HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 281, Issue 4, 2281-2288, January 27, 2006

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 281/4/2281    most recent M511695200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Flanagan, J. J. Articles by Barlowe, C. Search for Related Content PubMed PubMed Citation Articles by Flanagan, J. J. Articles by Barlowe, C. Social Bookmarking                      What's this?

Cysteine-Disulfide Cross-linking to Monitor SNARE Complex Assembly during Endoplasmic Reticulum-Golgi Transport^*

From the Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755

Assembly of cognate SNARE proteins into SNARE complexes is required for many intracellular membrane fusion reactions. However, the mechanisms that govern SNARE complex assembly and disassembly during fusion are not well understood. We have devised a new in vitro cross-linking assay to monitor SNARE complex assembly during fusion of endoplasmic reticulum (ER)-derived vesicles with Golgi-acceptor membranes. In Saccharomyces cerevisiae, anterograde ER-Golgi transport requires four SNARE proteins: Sec22p, Bos1p, Bet1p, and Sed5p. After tethering of ER-derived vesicles to Golgi-acceptor membranes, SNARE proteins are thought to assemble into a four-helix coiled-coil bundle analogous to the structurally characterized neuronal and endosomal SNARE complexes. Molecular modeling was used to generate a structure of the four-helix ER-Golgi SNARE complex. Based on this structure, cysteine residues were introduced into adjacent SNARE proteins such that disulfide bonds would form if assembled into a SNARE complex. Our initial studies focused on disulfide bond formation between the SNARE motifs of Bet1p and Sec22p. Expression of SNARE cysteine derivatives in the same strain produced a cross-linked heterodimer of Bet1p and Sec22p under oxidizing conditions. Moreover, this Bet1p-Sec22p heterodimer formed during in vitro transport reactions when ER-derived vesicles containing the Bet1p derivative fused with Golgi membranes containing the Sec22p derivative. Using this disulfide cross-linking assay, we show that inhibition of transport with anti-Sly1p antibodies blocked formation of the Bet1p-Sec22p heterodimer. In contrast, chelation of divalent cations did not inhibit formation of the Bet1p-Sec22p heterodimer during in vitro transport but potently inhibited Golgi-specific carbohydrate modification of glyco-pro- factor. This data suggests that Ca²⁺ is not directly required for membrane fusion between ER-derived vesicles and Golgi-acceptor membranes.

Received for publication, October 28, 2005 , and in revised form, November 21, 1005.

^* This work was supported by National Institutes of Health Grants GM52549 (to C. B.) and GM070096 (to J. J. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ To whom correspondence should be addressed: HB 7200, Hanover, NH 03755. Tel.: 603-650-6516; Fax: 603-650-1128; E-mail: barlowe{at}dartmouth.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				K. M. Collins and W. T. Wickner trans-SNARE complex assembly and yeast vacuole membrane fusion PNAS, May 22, 2007; 104(21): 8755 - 8760. [Abstract] [Full Text] [PDF]

				L. M. Welsh, A. H. Y. Tong, C. Boone, O. N. Jensen, and S. Otte Genetic and molecular interactions of the Erv41p-Erv46p complex involved in transport between the endoplasmic reticulum and Golgi complex J. Cell Sci., November 15, 2006; 119(22): 4730 - 4740. [Abstract] [Full Text] [PDF]