Elimination of Defective α-Factor Pheromone Receptors

Abstract

This report compares trafficking routes of a plasma membrane protein that was misfolded either during its synthesis or after it had reached the cell surface. A temperature-sensitive mutant form of the yeast ␣-factor pheromone receptor (ste2-3) was found to provide a model substrate for quality control of plasma membrane proteins. We show for the first time that a misfolded membrane protein is recognized at the cell surface and rapidly removed. When the ste2-3 mutant cells were cultured continuously at 34°C, the mutant receptor protein (Ste2-3p) failed to accumulate at the plasma membrane and was degraded with a half-life of 4 min, compared with a halflife of 33 min for wild-type receptor protein (Ste2p). Degradation of both Ste2-3p and Ste2p required the vacuolar proteolytic activities controlled by the PEP4 gene. At 34°C, Ste2-3p comigrated with glycosylated Ste2p on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that Ste2-3p enters the secretory pathway. Degradation of Ste2-3p did not require delivery to the plasma membrane as the sec1 mutation failed to block rapid turnover. Truncation of the C-terminal cytoplasmic domain of the mutant receptors did not permit accumulation at the plasma membrane; thus, the endocytic signals contained in this domain are unnecessary for intracellular retention. In the pep4 mutant, Ste2-3p accumulated as series of high-molecular-weight species, suggesting a potential role for ubiquitin in the elimination process. When ste2-3 mutant cells were cultured continuously at 22°C, Ste2-3p accumulated in the plasma membrane. When the 22°C culture was shifted to 34°C, Ste2-3p was removed from the plasma membrane and degraded by a PEP4-dependent mechanism with a 24-min half-life; the wild-type Ste2p displayed a 72-min half-life. Thus, structural defects in Ste2-3p synthesized at 34°C are recognized in transit to the plasma membrane, leading to rapid degradation, and Ste2-3p that is preassembled at the plasma membrane is also removed and degraded following a shift to 34°C.The plasma membrane separates the interior of the cell from its extracellular environment. Integral membrane proteins play vital roles in the function of the plasma membrane by mediating such processes as nutrient transport, maintenance of electrochemical gradients, and transduction of sensory information. Monitoring and maintaining the structural integrity of these proteins present special problems for the cell, since the proteins are exposed continuously to the external environment yet their function is monitored largely inside the cell. Specific signals are known to result in the elimination of plasma membrane proteins from the cell surface, that is, endocytosis in response to specific ligands or when the metabolic or developmental state of the cell has been altered (61). Degradation of defective membrane proteins in the endoplasmic reticulum (ER) has also been described (8,26,46,63). However, little is known about the fate of membrane proteins that suffer damage after they have been inserted into...

The First Extracellular Loop of the Saccharomyces cerevisiae G Protein-coupled Receptor Ste2p Undergoes a Conformational Change upon Ligand Binding

“…In our experiments the yeast cell background used for expression of the mutant constructs was deficient in the proteases Pep4p, Prc1p, and Prb1p. A banding pattern similar to that observed for the four receptor mutants was also documented previously for the mutant A52T when expressed in a pep4 mutant background, but not in the wild-type background (31). In our prior experiments in which protease competent strains of S. cerevisiae were used, the multiple glycosylation forms were not detected (37).…”

“…Based on the primary amino acid sequence of the FLAG-and His-tagged construct used in these studies, the predicted molecular mass for Cys-less Ste2p is 50.8 kDa. Glycosylation of the protein accounts for the observed higher molecular weights of the Ste2p monomer (31). For the poorly expressed mutants, the Ste2p signal on the immunoblot is more diffuse, with signal for the monomer ranging from 52 to 70 kDa.…”

“…Although residues Asn 46 , Asn 105 , and Asn 205 are all potential glycosylation sites, analysis by site-directed mutagenesis of these sites indicates that they were not utilized (32). When analyzed by SDS-PAGE, Ste2p migrates as a group of three bands, consisting of the two major glycosylated forms of Ste2p along with the unglycosylated form (31). To determine whether the banding pattern exhibited by the N105C, S108C, Y111C, and T114C mutants was because of differential glycosylation, membranes prepared from cells expressing these mutants were treated with PNGase F, which cleaves N-glycan chains from glycopeptides or glycoproteins.…”

Mechanisms Governing the Activation and Trafficking of Yeast G Protein-coupled Receptors

“…Various mutant forms of the ␣-factor receptor appear to have distinct targetting defects. Whereas constitutively active receptors accumulate in post-ER compartments without undergoing extensive degradation, temperature-sensitive receptors are targetted relatively efficiently to the vacuole and degraded (Jenness et al, 1997). Although the mechanisms responsible for achieving these different fates are unknown, there are several possibilities.…”

Sphingoid base synthesis is required for oligomerization and cell surface stability of the yeast plasma membrane ATPase, Pma1

“…Cell fractionation on Renografin density gradients was performed as described (23,24). Fourteen fractions were collected.…”