A yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum.

Abstract

Abstract. We have devised a genetic selection for mutant yeast cells that fail to translocate secretory protein precursors into the lumen of the endoplasmic reticulum (ER). Mutant cells are selected by a procedure that requires a signal peptide-containing cytoplasmic enzyme chimera to remain in contact with the cytosol. This approach has uncovered a new secretory mutant, sec61, that is thermosensitive for growth and that accumulates multiple secretory and vacuolar precursor proteins that have not acquired any detectable posttranslational modifications associated with translocation into the ER. Preproteins that accumulate at the sec61 block sediment with the particulate fraction, but are exposed to the cytosol as judged by sensitivity to proteinase K. Thus, the sec61 mutation defines a gene that is required for an early cytoplasmic or ER membrane-associated step in protein translocation.T hE first step in the biogenesis of proteins destined for the secretory pathway is their insertion into the membrane of the endoplasmic reticulum (ER).I This process has been studied intensively in mammalian cells through the use of an in vitro assay that faithfully reproduces cotranslational translocation of secretory proteins into the lumen of the ER (2). Dissection of the components required for this activity has revealed the existence of both soluble and membrane-bound factors that participate in protein translocation. The signal recognition particle is a soluble ribonucleoprotein particle consisting of six polypeptides (54) and one molecule of 7SL RNA (55). The signal recognition particle binds to the signal sequence of a nascent preprotein (28, 56), thereby forming a complex that interacts with an integral membrane protein of the ER known as docking protein or signal recognition particle receptor (13,33). This targeting event is followed by cotranslational translocation of the preprotein into the ER lumen. Either during or shortly after the translocation event, the signal sequence is cleaved by the enzyme signal peptidase (10) and core oligosaccharides are transferred to specific asparagine residues (44) of the translocated polypeptide. The mechanism of protein permeation across the hydrophobic core of the ER membrane is not understood. Experiments with intermediates artificially blocked at various stages of membrane penetration suggest that this process is mediated by proteins, though they have yet to be identified by the existing assays (14).Recently, several groups have reconstituted protein translocation into the yeast ER in vitro (15,42,57). A yeast translation extract programmed with prepro-ct-factor mRNA di- rects the synthesis of an intact precursor which can insert co-or posttranslationaUy into yeast microsomes and become core-glycosylated. The existence of a posttranslational reaction has allowed these investigators to demonstrate that protein translocation into the yeast ER is energy dependent (15,43,57). In addition, fractionation experiments suggest that the import reaction requires cytosolic components (58). T...

Probing the membrane topology of Candida tropicalis cytochrome P450

“…4D) or Hol-, is indicated. When the invHIS4C moiety is translocated to the ER lumen, the protein becomes accessible for glycosylation (symbolized in models A and C) and is rendered inactive [13].…”

The Sec61 complex is essential for the insertion of proteins into the membrane of the endoplasmic reticulum

“…Sec61p was first identified in Saccharomyces cerevisiae [10] and found to be a major cross-linking partner of secretory proteins artificially trapped during the translocation process (denoted 'translocation intermediates') [11,12]. Sec61~, the mammalian homologue of Sec61p, was isolated via its tight association with ribosomes and shown to interact with nascent secretory proteins during translocation across the ER membrane [13].…”

Les gènes et les protéines qui contrôlent la voie de sécrétion

“…Les mutations létales sensiblesà la température exercent souvent un effet partielà la température permissive, la rechercheétait donc celle de mutants qui poussent a 30 • C sur le substrat -dans notre cas l'histidinol, substrat de l'enzyme histidinol-déshydrogénase, dernièreétape dans la biosynthèse de l'histidinemais qui n'arrivent pasà former des coloniesà 37 • C sur un substrat riche. Le premier mutant de Ray fut appelé sec61 et, en utilisant la même technique de sélection, cinq autres gènes furent ensuite identifiés, codant pour des fonctions additionnelles essentielles pour la translocation, y compris pour d'autres sousunités du complexe canalaire et une sous-unité de la particule de reconnaissance du signal (SRP) (Deshaies & Schekman, 1987 ;Rothblatt et al, 1989). Le clonage ultérieur de ces gènes a révélé que Sec61 est homologue au gène PrlA/SecyY de E. coli (Stirling et al, 1992).…”