The polybasic juxtamembrane region of Sso1p is required for SNARE function in vivo.
Exocytosis in Saccharomyces cerevisiae requires the specific interaction between the plasma membrane t-SNARE complex (Sso1/2p;Sec9p)and a vesicular v-SNARE (Snc1/2p). While SNARE proteins drive membrane fusion, many aspects of SNARE assembly and regulation are ill defined. Plasma membrane syntaxin homologs (including Sso1p) contain a highly charged juxtamembrane region between the transmembrane helix and the "SNARE domain" or core complex domain. We examined this region in vitro and in vivo by targeted sequence modification, including insertions and replacements. These modified Sso1 proteins were expressed as the sole copy of Sso in S. cerevisiae and examined for viability. We found that mutant Sso1 proteins with insertions or duplications show limited function, whereas replacement of as few as three amino acids preceding the transmembrane domain resulted in a nonfunctional SNARE in vivo. Viability is also maintained when two proline residues are inserted in the juxtamembrane of Sso1p, suggesting that helical continuity between the transmembrane domain and the core coiled-coil domain is not absolutely required. Analysis of these mutations in vitro utilizing a reconstituted fusion assay illustrates that the mutant Sso1 proteins are only moderately impaired in fusion. These results suggest that the sequence of the juxtamembrane region of Sso1p is vital for function in vivo, independent of the ability of these proteins to direct membrane fusion.
Van Komen
Jeffrey S
JS
Department of Biochemistry and Cell Biology, Rice University, 6100 Main Street MS-140, Houston, TX 77005, USA.
Bai
Xiaoyang
X
Rodkey
Travis L
TL
Schaub
Johanna
J
McNew
James A
JA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D024361:Amino Acids, Basic / Q000737:chemistry; D003470:Culture Media / Q000737:chemistry; D004252:DNA Mutational Analysis; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism*; D018390:Gene Targeting; D005800:Genes, Fungal; D005818:Genetic Engineering; D008561:Membrane Fusion; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D017354:Point Mutation; D011392:Proline / Q000378:metabolism; D017434:Protein Structure, Tertiary; D050765:Qa-SNARE Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D050600:SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000254:growth & development / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion
D024361:Amino Acids, Basic; D003470:Culture Media; D005656:Fungal Proteins; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; C084006:SSO1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D011392:Proline