EFP1 is an ER stress-induced glycoprotein which interacts with the pro-apoptotic protein Par-4

We have isolated the rat ortholog of EFP1 (EF-hand binding protein 1) as a novel interaction partner of the pro-apoptotic protein Par-4 (prostate apoptosis response-4). Rat EFP1 contains two thioredoxin domains, the COOH-terminal one harboring a CGFC motif, and has a similar protein domain structure as members of the protein disulfide isomerase (PDI) family. In REF52.2 and CHO cells, EFP1 colocalized with the endoplasmic reticulum (ER) marker PDI. Furthermore, EFP1 possesses catalytic activity as demonstrated by an insulin disulfide reduction assay. Western blot analysis revealed two EFP1 protein bands of approximately 136 and 155 kDa, representing different glycosylation states of the protein. Complex formation between EFP1 and Par-4 was confirmed in vitro and in vivo by co-immunoprecipitation, dot blot overlay and pull-down experiments. In CHO cells, coexpression of EFP1 and Par-4 resulted in enhanced Par-4-mediated apoptosis, which required the catalytic activity of EFP1. Interestingly, EFP1 was specifically upregulated in NIH3T3 cells after induction of ER stress by thapsigargin, tunicamycin, and brefeldin A, but not by agents that induce oxidative stress or ER-independent apoptosis. Furthermore, we could show that the induction of apoptosis by Ca2+ stress-inducing agents was significantly decreased after siRNA oligonucleotide-mediated knockdown of Par-4. Our data suggest that EFP1 might represent a cell-protective enzyme that could play an important role in the decision between survival and initiation of Par-4-mediated apoptosis

A dual function for chaperones SSB–RAC and the NAC nascent polypeptide–associated complex on ribosomes

In addition to assisting with protein folding, SSB and NAC also regulate ribosome biogenesis (see also companion paper from Albanèse et al. in this issue)

Einblicke in Funktionen und Mechanismen Ribosom-assoziierter Chaperone in Saccharomyces cerevisiae

The folding of newly synthesized proteins into their native structures is a fundamental but failure prone process and therefore controlled by a network of molecular chaperones to assist and ensure correct protein folding events. Chaperones that guide the folding of newly synthesized proteins in the cytosol are classified into two groups: chaperones that are recruited to the ribosome are the first interaction partners of newborn proteins. They are assumed to protect nascent polypeptides against harmful conditions and to support initial cotranslational folding steps, while cytosolic chaperones act subsequently on a subset of newly synthesized proteins to mainly promote post-translational folding steps.Chaperones associating with the ribosome have been found in all kingdoms of life. While the Trigger Factor (TF) is the only known ribosome-associated chaperone in bacteria, two systems exist in yeast and higher eukaryotes. Both systems, the nascent chain-associated complex (NAC) and the yeast tripartite Ssb-system (Ssb/RAC) are unrelated to TF. In contrast to bacterial TF, the functions of ribosome-associated systems in yeast are still barely defined. The Hsp70/40-based Ssb/RAC-system is per definition a canonical chaperone system, however, its precise function and potential substrates are unknown. Moreover, it is unclear whether NAC contributes to the folding network for newly synthesized proteins since that far no function of NAC could be unambiguously assigned.This work adds to the knowledge about ribosome-associated chaperones in yeast and focuses on the functions and mechanisms of NAC and the Ssb/RAC-system during de novo protein folding. Genetic surveys were combined with biochemical approaches in order to gain insights into the complex cytosolic chaperone network of eukaryotes.The key-results are summarized below:The first main contribution of this study is the finding that the Ssb-system functionally cooperates with NAC in co-translational protein folding in vivo. Simultaneous deletions of genes encoding NAC and Ssb caused a severe synthetic sickness of cells grown at 30¡C and the loss of cell viability under protein folding stress conditions. Deprivation of Ssb impaired NAC association with translating ribosomes and provoked aggregation of newly synthesized proteins, which was enhanced by additional deletion of NAC. Further analysis discovered a second function of Ssb/RAC and NAC in regulating the amount of 60S and 40S ribosomal particles suggesting a profound role in the biogenesis of ribosomes. Nac!ssb! cells revealed the formation of ribosomal halfmers and a pronounced deficiency of ribosomal subunits accompanied by strongly reduced amounts of translating ribosomes. These data provide for the first time evidence that the two ribosome-associated systems NAC and Ssb/RAC are functionally interconnected and contribute to two major cellular processes: the folding of newly synthesized proteins and the production of actively translating ribosomes.The second main contribution of this study is the identification of the genetic and functional interaction of NAC and the Hsp110 Sse1. Sse1 functions as nucleotide exchange factor (NEF) for two types of Hsp70s, the cytosolic Ssa and the ribosome-associated Ssb. This study showed that NAC and Sse1 genetically interact and the simultaneous deletion caused a severe growth impairment at 30¡C, a sensitivity against drugs inducing protein folding stress and a mild induction of the cellular heat shock response. The phenotype could be complemented by expressing NAC or by overexpression of Fes1, the second NEF for Ssa and Ssb-type of Hsp70s. Loss of Sse1 was accompanied by protein aggregation including polyubiquitinated proteins which was enhanced by additional NAC deletion. Mass spectrometry identified 13 potential Sse1 and NAC substrates including the enzyme Glucose-6-phophate-dehydrogenase. These data further support the theory that NAC is a bonafide member of the cytosolic chaperone network.In summary, the findings of this work have led to a clearer picture of the mechanisms and functional interplay of ribosome-associated chaperones as decisive regulators at the birthplace of new proteins

Structural Analysis of the Ribosome-associated Complex (RAC) Reveals an Unusual Hsp70/Hsp40 Interaction

Yeast Zuotin and Ssz are members of the conserved Hsp40 and Hsp70 chaperone families, respectively, but compared with canonical homologs, they atypically form a stable heterodimer termed ribosome-associated complex (RAC). RAC acts as co-chaperone for another Hsp70 to assist de novo protein folding. In this study, we identified the molecular basis for the unusual Hsp70/Hsp40 pairing using amide hydrogen exchange (HX) coupled with mass spectrometry and mutational analysis. Association of Ssz with Zuotin strongly decreased the conformational dynamics mainly in the C-terminal domain of Ssz, whereas Zuotin acquired strong conformational stabilization in its N-terminal segment. Deletion of the highly flexible N terminus of Zuotin abolished stable association with Ssz in vitro and caused a phenotype resembling the loss of Ssz function in vivo. Thus, the C-terminal domain of Ssz, the N-terminal extension of Zuotin, and their mutual stabilization are the major structural determinants for RAC assembly. We furthermore found dynamic changes in the J-domain of Zuotin upon complex formation that might be crucial for RAC co-chaperone function. Taken together, we present a novel mechanism for converting Zuotin and Ssz chaperones into a functionally active dimer