In vitro fusion catalyzed by the sporulation-specific t-SNARE light-chain Spo20p is stimulated by phosphatidic acid.
Sec9p and Spo20p are two SNAP25 family SNARE proteins specialized for different developmental stages in yeast. Sec9p interacts with Sso1/2p and Snc1/2p to mediate intracellular trafficking between post-Golgi vesicles and the plasma membrane during vegetative growth. Spo20p replaces Sec9p in the generation of prospore membranes during sporulation. The function of Spo20p requires enzymatically active Spo14p, which is a phosphatidylcholine (PC)-specific phospholipase D that hydrolyzes PC to generate phosphatidic acid (PA). Phosphatidic acid is required to localize Spo20p properly during sporulation; however, it seems to have additional roles that are not fully understood. Here we compared the fusion mediated by all combinations of the Sec9p or Spo20p C-terminal domains with Sso1p/Sso2p and Snc1p/Snc2p. Our results show that Spo20p forms a less efficient SNARE complex than Sec9p. The combination of Sso2p/Spo20c is the least fusogenic t-SNARE complex. Incorporation of PA in the lipid bilayer stimulates SNARE-mediated membrane fusion by all t-SNARE complexes, likely by decreasing the energetic barrier during membrane merger. This effect may allow the weak SNARE complex containing Spo20p to function during sporulation. In addition, PA can directly interact with the juxtamembrane region of Sso1p, which contributes to the stimulatory effects of PA on membrane fusion. Our results suggest that the fusion strength of SNAREs, the composition of organelle lipids and lipid-SNARE interactions may be coordinately regulated to control the rate and specificity of membrane fusion.
Liu
Song
S
Department of Biochemistry and Cell Biology, Rice University, 6100 Main Street MS-140, Houston, TX 77251-1892, USA.
Wilson
Kirilee A
KA
Rice-Stitt
Travis
T
Neiman
Aaron M
AM
McNew
James A
JA
D000595:Amino Acid Sequence; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry; D008055:Lipids / Q000737:chemistry; D008561:Membrane Fusion; D008969:Molecular Sequence Data; D010712:Phosphatidic Acids / Q000737:chemistry*; D011189:Potassium Chloride / Q000737:chemistry; D017434:Protein Structure, Tertiary; D050766:Qb-SNARE Proteins / Q000737:chemistry*; D050767:Qc-SNARE Proteins / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry*; D050600:SNARE Proteins / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*
D008051:Lipid Bilayers; D008055:Lipids; D010712:Phosphatidic Acids; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C090117:SEC9 protein, S cerevisiae; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; C497084:Spo20 protein, S cerevisiae; D011189:Potassium Chloride