GTP hydrolysis by complexes of the signal recognition particle and the signal recognition particle receptor

Translocation of proteins across the endoplasmic reticulum membrane is a GTP-dependent process. The signal recognition particle (SRP) and the SRP receptor both contain subunits with GTP binding domains. One GTP-dependent reaction during protein translocation is the SRP receptor-mediated dissociation of SRP from the signal sequence of a nascent polypeptide. Here, we have assayed the SRP and the SRP receptor for GTP binding and hydrolysis activities. GTP hydrolysis by SRP was not detected, so the maximal GTP hydrolysis rate for SRP was estimated to be \u3c 0.002 mol GTP hydrolyzed x mol of SRP-1 x min-1. The intrinsic GTP hydrolysis activity of the SRP receptor ranged between 0.02 and 0.04 mol GTP hydrolyzed x mol of SRP receptor-1 x min-1. A 40-fold enhancement of GTP hydrolysis activity relative to that observed for the SRP receptor alone was obtained when complexes were formed between SRP and the SRP receptor. GTP hydrolysis activity was inhibited by GDP, but not by ATP. Extended incubation of the SRP or the SRP receptor with GTP resulted in substoichiometric quantities of protein-bound ribonucleotide. SRP-SRP receptor complexes engaged in GTP hydrolysis were found to contain a minimum of one bound guanine ribonucleotide per SRP-SRP receptor complex. We conclude that the GTP hydrolysis activity described here is indicative of one of the GTPase cycles that occur during protein translocation across the endoplasmic reticulum

The Tail End of Membrane Insertion

Many membrane proteins are inserted into cellular membranes via a carboxy-terminal tail-anchor segment, but the mechanism of insertion is poorly understood. In this issue of Cell, Stefanovic and Hegde (2007) report the identification and initial characterization of a soluble ATP-dependent receptor for the insertion of newly synthesized tail-anchored membrane proteins

Protein translocation across the ER requires a functional GTP binding site in the alpha subunit of the signal recognition particle receptor

The signal recognition particle (SRP)-mediated translocation of proteins across the RER is a GTP dependent process. Analysis of the primary amino acid sequence of one protein subunit of SRP (SRP54), as well as the alpha subunit of the SRP receptor (SR alpha), has indicated that these proteins contain predicted GTP binding sites. Several point mutations confined to the GTP binding consensus elements of SR alpha were constructed by site specific mutagenesis to define a role for the GTP binding site in SR alpha during protein translocation. The SR alpha mutants were analyzed using an in vitro system wherein SR alpha-deficient microsomal membranes were repopulated with SR alpha by in vitro translation of wild-type or mutant mRNA transcripts. SRP receptors containing SR alpha point mutants were analyzed for their ability to function in protein translocation and to form guanylyl-5\u27-imidodiphosphate (Gpp[NH]p) stabilized complexes with the SRP. Mutations in SR alpha produced SRP receptors that were either impaired or inactive in protein translocation. These SRP receptors were likewise unable to form Gpp(NH)p stabilized complexes with the SRP. One SR alpha point mutant, Thr 588 to Asn 588, required 50- to 100-fold higher concentrations of GTP relative to the wild-type SR alpha to function in protein translocation. This mutant has provided information on the reaction step in protein translocation that involves the GTP binding site in the alpha subunit of the SRP receptor

An evolving view of the eukaryotic oligosaccharyltransferase

Asparagine-linked glycosylation (ALG) is one of the most common protein modification reactions in eukaryotic cells, as many proteins that are translocated across or integrated into the rough endoplasmic reticulum (RER) carry N-linked oligosaccharides. Although the primary focus of this review will be the structure and function of the eukaryotic oligosaccharyltransferase (OST), key findings provided by the analysis of the archaebacterial and eubacterial OST homologues will be reviewed, particularly those that provide insight into the recognition of donor and acceptor substrates. Selection of the fully assembled donor substrate will be considered in the context of the family of human diseases known as congenital disorders of glycosylation (CDG). The yeast and vertebrate OST are surprisingly complex hetero-oligomeric proteins consisting of seven or eight subunits (Ost1p, Ost2p, Ost3p/Ost6p, Ost4p, Ost5p, Stt3p, Wbp1p, and Swp1p in yeast; ribophorin I, DAD1, N33/IAP, OST4, STT3A/STT3B, Ost48, and ribophorin II in mammals). Recent findings from several laboratories have provided overwhelming evidence that the STT3 subunit is critical for catalytic activity. Here, we will consider the evolution and assembly of the eukaryotic OST in light of recent genomic evidence concerning the subunit composition of the enzyme in diverse eukaryotes

Role of the cytoplasmic segments of Sec61alpha in the ribosome-binding and translocation-promoting activities of the Sec61 complex

The Sec61 complex performs a dual function in protein translocation across the RER, serving as both the high affinity ribosome receptor and the translocation channel. To define regions of the Sec61 complex that are involved in ribosome binding and translocation promotion, ribosome-stripped microsomes were subjected to limited digestions using proteases with different cleavage specificities. Protein immunoblot analysis using antibodies specific for the NH(2) and COOH terminus of Sec61alpha was used to map the location of proteolysis cleavage sites. We observed a striking correlation between the loss of binding activity for nontranslating ribosomes and the digestion of the COOH- terminal tail or cytoplasmic loop 8 of Sec61alpha. The proteolyzed microsomes were assayed for SRP-independent translocation activity to determine whether high affinity binding of the ribosome to the Sec61 complex is a prerequisite for nascent chain transport. Microsomes that do not bind nontranslating ribosomes at physiological ionic strength remain active in SRP-independent translocation, indicating that the ribosome binding and translocation promotion activities of the Sec61 complex do not strictly correlate. Translocation-promoting activity was most severely inhibited by cleavage of cytosolic loop 6, indicating that this segment is a critical determinant for this function of the Sec61 complex

Access of proteinase K to partially translocated nascent polypeptides in intact and detergent-solubilized membranes

We have used proteinase K as a probe to detect cytoplasmically and luminally exposed segments of nascent polypeptides undergoing transport across mammalian microsomal membranes. A series of translocation intermediates consisting of discrete-sized nascent chains was prepared by including microsomal membranes in cell-free translations of mRNAs lacking termination codons. The truncated mRNAs were derived from preprolactin and the G protein of vesicular stomatitis virus and encoded nascent chains ranging between 64 and 200 amino acid residues long. Partially translocated nascent chains of 100 amino acid residues or less were insensitive to protease digestion from the external surface of the membrane while longer nascent chains were susceptible to digestion by externally added protease. We conclude that the increased protease sensitivity of larger nascent chains is due to the exposure of a segment of the nascent polypeptide on the cytoplasmic face of the membrane. In contrast, low molecular weight nascent chains were remarkably resistant to protease digestion even after detergent solubilization of the membrane. The protease resistant behaviour of detergent solubilized nascent chains could be abolished by release of the polypeptide from the ribosome or by the addition of protein denaturants. We propose that the protease resistance of partially translocated nascent chains can be ascribed to components of the translocation apparatus that remain bound to the nascent chain after detergent solubilization of the membrane

Extreme C-terminal sites are posttranslocationally glycosylated by the STT3B isoform of the OST

Metazoan organisms assemble two isoforms of the oligosaccharyltransferase (OST) that have different catalytic subunits (STT3A or STT3B) and partially nonoverlapping roles in asparagine-linked glycosylation. The STT3A isoform of the OST is primarily responsible for co-translational glycosylation of the nascent polypeptide as it enters the lumen of the endoplasmic reticulum. The C-terminal 65-75 residues of a glycoprotein will not contact the translocation channel-associated STT3A isoform of the OST complex before chain termination. Biosynthetic pulse labeling of five human glycoproteins showed that extreme C-terminal glycosylation sites were modified by an STT3B-dependent posttranslocational mechanism. The boundary for STT3B-dependent glycosylation of C-terminal sites was determined to fall between 50 and 55 residues from the C terminus of a protein. C-terminal NXT sites were glycosylated more rapidly and efficiently than C-terminal NXS sites. Bioinformatics analysis of glycopeptide databases from metazoan organisms revealed a lower density of C-terminal acceptor sites in glycoproteins because of reduced positive selection of NXT sites and negative selection of NXS sites

ER translocation intermediates are adjacent to a nonglycosylated 34-kD integral membrane protein

We have used the homobifunctional cross-linking reagent disuccinimidyl suberate (DSS) to identify proteins that are adjacent to nascent polypeptides undergoing translocations across mammalian rough ER. Translocation intermediates were assembled by supplementing cell free translations of truncated mRNAs with the signal recognition particle (SRP) and microsomal membrane vesicles. Two prominent cross-linked products of 45 and 64 kD were detected. The 64-kD product was obtained when the cell free translation contained SRP, while formation of the 45-kD product required both SRP and translocation competent microsomal membrane vesicles. In agreement with previous investigators, we suggest that the 64-kD product arises by cross-linking of the nascent polypeptide to the 54-kD subunit of SRP. The 45-kD product resists alkaline extraction from the membrane, so we conclude that the 11-kD nascent polypeptide has been crosslinked to an integral membrane protein of approximately 34 kD (imp34). The cross-linked product does not bind to ConA Sepharose, nor is it sensitive to endoglycosidase H digestion; hence imp34 is not identical to the alpha or beta subunits of the signal sequence receptor (SSR). We propose that imp34 functions in concert with SSR to form a translocation site through which nascent polypeptides pass in traversing the membrane bilayer of the rough endoplasmic reticulum

Dual recognition of the ribosome and the signal recognition particle by the SRP receptor during protein targeting to the endoplasmic reticulum

We have analyzed the interactions between the signal recognition particle (SRP), the SRP receptor (SR), and the ribosome using GTPase assays, biosensor experiments, and ribosome binding assays. Possible mechanisms that could contribute to an enhanced affinity between the SR and the SRP-ribosome nascent chain complex to promote protein translocation under physiological ionic strength conditions have been explored. Ribosomes or 60S large ribosomal subunits activate the GTPase cycle of SRP54 and SRalpha by providing a platform for assembly of the SRP-SR complex. Biosensor experiments revealed high-affinity, saturable binding of ribosomes or large ribosomal subunits to the SR. Remarkably, the SR has a 100-fold higher affinity for the ribosome than for SRP. Proteoliposomes that contain the SR bind nontranslating ribosomes with an affinity comparable to that shown by the Sec61 complex. An NH2-terminal 319-residue segment of SRalpha is necessary and sufficient for binding of SR to the ribosome. We propose that the ribosome-SR interaction accelerates targeting of the ribosome nascent chain complex to the RER, while the SRP-SR interaction is crucial for maintaining the fidelity of the targeting reaction