Enhancement of protein secretion in Saccharomyces cerevisiae by overproduction of Sso protein, a late-acting component of the secretory machinery.
Increased production of secreted proteins in Saccharomyces cerevisiae was achieved by overexpressing the yeast syntaxins. Sso1 or Sso2 protein, the t-SNAREs functioning at the targeting/fusion of the Golgi-derived secretory vesicles to the plasma membrane. Up to four- or six-fold yields of a heterologous secreted protein, Bacillus alpha-amylase, or an endogenous secreted protein, invertase, were obtained respectively when expressing either one of the SSO genes, SSO1 or SSO2, from the ADH1 promoter on a multicopy plasmid. Direct correlation between the Sso protein level and the amount of secreted alpha-amylase was demonstrated by modulating the expression level of the SSO2 gene. Quantitation of the alpha-amylase activity in the culture medium, periplasmic space and cytoplasm suggests that secretion into the periplasmic space is the primary stage at which the SSO genes exert the secretion-enhancing function. Pulse-chase data also support enhanced secretion efficiently obtained by SSO overexpression. Our data suggest that the Sso proteins may be rate-limiting components of the protein secretion machinery at the exocytosis step in yeast.
Ruohonen
L
L
VTT Biotechnology and Food Research, Espoo, Finland.
Toikkanen
J
J
Tieaho
V
V
Outola
M
M
Soderlund
H
H
Keranen
S
S
D001407:Bacillus / Q000201:enzymology; D002480:Cellulase / Q000378:metabolism; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics; D015966:Gene Expression Regulation, Fungal / Q000502:physiology; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D050765:Qa-SNARE Proteins; D012331:RNA, Fungal / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D000516:alpha-Amylases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D043324:beta-Fructofuranosidase
D005656:Fungal Proteins; D008565:Membrane Proteins; D050765:Qa-SNARE Proteins; D012331:RNA, Fungal; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C084006:SSO1 protein, S cerevisiae; C084007:SSO2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D006026:Glycoside Hydrolases; D000516:alpha-Amylases; D043324:beta-Fructofuranosidase; D002480:Cellulase