Identification of domains required for developmentally regulated SNARE function in Saccharomyces cerevisiae.
Saccharomyces cerevisiae cells contain two homologues of the mammalian t-SNARE protein SNAP-25, encoded by the SEC9 and SPO20 genes. Although both gene products participate in post-Golgi vesicle fusion events, they cannot substitute for one another; Sec9p is active primarily in vegetative cells while Spo20p functions only during sporulation. We have investigated the basis for the developmental stage-specific differences in the function of these two proteins. Localization of the other plasma membrane SNARE subunits, Ssop and Sncp, in sporulating cells suggests that these proteins act in conjunction with Spo20p in the formation of the prospore membrane. In vitro binding studies demonstrate that, like Sec9p, Spo20p binds specifically to the t-SNARE Sso1p and, once bound to Sso1p, can complex with the v-SNARE Snc2p. Therefore, Sec9p and Spo20p interact with the same binding partners, but developmental conditions appear to favor the assembly of complexes with Spo20p in sporulating cells. Analysis of chimeric Sec9p/Spo20p molecules indicates that regions in both the SNAP-25 domain and the unique N terminus of Spo20p are required for activity during sporulation. Additionally, the N terminus of Spo20p is inhibitory in vegetative cells. Deletion studies indicate that activation and inhibition are separable functions of the Spo20p N terminus. Our results reveal an additional layer of regulation of the SNARE complex, which is necessary only in sporulating cells.
Department of Biochemistry and Cell Biology, Institute for Cell and Developmental Biology, State University of New York, Stony Brook, New York 11794-5215, USA. aneiman@ms.cc.sunysb.edu
A M
AM
Neiman
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L
Katz
P J
PJ
Brennwald
D000595:Amino Acid Sequence; D015153:Blotting, Western; D000252:Calcium-Transporting ATPases; D002462:Cell Membrane; D005455:Fluorescent Antibody Technique; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D005838:Genotype; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D050765:Qa-SNARE Proteins; D050767:Qc-SNARE Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050600:SNARE Proteins; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D050825:Synaptosomal-Associated Protein 25; D013696:Temperature; D033921:Vesicular Transport Proteins
D005656:Fungal Proteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; D050767:Qc-SNARE Proteins; D011993:Recombinant Fusion Proteins; C090117:SEC9 protein, S cerevisiae; D050600:SNARE Proteins; C101767:SSC1 protein, S cerevisiae; C084006:SSO1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D000252:Calcium-Transporting ATPases