Activity of Kex2 dibasic endoprotease is localized throughout the secretory pathway in Saccharomyces cerevisiae. An ultracytochemical study.

Abstract:

:The sites of calcium-dependent dibasic endoprotease (Kex2; yscF) activity have been ultracytochemically localized in exponential cultures of haploid (alpha) wild-type strain of Saccharomyces cerevisiae, in its pep4-3 mutant and in the kex2 mutant. The gently prefixed cells were thoroughly washed and incubated in a buffered mixture containing Ca2+, benzyloxycarbonyl-L-tyrosyl-L-lysyl-arginin-4-methoxy-2-naphthylam ide (Z-Tyr-Lys-Arg-MNA) as substrate and hexazotized p-rosaniline (HPR) as coupler of the liberated MNA. The precipitated azo-dye was osmicated, and the cells were embedded for ultrathin sectioning. In the pep4-3 mutant the reaction product labeled the periphery of lipoprotein condensates, the lumen of nuclear envelope plus endoplasmic membrane cisternae, the matrix of juvenile vacuoles and the lumen of microvesicles--the membrane vesicles and the smaller coated or uncoated globules. These were dispersed in the cytoplasm or in the senescent vacuoles. The reaction product labeled also both faces of the plasmalemma-restricted cells. In the presence of EDTA the reaction product appeared only in the lipoprotein condensates. In the absence of substrate and in the presence of HgCl2, no reaction product was formed. In the wild-type strain the enzyme activity was detectable in the cytoplasmic microvesicles and in the tonoplast of vacuoles. No reaction product formed in the kex2 mutant cells. Enzymic assay of total activity of dibasic endoprotease in investigated strains confirmed the substrate was hydrolyzed principally by calcium-dependent protease. The study was supplemented by ultracytochemical localization of glycoproteins in cells of secretory mutants cultivated under restrictive conditions. The results of both topochemical studies give further arguments against the established model of a polar compartmentalized Golgi apparatus in S. cerevisiae.

journal_name

Eur J Cell Biol

authors

Vorísek J

subject

Has Abstract

pub_date

1994-02-01 00:00:00

pages

130-9

issue

1

eissn

0171-9335

issn

1618-1298

journal_volume

63

pub_type

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