The N-terminal domain of the t-SNARE Vam3p coordinates priming and docking in yeast vacuole fusion.
Homotypic fusion of yeast vacuoles requires a regulated sequence of events. During priming, Sec18p disassembles cis-SNARE complexes. The HOPS complex, which is initially associated with the cis-SNARE complex, then mediates tethering. Finally, SNAREs assemble into trans-complexes before the membranes fuse. The t-SNARE of the vacuole, Vam3p, plays a central role in the coordination of these processes. We deleted the N-terminal region of Vam3p to analyze the role of this domain in membrane fusion. The truncated protein (Vam3 Delta N) is sorted normally to the vacuole and is functional, because the vacuolar morphology is unaltered in this strain. However, in vitro vacuole fusion is strongly reduced due to the following reasons: Assembly, as well as disassembly of the cis-SNARE complex is more efficient on Vam3 Delta N vacuoles; however, the HOPS complex is not associated well with the Vam3 Delta N cis-complex. Thus, primed SNAREs from Vam3 Delta N vacuoles cannot participate efficiently in the reaction because trans-SNARE pairing is substantially reduced. We conclude that the N-terminus of Vam3p is required for coordination of priming and docking during homotypic vacuole fusion.
Laage
R
R
University of Heidelberg, Biochemie Zentrum Heidelberg, 69120 Heidelberg, Germany.
Ungermann
C
C
D000251:Adenosine Triphosphatases; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology / Q000502:physiology; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000378:metabolism; D008561:Membrane Fusion / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism; D017434:Protein Structure, Tertiary; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D011817:Rabbits; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D014617:Vacuoles / Q000502:physiology*; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D005656:Fungal Proteins; C086796:GDP dissociation inhibitor 1; D020730:Guanine Nucleotide Dissociation Inhibitors; D008565:Membrane Proteins; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; C107706:VAM3 protein, S cerevisiae; C065454:VPS33 protein, S cerevisiae; C106736:VTI1 protein, S cerevisiae; D033921:Vesicular Transport Proteins; D004533:Egtazic Acid; D000251:Adenosine Triphosphatases; C064404:SEC18 protein, S cerevisiae; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid