The Unfolded Protein Response Regulates Multiple Aspects of Secretory and Membrane Protein Biogenesis and Endoplasmic Reticulum Quality Control

The unfolded protein response (UPR) is an intracellular signaling pathway that relays signals from the lumen of the ER to activate target genes in the nucleus. We devised a genetic screen in the yeast Saccharomyces cerevisiae to isolate mutants that are dependent on activation of the pathway for viability. Using this strategy, we isolated mutants affecting various aspects of ER function, including protein translocation, folding, glycosylation, glycosylphosphatidylinositol modification, and ER-associated protein degradation (ERAD). Extending results gleaned from the genetic studies, we demonstrate that the UPR regulates trafficking of proteins at the translocon to balance the needs of biosynthesis and ERAD. The approach also revealed connections of the UPR to other regulatory pathways. In particular, we identified SON1/RPN4, a recently described transcriptional regulator for genes encoding subunits of the proteasome. Our genetic strategy, therefore, offers a powerful means to provide insight into the physiology of the UPR and to identify novel genes with roles in many aspects of secretory and membrane protein biogenesis

Structure, function and regulation of a nuclear gene in Saccharomyces cerevisiae that specifies MRP13, a protein of the small subunit of the mitochondrial ribosome

MRP13 was defined by biochemical criteria as a 35-kilodalton small subunit protein of the yeast mitochondrial ribosome. The MRP13 gene was identified by immunological screening of a yeast genomic library in $\lambda$gt11 and a functional copy of the gene has been cloned on a 2.2-kilobase BglII fragment. Sequencing of this fragment showed that the MRP13 coding region specifies as 324-amino acid basic protein with a calculated M\sb{\rm r} of 37,366. Computer searches failed to reveal any significant sequence similarity to previously identified ribosomal proteins or to the sequences in the current National Biomedical Research Foundation data base. Cells carrying disrupted copies of MRP13 lacked the MRP13 protein but were not impaired for growth on nonfermentable carbon sources. However, in comparison to the wild type, mrp13-$\Delta$2::TRP1 mutant cells had a lower rate of whole cell respiration, an unusual profile on in vivo labeled mitochondrial translation products and an abnormal profile of ribosomal subunits in sucrose gradient centrifugation. Mutants lacking MRP13 were also impaired in their ability to undergo the transition from growth on high concentrations of glucose to growth on nonrepressing carbon sources. This mutant phenotype suggests an important role for the MRP13 protein under conditions where cells are actively increasing their capacity for the synthesis of mitochondrial encoded proteins. Analysis of the sequence in the MRP13 5\sp\prime-flanking region revealed the closely linked gene for the cytoplasmic ribosomal protein RP39A. The RP39A coding region begins at nucleotide $-$846 and ends at $-$325 with respect to the MRP13 translational start. The steady-state levels of the MRP13 mRNA were determined in response to carbon catabolite repression, variation in the mitochondrial genetic background, and increased gene dosage of MRP13. In \rho\sp+ cells, transcript levels were repressed severalfold by growth in glucose as compared with growth in either galactose or nonfermentable carbon sources. In respiratory-deficient strains (\rho\sp{\rm o,} mit\sp-), however, transcription appeared to be largely derepressed even in the presence of high concentrations of glucose. Thus, the MRP13 mRNA is a member of a class of yeast nucleus-encoded RNAs whose transcription responds to changes in the mitochondrial genetic background. Despite high levels of the MRP13 transcripts in \rho\sp{\rm o} cells, the MRP13 protein did not accumulate, suggesting that the protein is relatively unstable in the absence of ribosome assembly. Cells carrying the MRP13 gene on a multiple-copy plasmid overproduced the mRNA in rough proportion to the gene dosage and the protein accumulated to a significant but lesser extent. The results indicate that MRP13 expression is regulated predominantly at the transcriptional level in response to catabolite repression and the cellular capacity for respiration. In addition, the levels of the MRP13 protein appear to be modulated posttranscriptionally by degradation of excess, unassembled polypeptides

Computational study of protein specificity: The molecular basis of HIV-1 protease drug resistance

Drug resistance has sharply limited the effectiveness of HIV-1 protease inhibitors in AIDS therapy. It is critically important to understand the basis of this resistance for designing new drugs. We have evaluated the free energy contribution of each residue in the HIV protease in binding to one of its substrates and to the five FDA-approved protease drugs. Analysis of these free energy profiles and the variability at each sequence position suggests: (i) single drug resistance mutations are likely to occur at not well conserved residues if they interact more favorably with drugs than with the substrate; and (ii) resistance-evading drugs should have a free energy profile similar to the substrate and interact most favorably with well conserved residues. We also propose an empirical parameter, called the free energy/variability value, which combines free energy calculation and sequence analysis to suggest possible drug resistance mutations on the protease. The free energy/variability value is defined as the product of one residue's contribution to the binding free energy and the variability of that residue. This parameter can assist in designing resistance-evading drugs for any target