The Tlg SNARE complex is required for TGN homotypic fusion.
Using a new assay for membrane fusion between late Golgi/endosomal compartments, we have reconstituted a rapid, robust homotypic fusion reaction between membranes containing Kex2p and Ste13p, two enzymes resident in the yeast trans-Golgi network (TGN). Fusion was temperature, ATP, and cytosol dependent. It was inhibited by dilution, Ca+2 chelation, N-ethylmaleimide, and detergent. Coimmunoisolation confirmed that the reaction resulted in cointegration of the two enzymes into the same bilayer. Antibody inhibition experiments coupled with antigen competition indicated a requirement for soluble NSF attachment protein receptor (SNARE) proteins Tlg1p, Tlg2p, and Vti1p in this reaction. Membrane fusion also required the rab protein Vps21p. Vps21p was sufficient if present on either the Kex2p or Ste13p membranes alone, indicative of an inherent symmetry in the reaction. These results identify roles for a Tlg SNARE complex composed of Tlg1p, Tlg2p, Vti1p, and the rab Vps21p in this previously uncharacterized homotypic TGN fusion reaction.
Brickner
J H
JH
Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
Blanchette
J M
JM
Sipos
G
G
Fuller
R S
RS
D002352:Carrier Proteins / Q000378:metabolism*; D002474:Cell-Free System; D005647:Fucosyltransferases / Q000378:metabolism; D008561:Membrane Fusion / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D026901:Membrane Transport Proteins; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D015003:Yeasts; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism; D021601:trans-Golgi Network / Q000378:metabolism*; D000097631:Galactoside 2-alpha-L-fucosyltransferase
D002352:Carrier Proteins; D005647:Fucosyltransferases; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D000097631:Galactoside 2-alpha-L-fucosyltransferase; C112252:TLG2 protein, S cerevisiae; C106736:VTI1 protein, S cerevisiae