Biomedical subjects

T A Rapoport

Publications and source records attributed to T A Rapoport.

At least 19 recordsLinked to original sources

PubMed1996

Regulation by the ribosome of the GTPase of the signal-recognition particle during protein targeting.

The signal-recognition particle (SRP) is important for the targeting of many secretory and membrane proteins to the endoplasmic reticulum (ER). Targeting is regulated by three GTPases, the 54K subunit of SRP (SRP54), and the alpha- and beta-subunits of the SRP receptor. When a signal sequence emerges from the ribosome, SRP interacts with it and targets the resulting complex to the ER membrane by binding to the SRP receptor. Subsequently, SRP releases the signal sequence into the translocation channel. Here we use a complex of a ribosome with a nascent peptide chain, the SRP and its receptor, to investigate GTP binding to SRP54, and GTP hydrolysis. Our findings indicate that a ribosomal component promotes GTP binding to the SRP54 subunit of SRP. GTP-bound SRP54 is essential for high-affinity interaction between SRP and its receptor in the ER membrane. This interaction induces the release of the signal sequence from SRP, the insertion of the nascent polypeptide chain into the translocation channel, and GTP hydrolysis. The contribution of the ribosome had previously escaped detection because only synthetic signal peptides were used in the analysis.

Base Sequence↗

PubMed1996

A second trimeric complex containing homologs of the Sec61p complex functions in protein transport across the ER membrane of S. cerevisiae.

Yeast microsomes contain a heptameric Sec complex involved in post-translational protein transport that is composed of a heterotrimeric Sec61p complex and a tetrameric Sec62-Sec63 complex. The trimeric Sec61p complex also exists as a separate entity that probably functions in co-translational protein transport, like its homolog in mammals. We have now discovered in the yeast endoplasmic reticulum membrane a second, structurally related trimeric complex, named Ssh1p complex. It consists of Ssh1p1 (Sec sixty-one homolog 1), a rather distant relative of Sec61p, of Sbh2p, a homolog of the Sbh1p subunit of the Sec61p complex, and of Sss1p, a component common to both trimeric complexes. In contrast to Sec61p, Ssh1p is not essential for cell viability but it is required for normal growth rates. Sbh1p and Sbh2p individually are also not essential, but cells lacking both proteins are impaired in their growth at elevated temperatures and accumulate precursors of secretory proteins; microsomes isolated from these cells also exhibit a reduced rate of post-translational protein transport. Like the Sec61p complex, the Ssh1p complex interacts with membrane-bound ribosomes, but it does not associate with the Sec62-Sec63p complex to form a heptameric Sec complex. We therefore propose that it functions exclusively in the co-translational pathway of protein transport.

Amino Acid Sequence↗

PubMed1996

A 12-residue-long polyleucine tail is sufficient to anchor synaptobrevin to the endoplasmic reticulum membrane.

Synaptobrevin is a tail-anchored protein with a hydrophobic C-terminal transmembrane segment that inserts into the endoplasmic reticulum membrane independently of the SRP/Sec61p pathway. Here, we show that idealized hydrophobic segments composed of 11-17 leucines and 1 valine function as insertion signals in vitro, whereas shorter segments do not. These results suggest that there are no specific requirements beyond overall hydrophobicity for C-terminal endoplasmic reticulum insertion signals.

Amino Acid Sequence↗

PubMed1995

Signal sequence processing in rough microsomes.

Secretory proteins are synthesized with a signal sequence that is usually cleaved from the nascent protein during the translocation of the polypeptide chain into the lumen of the endoplasmic reticulum. To determine the fate of a cleaved signal sequence, we used a synchronized in vitro translocation system. We found that the cleaved signal peptide of preprolactin is further processed close to its COOH terminus. The resulting fragment accumulated in the microsomal fraction and with time was released into the cytosol. Signal sequence cleavage and processing could be reproduced with reconstituted vesicles containing Sec61, signal recognition particle receptor, and signal peptidase complex.

Amino Acid Sequence↗

PubMed1995

A posttargeting signal sequence recognition event in the endoplasmic reticulum membrane.

We have analyzed early phases of the cotranslational transport of the secretory protein preprolactin through the mammalian endoplasmic reticulum (ER) membrane. Following recognition of the signal sequence of the nascent polypeptide chain in the cytosol by the SRP, the chain is transferred into the membrane, where a second signal sequence recognition step takes place for which the presence in the lipid bilayer of the Sec61p complex is essential and sufficient. This step leads to a tight junction between the ribosomenascent chain complex and the Sec61p complex, and to the productive insertion of the nascent chain into the translocation site. These results show that a translocation substrate is subjected to two recognition events before being allowed to cross the ER membrane.

Amino Acid Sequence↗

PubMed1995

Posttranslational protein transport in yeast reconstituted with a purified complex of Sec proteins and Kar2p.

We have reproduced the posttranslational mode of protein translocation across the endoplasmic reticulum membrane with reconstituted proteoliposomes containing a purified complex of seven yeast proteins. This Sec complex includes a heterotrimeric Sec61p complex, homologous to that in mammals, as well as all other membrane proteins found in genetic screens for translocation components. Efficient posttranslational translocation also requires the addition of lumenal Kar2p (BiP) and ATP. The trimeric Sec61p complex also exists as a separate entity that, in contrast with the large Sec complex, is associated with membrane-bound ribosomes. We therefore hypothesize that distinct membrane protein complexes function in co- and posttranslational translocation pathways.

Adenosine Triphosphate↗

PubMed1995

Transport route for synaptobrevin via a novel pathway of insertion into the endoplasmic reticulum membrane.

Synaptobrevin/vesicle-associated membrane protein is one of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. It is proposed to provide specificity for the targeting and fusion of vesicles with the plasma membrane. It belongs to a class of membrane proteins which lack a signal sequence and contain a single hydrophobic segment close to their C-terminus, leaving most of the polypeptide chain in the cytoplasm (tail-anchored). We show that in neuroendocrine PC12 cells, synaptobrevin is not directly incorporated into the target organelle, synaptic-like vesicles. Rather, it is first inserted into the endoplasmic reticulum (ER) membrane and is then transported via the Golgi apparatus. Its insertion into the ER membrane in vitro occurs post-translationally, is dependent on ATP and results in a trans-membrane orientation of the hydrophobic tail. Membrane integration requires ER protein(s) different from the translocation components needed for proteins with signal sequences, thus suggesting a novel mechanism of insertion.

Adenosine Triphosphate↗

PubMed1994

Systematic probing of the environment of a translocating secretory protein during translocation through the ER membrane.

We have extended a previously developed photo-crosslinking approach to systematically probe the protein environment of the secretory protein preprolactin, trapped during its transfer through the endoplasmic reticulum membrane. Single photoreactive groups were placed at various positions of nascent polypeptide chains of various length, corresponding to different stages of the transport process, and photo-crosslinks to membrane proteins were analyzed. In all cases, the polypeptide segment extending from the ribosome was found to be located in a membrane environment that is formed almost exclusively from Sec61 alpha, the multi-spanning subunit of the Sec61p complex that is essential for translocation. At early stages of the translocation process, before cleavage of the signal sequence, almost the entire nascent chain emerged from the ribosome contacts Sec61 alpha. The 'translocating chain-associating membrane' protein interacts mainly with the region of the signal sequence preceding its hydrophobic core. Our results suggest that the nascent chain is transferred directly from the ribosome into a protein-conducting channel, the major constituent of which is Sec61 alpha.

Affinity Labels↗

PubMed1994

Protein translocation: common themes from bacteria to man.

Protein transport across the endoplasmic reticulum membrane in eukaryotes and across the cytoplasmic membrane in bacteria have turned out to be highly related. The core component of the translocation apparatus is the Sec61/SecYp complex; at least two of its subunits are conserved in evolution. The Sec61/SecYp complex is involved in both co- and post-translational transport pathways. The two modes require probably distinct additional components.

Bacteria↗

PubMed1994

Evolutionary conservation of components of the protein translocation complex.

Protein translocation into the mammalian endoplasmic reticulum requires the Sec61p complex, which consists of three membrane proteins. The alpha-subunit, the homologue of Sec61p of yeast, shows some similarity to SecYp, a key component of the protein export apparatus of bacteria. In Escherichia coli, SecYp is also associated with two other proteins (SecEp and band-1 protein). We have now determined the sequences of the beta- and gamma-subunits of the mammalian Sec61p complex. Sec61-gamma is homologous to SSS1p, a suppressor of sec61 mutants in Saccharomyces cerevisiae, and can functionally replace it in yeast cells. Moreover, Sec61-gamma and SSS1p are structurally related to SecEp of E. coli and to putative homologues in various other bacteria. At least two subunits of the Sec61/SecYp complex therefore seem to be key components of the protein translocation apparatus in all classes of organisms.

Amino Acid Sequence↗

PubMed1994

Binding of ribosomes to the rough endoplasmic reticulum mediated by the Sec61p-complex.

The cotranslational translocation of proteins across the ER membrane involves the tight binding of translating ribosomes to the membrane, presumably to ribosome receptors. The identity of the latter has been controversial. One putative receptor candidate is Sec61 alpha, a multi-spanning membrane protein that is associated with two additional membrane proteins (Sec61 beta and gamma) to form the Sec61p-complex. Other receptors of 34 and 180 kD have also been proposed on the basis of their ability to bind at low salt concentration ribosomes lacking nascent chains. We now show that the Sec61p-complex has also binding activity but that, at low salt conditions, it accounts for only one third of the total binding sites in proteoliposomes reconstituted from a detergent extract of ER membranes. Under these conditions, the assay has also limited specificity with respect to ribosomes. However, if the ribosome-binding assay is performed at physiological salt concentration, most of the unspecific binding is lost; the Sec61p-complex then accounts for the majority of specific ribosome-binding sites in reconstituted ER membranes. To study the membrane interaction of ribosomes participating in protein translocation, native rough microsomes were treated with proteases. The amount of membrane-bound ribosomes is only slightly reduced by protease treatment, consistent with the protease-resistance of Sec61 alpha which is shielded by these ribosomes. In contrast, p34 and p180 can be readily degraded, indicating that they are not essential for the membrane anchoring of ribosomes in protease-treated microsomes. These data provide further evidence that the Sec61p-complex is responsible for the membrane-anchoring of ribosomes during translocation and make it unlikely that p34 or p180 are essential for this process.

Animals↗

PubMed1993

Site-specific photocross-linking reveals that Sec61p and TRAM contact different regions of a membrane-inserted signal sequence.

A chemically charged amber suppressor tRNA was used to introduce the photoactivatable amino acid (Tmd)Phe at a selected position within the signal sequence of the secretory protein preprolactin. This allowed the interactions of the NH2-terminal, the central, and the COOH-terminal regions of the signal sequence to be investigated during insertion into the membrane of the endoplasmic reticulum (ER). We found that different regions of the nascent chains were photocross-linked to different ER proteins. The TRAM protein (translocating chain-associating membrane protein) contacts the NH2-terminal region of the signal sequence while the mammalian Sec61p contacts the hydrophobic core of the signal sequence and regions COOH-terminal of this. These results suggest that the ER translocation complex is composed of heterologous protein subunits which contact distinct regions of nascent polypeptides during their membrane insertion.

Acylation↗

PubMed1993

Protein translocation into proteoliposomes reconstituted from purified components of the endoplasmic reticulum membrane.

We have reproduced the process of protein transport across and of protein integration into the mammalian endoplasmic reticulum membrane by the use of proteoliposomes reconstituted from pure phospholipids and purified membrane proteins. The transport of some proteins requires only two membrane protein complexes: the signal recognition particle receptor, needed for targeting of a nascent chain to the membrane, and a novel complex, the Sec61p complex, that consists of Sec61p and two smaller polypeptides. The translocation of other proteins also needs the presence of the translocating chain-association membrane (TRAM) protein. The integration of two membrane proteins of different topologies into the membrane does not require additional components. These results indicate a surprising simplicity of the basic translocation machinery. They suggest that the Sec61p complex binds the ribosome during translocation and forms the postulated protein-conducting channel.

Animals↗

PubMed1993

DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) inhibits an early step of protein translocation across the mammalian ER membrane.

Protein translocation across the endoplasmic reticulum (ER) membrane of yeast can be inhibited by agents believed to specifically affect the transport of ATP through the membrane (Mayinger, P. and Meyer, D.I. (1993) EMBO J. 12, 659-666), suggesting the involvement of a translocation component in the lumen of the ER that binds ATP. We demonstrate that one of the inhibitors, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), also affects the translocation of proteins into mammalian microsomes. Translocation is blocked at the point of transfer of the nascent chain from the signal recognition particle (SRP) into the ER-membrane. We also confirm that photoaffinity-labelling of microsomes with 8-azido-ATP inhibits the same early step of protein translocation. Since this step is reported to not require ATP, these results raise the possibility that, in both cases, factor(s) other than ATP-binding components of the translocation machinery are perturbed.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1993

A tetrameric complex of membrane proteins in the endoplasmic reticulum.

The translocation site (translocon), at which nascent polypeptides pass through the endoplasmic reticulum membrane, contains a component previously called 'signal sequence receptor' that is now renamed as 'translocon-associated protein' (TRAP). Two glycosylated subunits of the TRAP complex have been identified before (alpha and beta subunits). We now show that TRAP complex is actually comprised of four membrane proteins (alpha, beta, gamma, delta), present in a stoichiometric relation, which are genuine neighbours in intact microsomes. The amino acid sequences of the additional, non-glycosylated subunits were deduced from cloning of the corresponding cDNAs. The delta subunit spans the membrane only once and has its major portion, containing a disulfide bridge, at the lumenal side. The gamma subunit is predicted to span the membrane four times.

Amino Acid Sequence↗

PubMed1993

Sec61p is adjacent to nascent type I and type II signal-anchor proteins during their membrane insertion.

We have identified membrane components which are adjacent to type I and type II signal-anchor proteins during their insertion into the membrane of the ER. Using two different cross-linking approaches a 37-38-kD nonglycosylated protein, previously identified as P37 (High, S., D. Görlich, M. Wiedmann, T. A. Rapoport, and B. Dobberstein. 1991. J. Cell Biol. 113:35-44), was found adjacent to all the membrane inserted nascent chains used in this study. On the basis of immunoprecipitation, this ER protein was shown to be identical to the recently identified mammalian Sec61 protein. Thus, Sec61p is the principal cross-linking partner of both type I and type II signal-anchor proteins during their membrane insertion (this work), and of secretory proteins during their translocation (Görlich, D., S. Prehn, E. Hartmann, K.-U. Kalies, and T. A. Rapoport. 1992. Cell. 71:489-503). We propose that membrane proteins of both orientations, and secretory proteins employ the same ER translocation sites, and that Sec61p is a core component of these sites.

Base Sequence↗

PubMed1992

Transport of proteins across the endoplasmic reticulum membrane.

The biosynthesis of many eukaryotic proteins requires their transport across the endoplasmic reticulum (ER) membrane. The process can be divided into two phases: (i) a targeting cycle, during which, by virtue of their signal sequences, nascent polypeptides are directed to translocation sites in the ER and (ii) the actual transfer of proteins across the membrane. The first phase has been well characterized, whereas the latter until recently was completely unresolved. Key components of the translocation apparatus have now been identified and it seems likely that they form a protein-conducting channel in the ER membrane. The transport process is similar to the process of protein export in bacteria.

Biological Transport↗

PubMed1992

A mammalian homolog of SEC61p and SECYp is associated with ribosomes and nascent polypeptides during translocation.

SEC61p is essential for protein translocation across the endoplasmic reticulum membrane of S. cerevisiae. We have found a mammalian homolog that shows more than 50% sequence identity with the yeast protein. Moreover, several regions of SEC61p have significant similarities with corresponding ones of SecYp of bacteria, indicating a strong evolutionary conservation of the mechanism of protein translocation. Mammalian Sec61p, like the yeast protein, is located in the immediate vicinity of nascent polypeptides during their membrane passage. It is tightly associated with membrane-bound ribosomes, suggesting that the nascent chain passes directly from the ribosome into a protein-conducting channel. These results define Sec61p as a ubiquitous key component of the protein translocation apparatus.

Amino Acid Sequence↗