Examination of Sec22 Homodimer Formation and Role in SNARE-dependent Membrane Fusion

John J Flanagan,Indrani Mukherjee,Charles Barlowe

doi:10.1074/jbc.m114.626911

Abstract

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein complexes play essential roles in catalyzing intracellular membrane fusion events although the assembly pathway and molecular arrangement of SNARE complexes in membrane fusion reactions are not well understood. Here we monitored interactions of the R-SNARE protein Sec22 through a cysteine scanning approach and detected efficient formation of cross-linked Sec22 homodimers in cellular membranes when cysteine residues were positioned in the SNARE motif or C terminus of the transmembrane domain. When specific Sec22 cysteine derivatives are present on both donor COPII vesicles and acceptor Golgi membranes, the formation of disulfide cross-links provide clear readouts on trans- and cis-SNARE arrangements during this fusion event. The Sec22 transmembrane domain was required for efficient homodimer formation and for membrane fusion suggesting a functional role for Sec22 homodimers. We propose that Sec22 homodimers promote assembly of higher-order SNARE complexes to catalyze membrane fusion. Sec22 is also reported to function in macroautophagy and in formation of endoplasmic reticulum-plasma membrane contact sites therefore homodimer assembly may regulate Sec22 activity across a range of cellular processes.

Highlights

Sec22 assembles into protein complexes to catalyze membrane fusion events in the early secretory pathway
In this study we examined sensitive factor attachment protein receptor (SNARE) protein assemblies and topological arrangements that accompany fusion of COPII vesicles with Golgi membranes
A cysteine cross-linking analysis of Sec22 indicated that this R-SNARE protein forms homodimers that are efficiently captured when cysteines are positioned at specific locations within the SNARE motif and surprisingly in transmembrane or luminal regions

Summary

Background

Sec assembles into protein complexes to catalyze membrane fusion events in the early secretory pathway. We monitored interactions of the R-SNARE protein Sec through a cysteine scanning approach and detected efficient formation of cross-linked Sec homodimers in cellular membranes when cysteine residues were positioned in the SNARE motif or C terminus of the transmembrane domain. There are specific limitations in approaches to detect short-lived intermediates during SNARE-dependent membrane fusion and to monitor low affinity protein-protein or protein-lipid interactions that are likely to drive SNARE complex assembly [8] To address these issues we are developing new probes and in vitro assays to monitor SNARE-catalyzed fusion in the yeast ER2Golgi transport stage. Sec Homodimer Formation bearing a specific cysteine derivative of Bet were fused with Golgi membranes containing a cysteine derivative of Sec22 Formation of this cross-linked heterodimer was temperature and time dependent and required the same components known to function in this fusion event. We propose that Sec and possibly other R-SNARE proteins connect SNARE complexes into higher ordered arrangements for efficient bilayer fusion

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION