Fatty acid-acylated proteins in secretory mutants of Saccharomyces cerevisiae.

Abstract

Yeast secretory (sec) mutants that are blocked in the transport of secretory proteins and accumulate membrane organelles were used to study the biosynthesis of fatty acid-acylated proteins. Four proteins were labeled with [3H]palmitate in sec mutants accumulating endoplasmic reticulum membranes. Three of these (molecular weights -20,000, 50,000, and 120,000) were N-linked glycoproteins, based on their ability to be labeled with [3H] Membrane-bound glycoproteins undergo a number of post-translational modifications in the process of forming mature, biologically active molecules from newly synthesized polypeptides. A recently discovered modification is the covalent attachment of long-chain fatty acid to eucaryotic cell membrane proteins (9). Although initially detected in the glycoproteins of two enveloped animal viruses, vesicular stomatitis virus and Sindbis virus (21, 22), fatty acid-acylated proteins have been found in several other enveloped viruses (20) as well as in cultured cells (19) and intact animals (1). In the study of vesicular stomatitis virus glycoprotein biosynthesis, Schmidt and Schlesinger (23) related the time of fatty acid incorporation to oligosaccharide processing in the glycoprotein and found that acylation occurred shortly before the protein became endoglycosidase H (endo H) resistant. Dunphy et al. (5) proposed that fatty acid was added to vesicular stomatitis virus glycoprotein just before the trimming of oligosaccharide chains, perhaps in transitional elements of endoplasmic reticulum (ER) or in Golgi membranes.To obtain further information about this unusual interaction between protein and lipid, we initiated a study of the acylation reaction in a set of Saccharomyces cerevisiae mutants carrying temperature-sensitive defects in the secretory pathway for this organism (16,17). Yeast sec mutants affect the maturation and localization of proteins destined for secretion (invertase and phosphatase), for the yeast vacuole (carboxypeptidase), and for the surface membrane and wall (10-13). Under the conditions in which secretion is blocked (37°C), intracellular organelles accumulate and intermediate forms of the "secreted" proteins are detected. The organelles appear to be analogous to those associated with the secretory pathway in higher eucaryotes and consist of ER, Golgi bodies, and secretory vesicles (10). From a genetic analysis, mutations in a minimum of 23 complementation groups can affect the yeast secretory pathway (11). With * Corresponding author. many of these mutants, secretion can be restored by shifting the organism to the permissive temperature of 25°C.These mutants offer a potentially valuable system for studying fatty acid acylation of membrane proteins, since acylation occurs during protein movement through intracellular organelles. Therefore, we examined acylation in sec mutants representing four distinct blocks in the secretory pathway: sec53 blocks penetration of nascent polypeptide into ER; secl8 blocks transport from ER to Golgi; sec7 blocks formation of secretory vesicl...

Myoinositol gets incorporated into numerous membrane glycoproteins of Saccharomyces cerevisiae; incorporation is dependent on phosphomannomutase (sec53).

“…In this study we characterize these proteins as membrane glycoproteins which contain myoinositol in the form of covalently attached glycophospholipids and correct an earlier view which considers these proteins as being acylated directly on the protein (Wen and Schlesinger, 1984). In this study we characterize these proteins as membrane glycoproteins which contain myoinositol in the form of covalently attached glycophospholipids and correct an earlier view which considers these proteins as being acylated directly on the protein (Wen and Schlesinger, 1984).…”

Processing and fatty acid acylation of RAS1 and RAS2 proteins in Saccharomyces cerevisiae.

“…Wen and Schlesinger (36) reported the accumulation of several fatty-acylated proteins in yeast secretory mutants (secl, sec7, and sec18) at the nonpermissive temperature. They also reported that no [3H]palmitic acid-labeled proteins were found in secS3 mutant cells, a mutant defective in translocation of nascent polypeptides into the endoplasmic reticulum (23).…”

Recombinant Soluble β‐1,4‐Galactosyltransferases Expressed in Saccharomyces cerevisiae

“…These results were consistent with other previous reports. Indeed influenza virus hemagglutinin (Wen and Schlesinger, 1984), envelope glycoprotein from Epstein-Barr virus (Schultz et al, 1987), lysozyme (Nakamura et al, 1993; Kato et al, 1994) and Geotrichum candidum lipase I1 (Vernet et al, 1993) have been shown to be hypergl ycosylated when expressed in S. cerevisiue. The N-linked mannan prevented recognition by polyclonal antibodies (Fig.…”