Immunochemical characterization of gp115, a yeast glycoprotein modulated by the cell cycle.
A cell cycle-modulated glycoprotein (gp115, 115 kDa, isoelectric point 4.8-5) of Saccharomyces cerevisiae has been purified by Concanavalin A-affinity chromatography, followed by preparative two-dimensional gel electrophoresis, from yeast membrane proteins solubilized in Triton X-100. Antisera have been generated against the electrophoretically purified protein. Their specificity has been established by immunoblot analysis and by comparison of the partial proteolytic map obtained for the immunoprecipitated 35S-labeled 115 kDa polypeptide with that of the in vivo [35S]methionine-labeled gp115 isolated from two-dimensional gels. In tunicamycin-treated cells the immunoblot analysis identifies an unglycosylated precursor (86-88 kDa) and in sec18 mutant cells at the restrictive temperature an intermediary precursor of about 100 kDa. Six to seven carbohydrate chains have been estimated to be present on the gp115 protein, accounting for an electrophoretic shift corresponding to about 27 to 29 kDa of its relative molecular mass. Affinity-purified antibodies against the unglycosylated precursor (86-88 kDa) of gp115 were prepared and used to localize gp115 by indirect immunofluorescence microscopy. The similarity between the pattern of fluorescence obtained with these antibodies and that obtained using anti-plasma membrane H+-ATPase antibodies suggests an association of gp115 with the plasma membrane.
Popolo
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L
Dipartimento di Fisiologia e Biochimica Generali, Università di Milano, Italy.
Grandori
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R
Vai
M
M
Lacanà
E
E
Alberghina
L
L
D000918:Antibody Specificity; D002241:Carbohydrates / Q000032:analysis; D002453:Cell Cycle; D005455:Fluorescent Antibody Technique; D005656:Fungal Proteins / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D007120:Immunochemistry; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D012441:Saccharomyces cerevisiae / Q000032:analysis* / Q000502:physiology
D002241:Carbohydrates; D005656:Fungal Proteins