Characterization of Munc-18c and Syntaxin-4 in 3T3-L1 Adipocytes

doi:10.1074/jbc.272.10.6179

Characterization of Munc-18c and Syntaxin-4 in 3T3-L1 Adipocytes

PUTATIVE ROLE IN INSULIN-DEPENDENT MOVEMENT OF GLUT-4*

From the ^‡ Centre for Molecular and Cellular Biology and Department of Physiology and Pharmacology, University of Queensland, St. Lucia 4072, Queensland, Australia and
^¶ CSIRO, Division of Biomolecular Engineering, 343 Royal Parade, Parkville 3052, Victoria, Australia

^∥Wellcome senior research fellow. To whom correspondence should be addressed. Tel.: 61-7-3365-4986; Fax: 61-7-3365-4388; E-mail: d.james{at}cmcb.uq.edu.au

Abstract

We have previously identified three mammalian Sec1/Munc-18 homologues in adipocytes (Tellam, J. T., McIntosh, S., and James, D. E. (1995) J. Biol. Chem. 270, 5857-5863). These proteins are thought to modulate the interaction between vesicle membrane and target membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and thus regulate intracellular vesicular transport. This study aimed to further characterize these Munc-18 isoforms and to define their potential role in the trafficking of GLUT-4 in adipocytes, a process reported to involve the vesicle membrane SNARE, VAMP-2. Using an in vitro binding assay with recombinant fusion proteins, we show that Munc-18a and Munc-18b bind to syntaxin-1A, −2, and −3, while Munc-18c binds only to syntaxin-2 and −4. The specific interaction between Munc-18c and syntaxin-4 is of interest because aside from syntaxin-1A, which is not expressed in adipocytes, syntaxin-4 is the only syntaxin that binds to VAMP-2. Using a three-way binding assay, it was shown that Munc-18c inhibits the binding of syntaxin-4 to VAMP-2. The subcellular distribution of syntaxin-4 and Munc-18c was almost identical, both being enriched in the plasma membrane, and both exhibiting an insulin-dependent movement out of an intracellular membrane fraction similar to that observed for GLUT-4. Munc-18b had a similar distribution to Munc-18c and so may also be involved in vesicle transport to the cell surface, whereas Munc-18a was undetectable by immunoblotting in adipocytes. Microinjection of a syntaxin-4 antibody into 3T3-L1 adipocytes blocked the insulin-dependent recruitment of GLUT-4 to the cell surface. These data suggest that syntaxin-4/Munc-18c/VAMP-2 may play a role in the docking/fusion of intracellular GLUT-4-containing vesicles with the cell surface in adipocytes.

Footnotes

↵^§ National Health and Medical Research Council (Dora Lush) postgraduate scholar.
↵* This work was supported in part by the National Health and Medical Research Council of Australia. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U76832[GenBank].
↵¹ The abbreviations used are:

v-SNARE

vesicle membrane SNARE

t-SNARE

target membrane SNARE

NSF

N-ethylmaleimide-sensitive factor

SNAPs

soluble NSF attachment proteins

SNAREs

soluble NSF attachment protein receptors

Munc-18

mammalian homologue of unc-18

VAMP

vesicle-associated membrane protein

PCR

polymerase chain reaction

CHO

Chinese hamster ovary

PAGE

polyacrylamide gel electrophoresis

BSA

bovine serum albumin

Ig

immunoglobulin

PM

plasma membrane(s)

LDM

low density microsome(s)

HDM

high density microsome(s)

M/N

mitochondria/nuclei.
↵² J. Tellam and D. James, unpublished results.
- Received October 3, 1996.
- Revision received November 27, 1996.