In Saccharomyces cerevisiae a short amino acid sequence facilitates excretion in the growth medium of periplasmic proteins.
In 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 beta-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.
Venturini
M
M
Dipartimento di Fisiologia e Biochimica Generali, Università degli Studi di Milano, Italy.
Morrione
A
A
Pisarra
P
P
Martegani
E
E
Vanoni
M
M
D001692:Biological Transport / Q000235:genetics; D015153:Blotting, Western; D002457:Cell Extracts / Q000032:analysis; D003001:Cloning, Molecular; D003470:Culture Media / Q000032:analysis / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D017384:Sequence Deletion; D015398:Signal Transduction / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*
D002457:Cell Extracts; D003470:Culture Media; D005656:Fungal Proteins; D010455:Peptides; D001616:beta-Galactosidase; D005087:Glucan 1,4-alpha-Glucosidase