with the plasma membrane in response to insulin stimulation. The insulin-induced translocation of Glut4 to the cell surface is essential for the maintenance of optimal blood glucose levels, and defects in this system are associated with insulin resistance and type II diabetes. Therefore, a major focus of recent research has been to identify and characterise proteins that regulate Glut4 translocation. Cysteine-string protein (Csp) is a secretory vesicle protein that functions in presynaptic neurotransmission and also in regulated exocytosis from non-neuronal cells. We show that Csp1 is expressed in 3T3-L1 adipocytes and that cellular levels of this protein are increased following cell differentiation. Combined fractionation and immunofluorescence analyses reveal that Csp1 is not a component of intracellular Glut4-storage vesicles (GSVs), but is associated with the adipocyte plasma membrane. This association is stable, and not affected by either insulin stimulation or chemical depalmitoylation of Csp1. We also demonstrate that Csp1 interacts with the t-SNARE syntaxin 4. As syntaxin 4 is an important mediator of insulin-stimulated GSV fusion with the plasma membrane, this suggests that Csp1 may play a regulatory role in this process. Syntaxin 4 interacts specifically with Csp1, but not with Csp2. In contrast, syntaxin 1A binds to both Csp isoforms, and actually exhibits a higher affinity for the Csp2 protein. The results described raise a number of interesting questions concerning the intracellular targeting of Csp in different cell types, and suggest that the composition and synthesis of GSVs may be different from synaptic and other secretory vesicles. In addition, the interaction of Csp1 with syntaxin 4 suggests that this Csp isoform may play a role in insulin-stimulated fusion of GSVs with the plasma membrane.

Benfenati
F.
,
Onorfi
F.
,
Giovedi
S.
(
1999
).
Protein-protein interactions and protein modules in the control of neurotransmitter release.
Phil. Trans. R. Soc. Lond. B
354
,
243
257
Bezprozvanny
I.
,
Scheller
R. H.
,
Tsien
R. W.
(
1995
).
Functional impact of syntaxin on gating of N-type and Q-type calcium channels.
Nature
378
,
623
626
Brown
H.
,
Larsson
O.
,
Branstrom
R.
,
Yang
S.-N.
,
Leibiger
B.
,
Leibiger
I.
,
Fried
G.
,
Moede
T.
,
Deeney
J. T.
,
Brown
G. R.
,
Jacobsson
G.
,
Rhodes
C. J.
,
Braun
J. E. A.
,
Scheller
R. H.
,
Corkey
B. E.
,
Berggren
P.-O.
,
Meister
B.
(
1998
).
Cysteine string protein (CSP) is an insulin secretory granule-associated protein regulating-cell exocytosis.
EMBO. J
17
,
5048
5058
Braun
J. E. A.
,
Scheller
R. H.
(
1995
).
Cysteine string protein, a DnaJ family member, is present on diverse secretory vesicles.
Neuropharmacology
34
,
1361
1369
Braun
J. E. A.
,
Wilbanks
S. M.
,
Scheller
R. H.
(
1996
).
The cysteine string secretory vesicle protein activates Hsc70 ATPase.
J. Biol. Chem
271
,
25989
25993
Burgoyne
R. D.
,
Morgan
A.
(
1995
).
Ca2+and secretory vesicle dynamics.
Trends in Neurosci
18
,
191
196
Chamberlain
L. H.
,
Burgoyne
R. D.
(
1996
).
Identification of a novel cysteine string protein variant and expression of cysteine string proteins in non-neuronal cells.
J. Biol. Chem
271
,
7320
7323
Chamberlain
L. H.
,
Burgoyne
R. D.
(
1997
).
Activation of the ATPase activity of heat-shock proteins Hsc70/Hsp70 by cysteine-string protein.
Biochem. J
322
,
853
858
Chamberlain
L. H.
,
Burgoyne
R. D.
(
1997
).
The molecular chaperone function of the secretory vesicle cysteine string proteins.
J. Biol. Chem
272
,
31420
31426
Chamberlain
L. H.
,
Burgoyne
R. D.
(
1998
).
Cysteine string protein functions directly in regulated exocytosis.
Mol. Biol. Cell
9
,
2259
2267
Chamberlain
L. H.
,
Burgoyne
R. D.
(
1998
).
The cysteine-string domain of the secretory vesicle cysteine-string protein is required for membrane targeting.
Biochem. J
335
,
205
209
Chamberlain
L. H.
,
Burgoyne
R. D.
(
2000
).
Cysteine-string protein: the chaperone at the synapse.
J. Neurochem
74
,
1781
1789
Chamberlain
L. H.
,
Henry
J.
,
Burgoyne
R. D.
(
1996
).
Cysteine string proteins are associated with chromaffin granules.
J. Biol. Chem
271
,
19514
19517
Chamberlain
L. H.
,
Roth
D.
,
Morgan
A.
,
Burgoyne
R. D.
(
1995
).
Distinct effects of-SNAP, 14–3-3 proteins, and calmodulin on priming and triggering of regulated exocytosis.
J. Cell Biol
130
,
1063
1071
Cheatham
B.
,
Volchuk
A.
,
Kahn
C. R.
,
Wang
L.
,
Rhodes
C. J.
,
Klip
A.
(
1996
).
Insulin-stimulated translocation of GLUT4 glucose transporters requires SNARE-complex proteins.
Proc. Natl. Acad. Sci. USA
93
,
15169
15173
Coppola
T.
,
Gundersen
C. B.
(
1996
).
Widespread expression of human cysteine string proteins.
FEBS Lett
391
,
269
272
Dawson-Scully
K.
,
Bronk
P.
,
Atwood
H. L.
,
Zinsmaier
K. E.
(
2000
).
Cysteine-string protein increases the calcium sensitivity of neurotransmitter exocytosis in Drosophila.
J. Neurosci
20
,
6039
6047
Frost
S. C.
,
Lane
M. D.
(
1988
).
Evidence for the involvement of vicinal sulphydryl groups in insulin-activated hexose transport in 3T3-L1 adipocytes.
J. Biol. Chem
260
,
2646
2652
Gonzalo
S.
,
Linder
M. E.
(
1998
).
SNAP-25 palmitoylation and plasma membrane targeting require a functional secretory pathway.
Mol. Biol. Cell
9
,
585
597
Gould
G. W.
,
Lienhard
G. E.
,
Tanner
L. I.
,
Gibbs
E. M.
(
1989
).
Phenylarsine oxide stimulates hexose transport in 3T3-L1 adipocytes by amechanism other than an increase in surface transporters.
Arch. Biochem. Biophys
268
,
264
275
Graham
M. E.
,
Burgoyne
R. D.
(
2000
).
Comparison of cysteine string protein (Csp) and mutant-SNAP overexpression reveals a role for Csp in late steps of membrane fusion in dense-core granule exocytosis in adrenal chromaffin cells.
J. Neurosci
20
,
1281
1289
Gundersen
C. B.
,
Mastrogiacomo
A.
,
Faull
K.
,
Umbach
J. A.
(
1994
).
Extensive lipidation of a Torpedo cysteine string protein.
J. Biol. Chem
269
,
19197
19199
Gundersen
C. B.
,
Umbach
J. A.
(
1992
).
Suppression cloning of the cDNA for a candidate subunit of a presynaptic calcium channel.
Neuron
9
,
527
537
Hashiramoto
M.
,
James
D. E.
(
2000
).
Characterization of insulin-responsive Glut4 storage vesicles isolated from 3T3-L1 adipocytes.
Mol. Cell Biol
20
,
416
427
Kelley
W. L.
(
1998
).
The J-domain family and the recruitment of chaperone power.
Trends Biochem. Sci
23
,
222
227
Kwon
O.-J.
,
Kim
C.
,
Kim
H.
,
Chin
H.
(
1996
).
Differential expression of the alternatively spliced variants of the rat cysteine string protein.
Mol. Biol. Cell
7
,
2599
–.
Langer
T.
,
Lu
C.
,
Echols
H.
,
Flanagan
J.
,
Hayer
M. K.
,
Hartl
F.-U.
(
1992
).
Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated protein folding.
Nature
356
,
683
689
Leveque
C.
,
El Far
O.
,
Martin-Moutot
N.
,
Sato
K.
,
Kato
R.
,
Takahashi
M.
,
Seagar
M. J.
(
1994
).
Purification of the N-type calcium channel associated with syntaxin and synaptotagmin.
J. Biol. Chem
269
,
6306
6312
Leveque
C.
,
Pupier
S.
,
Marqueze
B.
,
Geslin
L.
,
Kataoka
M.
,
Takahashi
M.
,
De Waard
M.
,
Seagar
M.
(
1998
).
Interaction of cysteine string proteins with the1Asubunit of the P/Q-type calcium channel.
J. Biol. Chem
273
,
13488
13492
Macaulay
S. L.
,
Hewish
D. R.
,
Gough
K. H.
,
Stoichevska
V.
,
MacPherson
S. F.
,
Jagadish
M.
,
Ward
C. W.
(
1997
).
Functional studies in 3T3L1 cells support a role for SNARE proteins in insulin stimulation of GLUT4 translocation.
Biochem. J
324
,
217
224
Martin
L. B.
,
Shewan
A.
,
Millar
C. A.
,
Gould
G. W.
,
James
D. E.
(
1998
).
Vesicle-associated membrane protein 2 plays a specific role in the insulin-dependent trafficking of the facilitative glucose transporter GLUT4 in 3T3-L1 adipocytes.
J. Biol. Chem
273
,
1444
1452
Martin
S.
,
Tellam
J.
,
Livingstone
C.
,
Slot
J. W.
,
Gould
G. W.
,
James
D. E.
(
1996
).
The glucose transporter (GLUT-4) and vesicle-associated membrane protein-2 (VAMP-2) are segregated from recycling endosomes in insulin-sensitive cells.
J. Cell Biol
134
,
625
635
Martin-Moutot
N.
,
Charvin
N.
,
Leveque
C.
,
Sato
K.
,
Nishiki
T.
,
Kozaki
S.
,
Takahashi
M.
,
Seagar
M. J.
(
1996
).
Interaction of SNARE complexes with P/Q-type calcium channels in rat cerebellar synaptosomes.
J. Biol. Chem
271
,
6567
6570
Mastrogiacomo
A.
,
Evans
C. J.
,
Gundersen
C. B.
(
1994
).
Antipeptide antibodies against a Torpedo cysteine-string protein.
J. Neurochem
62
,
873
880
Mastrogiacomo
A.
,
Kohan
S. A.
,
Whitelegge
J. P.
,
Gundersen
C. B.
(
1998
).
Intrinsic membrane association of Drosophila cysteine string proteins.
FEBS Lett
436
,
85
91
Mastrogiacomo
A.
,
Parsons
S. M.
,
Zampighi
G. A.
,
Jenden
D. J.
,
Umbach
J. A.
,
Gundersen
C. B.
(
1994
).
Cysteine string proteins: A potential link between synaptic vesicles and presynaptic Ca2+channels.
Science
263
,
981
982
Min
J.
,
Okada
S.
,
Kanzaki
M.
,
Elmendorf
J. S.
,
Coker
K. J.
,
Ceresa
B. P.
,
Syu
L.-J.
,
Noda
Y.
,
Saltiel
A. R.
,
Pessin
J. E.
(
1999
).
Synip: A novel insulin-regulated syntaxin-4 binding protein mediating GLUT4 translocation in adipocytes.
Mol. Cell
3
,
751
760
Minami
Y.
,
Hohfeld
J.
,
Ohtsuka
K.
,
Hartl
F.-U.
(
1996
).
Regulation of the heat-shock protein 70 reaction cycle by the mammalian DnaJ homologue, Hsp40.
J. Biol. Chem
271
,
19617
19624
Morales
M.
,
Ferrus
A.
,
Martinez-Pardon
M.
(
1999
).
Presynaptic calcium channel currents in normal and csp mutant Drosophila peptidergic terminals.
Eur. J. Neurosci
11
,
1818
1826
Nie
Z.
,
Ranjan
R.
,
Wenniger
J. J.
,
Hong
S. N.
,
Bronk
P.
,
Zinsmaier
K. E.
(
1999
).
Overexpression of cysteine-string proteins in Drosophila reveals interactions with syntaxin.
J. Neurosci
19
,
10270
10279
Olson
A. L.
,
Knight
J. B.
,
Pessin
J. E.
(
1997
).
Syntaxin 4, VAMP2, and/or VAMP3/Cellubrevin are functional target membrane and vesicleSNAP receptors for insulin-stimulated GLUT4 translocation in adipocytes.
Mol. Cell Biol
17
,
2425
2435
Piper
R. C.
,
Hess
L. J.
,
James
D. E.
(
1991
).
Differential sorting of two glucose transporters expressed in insulin-sensitive cells.
Am. J. Physiol
260
,
570
–.
Pupier
S.
,
Leveque
C.
,
Marqueze
B.
,
Kataoka
M.
,
Takahashi
M.
,
Seagar
M. J.
(
1997
).
Cysteine string proteins are associated with secretory granules of the rat neurohypophysis.
J. Neurosci
17
,
2722
2727
Rea
S.
,
Martin
L. B.
,
McIntosh
S.
,
Macaulay
S. L.
,
Ramsdale
T.
,
Baldini
G.
,
James
D. E.
(
1998
).
Syndet, an adipocyte target SNARE involved in the insulin-induced translocation of GLUT4 to the cell surface.
J. Biol. Chem
273
,
18784
18792
Satoh
S.
,
Nishimura
H.
,
Clark
A. E.
,
Kozka
I. J.
,
Quon
M. J.
,
Cushman
S. W.
,
Holman
G. D.
(
1993
).
Use of the bismannose photolabel to elucidate insulin regulated GLUT4 subcellular trafficking in rat adipose cells: Evidence that exocytosis is a critical site of hormone action.
J. Biol. Chem
268
,
17820
17829
Sheng
Z.-H.
,
Rettig
J.
,
Takahashi
M.
,
Catterall
W. A.
(
1994
).
Identification of a syntaxin-binding site on N-type calcium channels.
Neuron
13
,
1303
1313
Sollner
T.
,
Whiteheart
S. W.
,
Brunner
M.
,
Erdjument-Bromage
H.
,
Geromanos
S.
,
Tempst
P.
,
Rothman
J. E.
(
1993
).
SNAP receptors implicated in vesicle targeting and fusion.
Nature
362
,
318
323
Sudhof
T. C.
(
1995
).
The synaptic vesicle cycle: a cascade of protein-protein interactions.
Nature
375
,
645
653
Tellam
J. T.
,
Macaulay
S. L.
,
McIntosh
S.
,
Hewish
D. R.
,
Ward
C. W.
,
James
D. E.
(
1997
).
Characterisation of Munc-18c and Syntaxin-4 in 3T3-L1 adipocytes.
J. Biol. Chem
272
,
6179
6186
Thurmond
D. C.
,
Ceresa
B. P.
,
Okada
S.
,
Elmendorf
J. S.
,
Coker
K.
,
Pessin
J. E.
(
1998
).
Regulation of insulin-stimulated GLUT4 translocation by Munc18c in 3T3L1 adipocytes.
J. Biol. Chem
273
,
33876
33883
Umbach
J. A.
,
Saitoe
M.
,
Kidokoro
Y.
,
Gundersen
C. B.
(
1998
).
Attenuated influx of calcium ions at nerve endings of csp and shibire mutant Drosophila.
J. Neurosci
18
,
3521
3529
Umbach
J. A.
,
Zinsmaier
K. E.
,
Eberle
K. K.
,
Buchner
E.
,
Benzer
S.
,
Gundersen
C. B.
(
1994
).
Presynaptic dysfunction in Drosophila csp mutants.
Neuron
13
,
899
907
Van de Goor
J.
,
Kelly
R. B.
(
1996
).
Association of Drosophila cysteine string proteins with membranes.
FEBS Lett
380
,
251
256
Van de Goor
J.
,
Ramaswami
M.
,
Kelly
R. B.
(
1995
).
Redistribution of synaptic vesicles and their proteins in temperature-sensitive shibirets1 mutant Drosophila.
Proc. Natl. Acad. Sci. USA
92
,
5739
5743
Vogel
K.
,
Cabaniols
J.-P.
,
Roche
P. A.
(
2000
).
Targeting of SNAP-25 to membranes is mediated by its association with the target SNARE syntaxin.
J. Biol. Chem
275
,
2959
2965
Volchuk
A.
,
Sargeant
R.
,
Sumitani
S.
,
Liu
Z.
,
He
L.
,
Klip
A.
(
1996
).
Cellubrevin is a resident protein of insulin-sensitive GLUT4 glucose transporter vesicles in 3T3-L1 adipocytes.
J. Biol. Chem
270
,
8233
8240
Volchuk
A.
,
Wang
Q.
,
Ewart
H. S.
,
Liu
Z.
,
He
L.
,
Bennett
M. K.
,
Klip
A.
(
1995
).
Syntaxin 4 in 3T3-L1 adipocytes: regulation by insulin and participation in insulin-dependent glucose transport.
Mol. Biol. Cell
7
,
1075
1082
Wiser
O.
,
Bennett
M. K.
,
Atlas
D.
(
1996
).
Functional interaction of syntaxin and SNAP-25 with voltage-sensitive L-and N-type Ca2+channels.
EMBO J
15
,
4100
4110
Wu
M. N.
,
Fergestad
T.
,
Lloyd
T. E.
,
He
Y.
,
Broadie
K.
,
Bellen
H. J.
(
1999
).
Syntaxin 1A interacts with multiple exocytotic proteins to regulate neurotransmitter release in vivo.
Neuron
23
,
593
605
Xu
T.
,
Ashery
U.
,
Burgoyne
R. D.
,
Neher
E.
(
1999
).
Early requirement for-SNAP and NSF in the secretory cascade in chromaffin cells.
EMBO J
18
,
3293
3304
Yang
J.
,
Clark
A. E.
,
Harrison
R.
,
Kozka
I. J.
,
Holman
G. D.
(
1992
).
Trafficking of glucose transporters in 3T3-L1 cells.
Biochem. J
281
,
809
814
Zhang
H.
,
Kelley
W. L.
,
Chamberlain
L. H.
,
Burgoyne
R. D.
,
Wollheim
C. B.
,
Lang
J.
(
1998
).
Cysteine-string proteins regulate exocytosis of insulin independent from transmembrane ion fluxes.
FEBS Lett
437
,
267
272
Zhang
H.
,
Kelley
W. L.
,
Chamberlain
L. H.
,
Burgoyne
R. D.
,
Wollheim
C. B.
,
Lang
J.
(
1999
).
Mutational analysis of cysteine-string protein function in insulin exocytosis.
J. Cell Sci
112
,
1345
1351
Zinsmaier
K. E.
,
Eberle
K. K.
,
Buchner
E.
,
Walter
N.
,
Benzer
S.
(
1994
).
Paralysis and early death in cysteine string protein mutants in Drosophila.
Science
263
,
977
980
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