Reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex in yeast: the acceptor Golgi compartment is defective in the sec23 mutant.

Abstract

Abstract. Using either permeabilized cells or microsomes we have reconstituted the early events of the yeast secretory pathway in vitro. In the first stage of the reaction ,'o50-70% of the prepro-ct-factor, synthesized in a yeast translation lysate, is translocated into the endoplasmic reticulum (ER) of permeabilized yeast cells or directly into yeast microsomes. In the second stage of the reaction 48-66 % of the ER form of a-factor (26,000 D) is then converted to the high molecular weight Golgi form in the presence of ATP, soluble factors and an acceptor membrane fraction; GTPyS inhibits this transport reaction. Donor, acceptor, and soluble fractions can be separated in this assay. This has enabled us to determine the defective fraction in sec23, a secretory mutant that blocks ER to Golgi transport in vivo. When fractions were prepared from mutant cells grown at the permissive or restrictive temperature and then assayed in vitro, the acceptor Golgi fraction was found to be defective.

Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway

“…Classic epistasis analyses of the Novick and Schekman sec mutants (Novick et al 1980), placed the early sec genes in order within the secretory pathway: sec12, sec13, sec16, and sec23 mutants blocked formation of transport vesicles and induced proliferation of the ER, whereas sec17, sec18, and sec22 mutants blocked vesicle fusion and caused accumulation of vesicles (Novick et al 1981;Kaiser and Schekman 1990). The subsequent development of in vitro assays relied in part on the use of these mutants in biochemical complementation assays (Baker et al 1988;Ruohola et al 1988). Recapitulation of ER-Golgi traffic in permeabilized yeast cells was perturbed in sec23 mutants, but could be restored by incubation with cytosol prepared from wild-type cells, placing Sec23 as a soluble factor required for transport vesicle formation (Baker et al 1988).…”

Distribution and role of heterotrimeric G proteins in the secretory pathway of polarized epithelial cells

“…The use of non-hydrolyzable nucleotide analogs such as GTPyS provided the first clue that GTP-binding proteins were involved in various steps of vesicle trafficking (Balch, 1990;Beckers and Balch, 1989;Melancon et al, 1987;Ruohola et al, 1988;Tooze et al, 1990). Addition of GTPyS to permeabilized cells and or cell-free assays was generally found to block constitutive protein transport between membrane-bound compartments, by inhibiting either fusion or formation of vesicle carriers.…”

Retention of secretory proteins in an intermediate compartment and disappearance of the Golgi complex in an END4 mutant of Chinese hamster ovary cells

“…Major contributions to understanding secretory transport at the molecular level have been made by combining the genetic analysis of the secretory pathway in yeast (21,32,46) with the in vitro biochemical study of secretory transport in both mammalian cells (for example, see 2,6,58) and yeast (for example, see 1,42,51). Progress in understanding secretion would also be advanced by isolating and analyzing secretion-defective mutants in animal cells.…”