Examination of Sec22 Homodimer Formation and Role in SNARE-Dependent Membrane Fusion [Membrane Biology]

March 6th, 2015 by Flanagan, J. J., Mukherjee, I., Barlowe, C.

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 crosslinked 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 crosslinks 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 ER-plasma membrane contact sites therefore homodimer assembly may regulate Sec22 activity across a range of cellular processes.