Ste6p Mutants Defective in Exit from the Endoplasmic Reticulum (ER) Reveal Aspects of an ER Quality Control Pathway in Saccharomyces cerevisiae

We are studying the intracellular trafficking of the multispanning membrane protein Ste6p, the a-factor transporter in Saccharomyces cerevisiae and a member of the ATP-binding cassette superfamily of proteins. In the present study, we have used Ste6p as model for studying the process of endoplasmic reticulum (ER) quality control, about which relatively little is known in yeast. We have identified three mutant forms of Ste6p that are aberrantly ER retained, as determined by immunofluorescence and subcellular fractionation. By pulse-chase metabolic labeling, we demonstrate that these mutants define two distinct classes. The single member of Class I, Ste6–166p, is highly unstable. We show that its degradation involves the ubiquitin–proteasome system, as indicated by its in vivo stabilization in certain ubiquitin–proteasome mutants or when cells are treated with the proteasome inhibitor drug MG132. The two Class II mutant proteins, Ste6–13p and Ste6–90p, are hyperstable relative to wild-type Ste6p and accumulate in the ER membrane. This represents the first report of a single protein in yeast for which distinct mutant forms can be channeled to different outcomes by the ER quality control system. We propose that these two classes of ER-retained Ste6p mutants may define distinct checkpoint steps in a linear pathway of ER quality control in yeast. In addition, a screen for high-copy suppressors of the mating defect of one of the ER-retained ste6 mutants has identified a proteasome subunit, Hrd2p/p97, previously implicated in the regulated degradation of wild-type hydroxymethylglutaryl-CoA reductase in the ER membrane.

Implications of macromolecular crowding for signal transduction and metabolite channeling

The effect of different total enzyme concentrations on the flux through the bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in vitro was determined by measuring PTS-mediated carbohydrate phosphorylation at different dilutions of cell-free extract of Escherichia coli. The dependence of the flux on the protein concentration was more than linear but less than quadratic. The combined flux–response coefficient of the four enzymes constituting the glucose PTS decreased slightly from values of ≈1.8 with increasing protein concentrations in the assay. Addition of the macromolecular crowding agents polyethylene glycol (PEG) 6000 and PEG 35000 led to a sharper decrease in the combined flux–response coefficient, in one case to values of ≈1. PEG 6000 stimulated the PTS flux at lower protein concentrations and inhibited the flux at higher protein concentrations, with the transition depending on the PEG 6000 concentration. This suggests that macromolecular crowding decreases the dissociation rate constants of enzyme complexes. High concentrations of the microsolute glycerol did not affect the combined flux–response coefficient. The data could be explained with a kinetic model of macromolecular crowding in a two-enzyme group-transfer pathway. Our results suggest that, because of the crowded environment in the cell, the different PTS enzymes form complexes that live long on the time-scale of their turnover. The implications for the metabolic behavior and control properties of the PTS, and for the effect of macromolecular crowding on nonequilibrium processes, are discussed.