Identification of Domains Required for Developmentally Regulated SNARE Function in Saccharomyces cerevisiae

Abstract

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.

Prospore membrane formation linked to the leading edge protein (LEP) coat assembly

“…This meiotic differentiation process involves the formation of the so‐called prospore membranes (PSMs), the plasma membrane equivalent encapsulating the spores. PSM formation is dependent on a developmentally controlled reprogramming of the secretory pathway and was shown to rely on secretory processes equivalent to the fusion of vesicles with the plasma membrane (Neiman, 1998; Neiman et al ., 2000). The differentiation process starts with the formation of cytosolic precursors of the PSMs during meiosis I (Knop and Strasser, 2000).…”

Triosephosphate Isomerase and Its Product Glyceraldehyde-3-Phosphate Are Involved in the Regulatory Mechanism That Suppresses Exit from the Quiescent State in Yeast Cells

“…The genotypes and sources of yeast and E. coli strains used in this study are listed in Table S2 in the supplemental material. The S. cerevisiae strains used in this research were the rapidly sporulating SK-1 background strains AN120 (diploid) and AN117-16D (haploid) ( 38 ). To create the CLB2-GFP -expressing strain, pFA6a-GFP(S65T)-HIS3MX6 was amplified by PCR using primers clb 2 -F2 and clb 2 -R1 and then inserted at the 3′ end of the CLB2 coding sequence locus of haploid segregants of AN120 ( 38 ) by homologous recombination.…”

Receptor for advanced glycation end-products (RAGE) mediates phagocytosis in nonprofessional phagocytes

“…The yeast strains used in this study are AN120 (wild-type) ( MATα/MATa ARG4/arg4-NspI his3∆SK/his3∆SK ho::LYS2/ho::LYS2 leu2/leu2 lys2/lys2 RME1/rme1::LEU2 trp1::hisG/trp1::hisG ura3/ura3 ) 49 and HW3 ( dit1∆ ) ( MATα/MATa ARG4/arg4-NspI his3∆SK/his3∆SK ho::LYS2/ho::LYS2 leu2/leu2 lys2/lys2 RME1/rme1::LEU2 trp1::hisG/trp1::hisG ura3/ura3 dit1∆::his5 + /dit1∆::his5 + ) 50 . These strains are SK-1 strain background that sporulates with high efficiency 51 .…”

“…These strains are SK-1 strain background that sporulates with high efficiency 51 . To express GFP in AN120, the haploid strains 49 were transformed with pRS306TEF-GFP digested with Eco RV and the transformants were mated.…”