Role of the Vam3p transmembrane segment in homodimerization and SNARE complex formation.
Intracellular membrane fusion in eukaryotic cells is mediated by SNARE (soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor) proteins and is known to involve assembly of cognate subunits to heterooligomeric complexes. For synaptic SNAREs, it has previously been shown that the transmembrane segments drive homotypic and support heterotypic interactions. Here, we demonstrate that a significant fraction of the yeast vacuolar SNARE Vam3p is a homodimer in detergent extracts of vacuolar membranes. This homodimer exists in parallel to the heterooligomeric SNARE complex. A Vam3p homodimer also formed from the isolated recombinant protein. Interestingly, homodimerization depended on the transmembrane segment. In contrast, formation of the quaternary SNARE complex from recombinant Vam3p, Nyv1p, Vti1p, and Vam7p subunits did not depend on the transmembrane segment of Vam3p nor on the transmembrane segments of its partner proteins. We conclude that Vam3p homodimerization, but not quaternary SNARE complex formation, is promoted by TMS-TMS interaction. As the transmembrane segments of Vam3p and other SNARE homologues were previously shown to be critical for membrane fusion downstream of membrane apposition, our results may shed light on the functional significance of SNARE TMS-TMS interactions.
Roy
R
R
Lehrstuhl Chemie der Biopolymere, Technische Universität München, Weihenstephaner Berg 3, 85354 Freising, Germany.
Peplowska
K
K
Rohde
J
J
Ungermann
C
C
Langosch
D
D
D019281:Dimerization; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D050765:Qa-SNARE Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins / Q000235:genetics / Q000378:metabolism*
D005656:Fungal Proteins; D008565:Membrane Proteins; D050765:Qa-SNARE Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; C107706:VAM3 protein, S cerevisiae