The action of molecular chaperones in the early secretory pathway.

The endoplasmic reticulum (ER) serves as a way-station during the biogenesis of nearly all secreted proteins, and associated with or housed within the ER are factors required to catalyze their import into the ER and facilitate their folding. To ensure that only properly folded proteins are secreted and to temper the effects of cellular stress, the ER can target aberrant proteins for degradation and/or adapt to the accumulation of misfolded proteins. Molecular chaperones play critical roles in each of these phenomena.

Molecular chaperones in the yeast endoplasmic reticulum maintain the solubility of proteins for retrotranslocation and degradation.

Endoplasmic reticulum (ER)-associated degradation (ERAD) is the process by which aberrant proteins in the ER lumen are exported back to the cytosol and degraded by the proteasome. Although ER molecular chaperones are required for ERAD, their specific role(s) in this process have been ill defined. To understand how one group of interacting lumenal chaperones facilitates ERAD, the fates of pro-alpha-factor and a mutant form of carboxypeptidase Y were examined both in vivo and in vitro. We found that these ERAD substrates are stabilized and aggregate in the ER at elevated temperatures when BiP, the lumenal Hsp70 molecular chaperone, is mutated, or when the genes encoding the J domain-containing proteins Jem1p and Scj1p are deleted. In contrast, deletion of JEM1 and SCJ1 had little effect on the ERAD of a membrane protein. These results suggest that one role of the BiP, Jem1p, and Scj1p chaperones is to maintain lumenal ERAD substrates in a retrotranslocation-competent state.

Identification of an inhibitor of hsc70-mediated protein translocation and ATP hydrolysis.

Members of the hsc70 family of molecular chaperones are critical players in the folding and quality control of cellular proteins. Because several human diseases arise from defects in protein folding, the activity of hsc70 chaperones is a potential therapeutic target for these disorders. By using a known hsc70 modulator, 15-deoxyspergualin, as a seed, we identified a novel inhibitor of hsc70 activity. This compound, R/1, inhibits the endogenous and DnaJ-stimulated ATPase activity of hsc70 by 48 and 51%, respectively, and blocks the hsc70-mediated translocation of a preprotein into yeast endoplasmic reticulum-derived microsomal vesicles. Biochemical studies demonstrate that R/1 most likely exerts these effects by altering the oligomeric state of hsc70.

Species-specific elements in the large T-antigen J domain are required for cellular transformation and DNA replication by simian virus 40.

The J domain of simian virus 40 (SV40) large T antigen is required for efficient DNA replication and transformation. Despite previous reports demonstrating the promiscuity of J domains in heterologous systems, results presented here show the requirement for specific J-domain sequences in SV40 large-T-antigen-mediated activities. In particular, chimeric-T-antigen constructs in which the SV40 T-antigen J domain was replaced with that from the yeast Ydj1p or Escherichia coli DnaJ proteins failed to replicate in BSC40 cells and did not transform REF52 cells. However, T antigen containing the JC virus J domain was functional in these assays, although it was less efficient than the wild type. The inability of some large-T-antigen chimeras to promote DNA replication and elicit cellular transformation was not due to a failure to interact with hsc70, since a nonfunctional chimera, containing the DnaJ J domain, bound hsc70. However, this nonfunctional chimeric T antigen was reduced in its ability to stimulate hsc70 ATPase activity and unable to liberate E2F from p130, indicating that transcriptional activation of factors required for cell growth and DNA replication may be compromised. Our data suggest that the T-antigen J domain harbors species-specific elements required for viral activities in vivo.

Ribosomal protein S14 of Saccharomyces cerevisiae regulates its expression by binding to RPS14B pre-mRNA and to 18S rRNA.

Production of ribosomal protein S14 in Saccharomyces cerevisiae is coordinated with the rate of ribosome assembly by a feedback mechanism that represses expression of RPS14B. Three-hybrid assays in vivo and filter binding assays in vitro demonstrate that rpS14 directly binds to an RNA stem-loop structure in RPS14B pre-mRNA that is necessary for RPS14B regulation. Moreover, rpS14 binds to a conserved helix in 18S rRNA with approximately five- to sixfold-greater affinity. These results support the model that RPS14B regulation is mediated by direct binding of rpS14 either to its pre-mRNA or to rRNA. Investigation of these interactions with the three-hybrid system reveals two regions of rpS14 that are involved in RNA recognition. D52G and E55G mutations in rpS14 alter the specificity of rpS14 for RNA, as indicated by increased affinity for RPS14B RNA but reduced affinity for the rRNA target. Deletion of the C terminus of rpS14, where multiple antibiotic resistance mutations map, prevents binding of rpS14 to RNA and production of functional 40S subunits. The emetine-resistant protein, rpS14-EmRR, which contains two mutations near the C terminus of rpS14, does not bind either RNA target in the three-hybrid or in vitro assays. This is the first direct demonstration that an antibiotic resistance mutation alters binding of an r protein to rRNA and is consistent with the hypothesis that antibiotic resistance mutations can result from local alterations in rRNA structure.