Martin Wiedmann

YidC mediates membrane protein insertion in bacteria.

The basic machinery for the translocation of proteins into or across membranes is remarkably conserved from Escherichia coli to humans. In eukaryotes, proteins are inserted into the endoplasmic reticulum using the signal recognition particle (SRP) and the SRP receptor, as well as the integral membrane Sec61 trimeric complex (composed of alpha, beta and gamma subunits). In bacteria, most proteins are inserted by a related pathway that includes the SRP homologue Ffh, the SRP receptor FtsY, and the SecYEG trimeric complex, where Y and E are related to the Sec61 alpha and gamma subunits, respectively. Proteins in bacteria that exhibit no dependence on the Sec translocase were previously thought to insert into the membrane directly without the aid of a protein machinery. Here we show that membrane insertion of two Sec-independent proteins requires YidC. YidC is essential for E. coli viability and homologues are present in mitochondria and chloroplasts. Depletion of YidC also interferes with insertion of Sec-dependent membrane proteins, but it has only a minor effect on the export of secretory proteins. These results provide evidence for an additional component of the translocation machinery that is specialized for the integration of membrane proteins.

Oncogenic Ras and Akt Signaling Contribute to Glioblastoma Formation by Differential Recruitment of Existing mRNAs to Polysomes

In order to determine the global effects of oncogenic Ras and Akt signaling pathways on translational efficiencies, we compared the gene expression profiles of total cellular mRNA and mRNA associated with polysomes. We found that the immediate effect of Ras and Akt signaling blockade on transcription was relatively modest; however, the profile of mRNA associated with polysomes was substantially altered. These observations indicate that the immediate effect of Ras and Akt signaling regulates the recruitment of specific mRNAs to ribosomes to a far greater extent than they regulate the production of mRNAs by transcriptional effects. The mRNAs most affected are those encoding proteins that regulate growth, transcription regulation, cell to cell interactions, and morphology. These data support a model whereby Ras and Akt signaling primarily lead to cellular transformation by altering the transcriptome and producing a radical shift in the composition of mRNAs associated with actively translating polysomes.

The Organizing Principle in the Formation of the T Cell Receptor-CD3 Complex

The T cell receptor (TCR) serves a critical function in the immune system and represents one of the most complex receptor structures. A striking feature is the presence of nine highly conserved, potentially charged residues in the transmembrane helices. Previous models have attempted to explain assembly based on pairwise interactions of these residues. Using a novel method for the isolation of intact radiolabeled protein complexes, we demonstrate that one basic and two acidic transmembrane residues are required for the assembly of each of the three signaling dimers with the TCR. This remarkable three-helix arrangement applies to all three assembly steps and represents the organizing principle for the formation of this intricate receptor structure.

The molecular chaperone Ssb from Saccharomyces cerevisiae is a component of the ribosome–nascent chain complex

The 70 kDa heat shock proteins (Hsp70s) are a ubiquitous class of molecular chaperones. The Ssbs of Saccharomyces cerevisiae are an abundant type of Hsp70 found associated with translating ribosomes. To understand better the function of Ssb in association with ribosomes, the Ssb–ribosome interaction was characterized. Incorporation of the aminoacyl‐tRNA analog puromycin by translating ribosomes caused the release of Ssb concomitant with the release of nascent chains. In addition, Ssb could be cross‐linked to nascent chains containing a modified lysine residue with a photoactivatable cross‐linker. Together, these results suggest an interaction of Ssb with the nascent chain. The interaction of Ssb with the ribosome–nascent chain complex was stable, as demonstrated by resistance to treatment with high salt; however, Ssb interaction with the ribosome in the absence of nascent chain was salt sensitive. We propose that Ssb is a core component of the translating ribosome which interacts with both the nascent polypeptide chain and the ribosome. These interactions allow Ssb to function as a chaperone on the ribosome, preventing the misfolding of newly synthesized proteins.

The in vivo function of the ribosome-associated Hsp70, Ssz1, does not require its putative peptide-binding domain.

Two proteins of the Hsp70 class (Ssb and Ssz1) and one of the J-type class (Zuo1) of molecular chaperones reside on the yeast ribosome, with Ssz1 forming a stable heterodimer with Zuo1. We designed experiments to address the roles of these two distantly related ribosome-associated Hsp70s and their functional relationship to Zuo1. Strains lacking all three proteins have the same phenotype as those lacking only one, suggesting that these chaperones all function in the same pathway. The Hsp70 Ssb, whose peptide-binding domain is essential for its in vivo function, can be crosslinked to nascent chains on ribosomes that are as short as 54 amino acids, suggesting that Ssb interacts with nascent chains that extend only a short distance beyond the tunnel of the ribosome. A ssz1 mutant protein lacking its putative peptide-binding domain allows normal growth. Thus, binding of unfolded protein substrates in a manner similar to that of typical Hsp70s is not critical for Ssz1s in vivo function. The three chaperones are present in cells in approximately equimolar amounts compared with ribosomes. The level of Ssb can be reduced only a few-fold before growth is affected. However, a 50- to 100-fold reduction of Ssz1 and Zuo1 levels does not have a substantial effect on cell growth. On the basis of these results, we propose that Ssbs function as the major Hsp70 chaperone for nascent chains on the ribosome, and that Ssz1 has evolved to perform a nonclassical function, perhaps modulating Zuo1s ability to function as a J-type chaperone partner of Ssb.

Initial characterization of the nascent polypeptide-associated complex in yeast.

The three subunits of the nascent polypeptide‐associated complex (α, β1, β3) in Saccharomyces cerevisiae are encoded by three genes (EGD2, EGD1, BTT1). We found the complex bound to ribosomes via the β‐subunits in a salt‐sensitive manner, in close proximity to nascent polypeptides. Estimation of the molecular weight of the complex of wild‐type cells and cells lacking one or two subunits revealed that the composition of the complex is variable and that as yet unknown proteins might be included. Regardless of the variability, a certain balance of the subunits has to be maintained: the deletion of one subunit causes downregulation of the remaining subunits at physiological growth temperature. Cells lacking both β‐subunits are unable to grow at 37°C, most likely due to a toxic effect of the α‐subunit. Based on in vitro experiments, it has been proposed that the function of mammalian nascent‐polypeptide associated complexes (NAC) is to prevent inappropriate targeting of non‐secretory nascent polypeptides. In vivo, however, the lack of NAC does not cause secretion of signal‐less invertase in yeast. This result and the lack of a drastic phenotype of cells missing one, two or three subunits at optimal conditions (28°C, YPD‐medium) suggest either the existence of a substitute for NAC or that cells tolerate or ‘repair’ the damage caused by the absence of NAC. Copyright

Polypeptide‐binding proteins mediate completion of co‐translational protein translocation into the mammalian endoplasmic reticulum

The first step in the secretion of most mammalian proteins is their transport into the lumen of the endoplasmic reticulum (ER). Transport of pre‐secretory proteins into the mammalian ER requires signal peptides in the precursor proteins and a protein translocase in the ER membrane. In addition, hitherto unidentified lumenal ER proteins have been shown to be required for vectorial protein translocation. This requirement was confirmed in this study by using proteoliposomes that were made from microsomal detergent extracts and contained either low or high concentrations of lumenal ER proteins. Furthermore, immunoglobulin‐heavy‐chain‐binding protein (BiP) was shown to be able to substitute for the full set of lumenal proteins and, in the case of biotinylated precursor proteins, avidin was found to be able to substitute for lumenal proteins. Thus, the polypeptide‐chain‐binding protein BiP was identified as one lumenal protein that is involved in efficient vectorial protein translocation into the mammalian ER.

A membrane component of the endoplasmic reticulum that may be essential for protein translocation.

We have purified a glycosylated, membrane‐spanning protein of relative molecular mass approximately 34,000 (Mr approximately 34 K) from canine microsomes that appears to be essential for protein translocation across the endoplasmic reticulum (ER) as shown by the inhibitory action of antibodies directed against it and of monovalent Fab‐fragments produced from them. The ER membrane contains at least as many molecules of the 34 K membrane protein as bound ribosomes. The protein can be detected immunologically in tissues of various organisms, indicating an universal function.

Cloning of Novel Injury-regulated Genes IMPLICATIONS FOR AN IMPORTANT ROLE OF THE MUSCLE-SPECIFIC PROTEIN skNAC IN MUSCLE REPAIR

To gain insight into the molecular mechanisms underlying the wound repair process, we searched for genes that are regulated by skin injury. Using the differential display reverse transcription-polymerase chain reaction technique, we identified a gene that was strongly induced as early as 12 h after wounding. Sequence analysis revealed the identity of the corresponding protein with skeletal muscle nascent polypeptide-associated complex (skNAC), a recently identified muscle-specific transcription factor. By in situ hybridization and immunohistochemistry, we demonstrated the specific expression of skNAC in skeletal muscle cells of the panniculus carnosus at the wound edge. Furthermore, in vitro studies with cultured myoblasts revealed expression of skNAC in differentiating and differentiated, but not in proliferating, nondifferentiated cells. Differentiation of cultured myoblasts was accompanied by simultaneous expression of skNAC and the muscle-specific transcription factor myogenin. Our results provide the first evidence for a role of skNAC in muscle repair processes. Furthermore, they demonstrate the usefulness of our approach in identifying new players in wound repair.

Unregulated exposure of the ribosomal M-site caused by NAC depletion results in delivery of non-secretory polypeptides to the Sec61 complex

Nascent polypeptide associated complex (NAC) interacts with nascent polypeptides emerging from ribosomes. Both signal recognition particle (SRP) and NAC work together to ensure specificity in co‐translational targeting by competing for binding to the ribosomal membrane attachment site. While SRP selects signal‐containing ribosomes for targeting, NAC prevents targeting of signal peptide‐less nascent chains to the endoplasmic reticulum membrane. Here we show that the ribosome binding that occurs in NACs absence delivers signal‐less nascent chains to the Sec61 complex, underscoring the danger of unregulated exposure of the ribosomal M‐site. Recently, the idea that NAC prevents ribosome binding has been challenged. By carefully examining the physiologic NAC concentration in a variety of tissues from different species we here demonstrate that the discrepancy resulted from subphysiologic NAC concentrations.