In Saccharomyces cerevisiae a short amino acid sequence facilitates excretion in the growth medium of periplasmic proteins

Abstract

SummaryIn Saccharomyces cerevisiae the cell wall is a barrier to excretion of proteins in the growth medium. Although small proteins are more easily released than bigger ones, other factors besides molecular sieving may play a role in partitioning of periplasmic proteins. By using several complementary approaches including enzyme-activity assays, quantitative immunoblotting on subcellular fractions and growth media, as well as a novel approach involving the use of flow cytometry and specific antibodies, we show that residues 1-8 of mature glucoamylase greatly enhance excretion of both glucoamylase and ␤-galactosidase in vivo and facilitate extraction of periplasmic proteins in vitro. Immunological data obtained by flow cytometry on whole cells indicate that this amino acid sequence increases the fraction of enzyme reaching the outer cell-wall layers. This amino acid sequence may define a novel type of topogenic sequence, facilitating the crossing of the yeast cell wall in vivo and facilitating extraction of periplasmic proteins by non-disruptive means in vitro.

Metabolic engineering ofSaccharomyces cerevisiaefor lactose/whey fermentation

“…The fusion of glucoamylase residues with E. coli β-galactosidase was shown to facilitate its secretion although the secretion was not as efficient as with the glucoamylase gene. 47 However, the authors do not mention if the recombinants were able to grow on lactose. Using the signal sequence of the membranar protein GgpI (the major yeast glycosylphosphatidylinositol-containing protein), it was possible to direct the E. coli β-galactosidase to the extracellular medium and for the first time, positive growth on lactose was observed.…”

The yeast : a new host for heterologous protein production, secretion and for metabolic engineering applications

“…a: pZ 3 : the backbone of the plasmid is the pYX022 S. cerevisiae expression plasmid; the expression cassette is based on the constitutive S. cerevisiae TPI promoter and the corresponding polyA signal, as indicated in the ¢gure. For the construction of plasmid pZ 3 klSTA2, the coding sequence of the same amylase but functionally linked to the K. lactis killer toxin leader sequence was cut with XhoI/AseI-blunt from plasmid pMV57 [41] and inserted in plasmid pZ 3 opened with EcoRI-blunt. b: pZ 3 bT: a pZ 3 vector where the S. cerevisiae TPI promoter was replaced by the Z. bailii TPI promoter.…”

“…Recombinant L-galactosidase and glucoamylase/amylase enzymatic activities from the di¡erent transformed yeasts were determined as previously described [41,43].…”

Functional analysis of multiple AUG codons in the transcripts of the STA2 glucoamylase gene from Saccharomyces cerevisiae

“…However, when the additional 10 aa peptide (signal II) is present in conjunction with signal I, it seems that secretion to the culture medium can proceed more eciently. Compatible with these results is the observation that heterologous proteins, when fused to signal I of STA2, are delivered to the periplasmic space, whereas these proteins are secreted into the culture medium when a stretch corresponding to signal II is also fused to the heterologous protein (Venturini et al 1997). Fittingly, the MUC1 gene, which encodes a putative integral membrane protein, only has AUG 2 , which will yield a protein with only signal I to deliver it to the cell membrane where its structural features will cause it to integrate into the membrane.…”