Reid Gilmore

Protein translocation across the endoplasmic reticulum

In the past year, dramatic progress has been made in our understanding of protein biogenesis at the initial steps of the eukaryotic secretory pathway. New insights have refined our view of protein targeting to the endoplasmic reticulum membrane and provided the best glimpse so far of the subsequent translocation step. The interactions of three GTP-binding proteins have been found to result in a novel cycle of GTP binding and hydrolysis to regulate protein targeting. Experiments with fluorescent probes have revealed that the nascent chain enters an aqueous environment within the membrane sealed off from the cytosol. In vitro reconstitution experiments have shown surprising simplicity in the number of polypeptides required to facilitate translocation across a synthetic membrane and to promote the integration of membrane proteins. Furthermore, new genetic and functional similarities between divergent organisms have been discovered, providing convincing evidence of the evolutionary conservation of strategies used in the targeting and translocation of polypeptides.

Oligosaccharyltransferase activity is associated with a protein complex composed of ribophorins i and ii and a 48 kd protein

Oligosaccharyltransferase catalyzes the N-linked glycosylation of asparagine residues on nascent polypeptides in the lumen of the rough endoplasmic reticulum (RER). A protein complex composed of 66, 63, and 48 kd subunits copurified with oligosaccharyltransferase from canine pancreas. The 66 and 63 kd subunits were shown by protein immunoblotting to be identical to ribophorin I and II, two previously identified RER glycoproteins that colocalize with membrane-bound ribosomes. The transmembrane segment of ribophorin I was found to be homologous to a recently proposed dolichol recognition consensus sequence. Based on a revision of the consensus sequence, we propose a model for the interaction of dolichol with the glycosyltransferases that catalyze the assembly and transfer of lipid-linked oligosaccharides.

The signal recognition particle receptor mediates the GTP-dependent displacement of SRP from the signal sequence of the nascent polypeptide

The signal recognition particle (SRP)-mediated transport of proteins across mammalian endoplasmic reticulum requires GTP in a capacity distinct from polypeptide elongation. We defined the role of GTP by a molecular characterization of translocation intermediates that accumulate after incubation of SRP-ribosome complexes with microsomal membranes. SRP receptor-catalyzed displacement of SRP from ribosomes was GTP-dependent both with intact membranes and with the purified SRP receptor. GTP-specific binding was localized to the alpha subunit of the receptor by photoaffinity labeling and by probing nitrocellulose blots of the receptor with GTP. Analysis of the alpha subunit of the SRP receptor revealed amino acid sequences that are similar to guanine ribonucleotide binding site consensus sequence elements.

Structure of Monomeric Yeast and Mammalian Sec61 Complexes Interacting with the Translating Ribosome

Nascent Chains Revealed Detailed analysis of protein translation and translocation across membranes requires the identification and structural analysis of intermediates involved in these processes (see the Perspective by Kampmann and Blobel). Seidelt et al. (p. 1412, published online 29 October) report the visualization by cryo-electron microscopy of a nascent polypeptide chain in the tunnel of the ribosome at 5.8 angstroms. This resolution allows analysis of the conformation and distinct contacts of the nascent chain within the ribosomal tunnel, which suggests a mechanism by which translational stalling is induced by this peptide. Protein translocation across cellular membranes involves the Sec61 protein, a component of a protein-conducting channel. Whether Sec61 acts as a monomer or as an oligomer during protein translocation has been unclear. Becker et al. (p. 1369, published online 29 October) describe active yeast and mammalian ribosome-Sec61 structures that show the Sec61 complex interacting with the ribosome and a nascent secretory protein signal sequence. The analysis unambiguously reveals that the active protein-conducting channel is a single Sec61 copy with its central pore serving as conduit for the nascent polypeptide. A single copy of a protein-conducting channel molecule provides a conduit for polypeptide translocation across membranes. The trimeric Sec61/SecY complex is a protein-conducting channel (PCC) for secretory and membrane proteins. Although Sec complexes can form oligomers, it has been suggested that a single copy may serve as an active PCC. We determined subnanometer-resolution cryo–electron microscopy structures of eukaryotic ribosome-Sec61 complexes. In combination with biochemical data, we found that in both idle and active states, the Sec complex is not oligomeric and interacts mainly via two cytoplasmic loops with the universal ribosomal adaptor site. In the active state, the ribosomal tunnel and a central pore of the monomeric PCC were occupied by the nascent chain, contacting loop 6 of the Sec complex. This provides a structural basis for the activity of a solitary Sec complex in cotranslational protein translocation.

Biochemistry, molecular biology, and genetics of the oligosaccharyltransferase.

Asparagine‐linked glycosylation is a highly conserved protein Modification reaction that occurs in all eukaryotes. The initial stage in the biosynthesis of N‐linked glycoproteins, catalyzed by the enzyme oligosaccharyltransferase (OST), involves the transfer of a preassembled high‐mannose oligosaccharide from a dolichol‐linked oligosaccharide donor onto asparagine acceptor sites in nascent proteins in the lumen of the rough endoplasmic reticulum. Biochemical, molecular biological, and genetic studies conducted during the past 5 years have resulted in an explosive growth in our knowl‐edge concerning the OST. Although the basic biochemical properties of the enzyme were determined more than a decade ago using intact microsomal membranes, recent studies provide novel insight into the catalytic mechanism of the enzyme. The OST was recently purified as a large heteroligomeric membrane protein complex; the sequences of many of the subunits have been determined from both fungal and vertebrate sources. Consistent with the evolutionary conservation of N‐linked glycosylation, protein sequence comparisons reveal significant homologies between vertebrate, invertebrate, plant, and fungal OST subunits. Yeast molecular genetic methods have been instrumental in the functional characterization of the OST subunits, and halve proven to be powerful tools for the identification of novel gene products that influence oligosaccharide transfer in vivo.—Sil‐ berstein, S., Gilmore, R. Biochemistry, molecular biology, and genetics of the oligosaccharyltransferase. FASEB J. 10,849‐858 (1996)

Translocation of secretory proteins across the microsomal membrane occurs through an environment accessible to aqueous perturbants

We have characterized the association of a nascent secretory protein with the microsomal membrane at two distinct stages in cell-free synthesis and translocation. Stage one corresponded to a nascent chain of approximately 70 residues generated via elongation arrest by the signal recognition particle (SRP). Binding to microsomal membranes occurred independently of chain elongation and required SRP receptor. Following binding, the 70-mer remained attached to the membrane after extraction of the ribosome. However, protein denaturants (4 M urea or alkaline pH) extracted the 70-mer from the membrane. Stage two of synthesis corresponded to nascent chains of approximately 158 residues generated by oligonucleotide-mediated hybrid arrest of translation. Again, these partially translocated nascent chains were extracted by 4 M urea. Therefore, the initial interaction of the signal sequence with the membrane as well as subsequent chain conductance occur in a microenvironment that is accessible to aqueous reagents. Thus, both processes probably require integral membrane proteins.

Cotranslational and Posttranslational N-Glycosylation of Polypeptides by Distinct Mammalian OST Isoforms

Asparagine-linked glycosylation of polypeptides in the lumen of the endoplasmic reticulum is catalyzed by the hetero-oligomeric oligosaccharyltransferase (OST). OST isoforms with different catalytic subunits (STT3A versus STT3B) and distinct enzymatic properties are coexpressed in mammalian cells. Using siRNA to achieve isoform-specific knockdowns, we show that the OST isoforms cooperate and act sequentially to mediate protein N-glycosylation. The STT3A OST isoform is primarily responsible for cotranslational glycosylation of the nascent polypeptide as it enters the lumen of the endoplasmic reticulum. The STT3B isoform is required for efficient cotranslational glycosylation of an acceptor site adjacent to the N-terminal signal sequence of a secreted protein. Unlike STT3A, STT3B efficiently mediates posttranslational glycosylation of a carboxyl-terminal glycosylation site in an unfolded protein. These distinct and complementary roles for the OST isoforms allow sequential scanning of polypeptides for acceptor sites to insure the maximal efficiency of N-glycosylation.

Requirement of GTP hydrolysis for dissociation of the signal recognition particle from its receptor

The signal recognition particle (SRP) directs signal sequence specific targeting of ribosomes to the rough endoplasmic reticulum. Displacement of the SRP from the signal sequence of a nascent polypeptide is a guanosine triphosphate (GTP)-dependent reaction mediated by the membrane-bound SRP receptor. A nonhydrolyzable GTP analog can replace GTP in the signal sequence displacement reaction, but the SRP then fails to dissociate from the membrane. Complexes of the SRP with its receptor containing the nonhydrolyzable analog are incompetent for subsequent rounds of protein translocation. Thus, vectorial targeting of ribosomes to the endoplasmic reticulum is controlled by a GTP hydrolysis cycle that regulates the affinity between the SRP, signal sequences, and the SRP receptor.

Oligosaccharyltransferase Isoforms that Contain Different Catalytic STT3 Subunits Have Distinct Enzymatic Properties

Oligosaccharyltransferase (OST) is an integral membrane protein that catalyzes N-linked glycosylation of nascent proteins in the lumen of the endoplasmic reticulum. Although the yeast OST is an octamer assembled from nonhomologous subunits (Ost1p, Ost2p, Ost3p/Ost6p, Ost4p, Ost5p, Wbp1p, Swp1p, and Stt3p), the composition of the vertebrate OST was less well defined. The roles of specific OST subunits remained enigmatic. Here we show that genomes of most multicellular eukaryotes encode two homologs of Stt3p and mammals express two homologs of Ost3p. The Stt3p and Ost3p homologs are assembled together with the previously described mammalian OST subunits (ribophorins I and II, OST48, and DAD1) into complexes that differ significantly in enzymatic activity. Tissue and cell type-specific differences in expression of the Stt3p homologs suggest that the enzymatic properties of oligosaccharyltransferase are regulated in eukaryotes to respond to alterations in glycoprotein flux through the secretory pathway and may contribute to tissue-specific glycan heterogeneity.

Role of Sec61α in the Regulated Transfer of the Ribosome–Nascent Chain Complex from the Signal Recognition Particle to the Translocation Channel

Targeting of ribosome-nascent chain complexes to the translocon in the endoplasmic reticulum is mediated by the concerted action of the signal recognition particle (SRP) and the SRP receptor (SR). Ribosome-stripped microsomes were digested with proteases to sever cytoplasmic domains of SRalpha, SRbeta, TRAM, and the Sec61 complex. We characterized protein translocation intermediates that accumulate when Sec61alpha or SRbeta is inactivated by proteolysis. In the absence of a functional Sec61 complex, dissociation of SRP54 from the signal sequence is blocked. Experiments using SR proteoliposomes confirmed the assembly of a membrane-bound posttargeting intermediate. These results strongly suggest that the Sec61 complex regulates the GTP hydrolysis cycle of the SRP-SR complex at the stage of signal sequence dissociation from SRP54.