Biomedical subjects

B Jungnickel

Publications and source records attributed to B Jungnickel.

13 recordsLinked to original sources

PubMed1998

Signal sequence recognition in cotranslational translocation by protein components of the endoplasmic reticulum membrane.

We have investigated the role of membrane proteins and lipids during early phases of the cotranslational insertion of secretory proteins into the translocation channel of the endoplasmic reticulum (ER) membrane. We demonstrate that all steps, including the one during which signal sequence recognition occurs, can be reproduced with purified translocation components in detergent solution, in the absence of bulk lipids or a bilayer. Photocross-linking experiments with native membranes show that upon complete insertion into the channel signal sequences are both precisely positioned with respect to the protein components of the channel and contact lipids. Together, these results indicate that signal sequences are bound to a specific binding site at the interface between the channel and the surrounding lipids, and are recognized ultimately by protein-protein interactions. Our data also suggest that at least some signal sequences reach the binding site by transfer through the interior of the channel.

Animals↗

PubMed1998

Binding of signal recognition particle gives ribosome/nascent chain complexes a competitive advantage in endoplasmic reticulum membrane interaction.

Most secretory and membrane proteins are sorted by signal sequences to the endoplasmic reticulum (ER) membrane early during their synthesis. Targeting of the ribosome-nascent chain complex (RNC) involves the binding of the signal sequence to the signal recognition particle (SRP), followed by an interaction of ribosome-bound SRP with the SRP receptor. However, ribosomes can also independently bind to the ER translocation channel formed by the Sec61p complex. To explain the specificity of membrane targeting, it has therefore been proposed that nascent polypeptide-associated complex functions as a cytosolic inhibitor of signal sequence- and SRP-independent ribosome binding to the ER membrane. We report here that SRP-independent binding of RNCs to the ER membrane can occur in the presence of all cytosolic factors, including nascent polypeptide-associated complex. Nontranslating ribosomes competitively inhibit SRP-independent membrane binding of RNCs but have no effect when SRP is bound to the RNCs. The protective effect of SRP against ribosome competition depends on a functional signal sequence in the nascent chain and is also observed with reconstituted proteoliposomes containing only the Sec61p complex and the SRP receptor. We conclude that cytosolic factors do not prevent the membrane binding of ribosomes. Instead, specific ribosome targeting to the Sec61p complex is provided by the binding of SRP to RNCs, followed by an interaction with the SRP receptor, which gives RNC-SRP complexes a selective advantage in membrane targeting over nontranslating ribosomes.

Animals↗

PubMed1996

Oligomeric rings of the Sec61p complex induced by ligands required for protein translocation.

The heterotrimeric Sec61p complex is a major component of the protein-conducting channel of the endoplasmic reticulum (ER) membrane, associating with either ribosomes or the Sec62/63 complex to perform co- and posttranslational transport, respectively. We show by electron microscopy that purified mammalian and yeast Sec61p complexes in detergent form cylindrical oligomers with a diameter of approximately 85 A and a central pore of approximately 20 A. Each oligomer contains 3-4 heterotrimers. Similar ring structures are seen in reconstituted proteoliposomes and native membranes. Oligomer formation by the reconstituted Sec61p complex is stimulated by its association with ribosomes or the Sec62/63p complex. We propose that these cylindrical oligomers represent protein-conducting channels of the ER, formed by ligands specific for co- and posttranslational transport.

Animals↗

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

Approaching the mechanism of protein transport across the ER membrane.

Since the identification of essential protein-translocation components in the endoplasmic reticulum membrane, research efforts have concentrated on the elucidation of the molecular mechanism of protein transport across this membrane. Recent results have provided new information as to how proteins are targeted to, and inserted into, the translocation site during translation. Post-translational translocation has also been examined and is distinct from cotranslational translocation with respect to the mechanism and membrane protein components involved.

Biological Transport↗

PubMed1996

Signal sequence-dependent function of the TRAM protein during early phases of protein transport across the endoplasmic reticulum membrane.

Cotranslational translocation of proteins across the mammalian ER membrane involves, in addition to the signal recognition particle receptor and the Sec61p complex, the translocating chain-associating membrane (TRAM) protein, the function of which is still poorly understood. Using reconstituted proteoliposomes, we show here that the translocation of most, but not all, secretory proteins requires the function of TRAM. Experiments with hybrid proteins demonstrate that the structure of the signal sequence determines whether or not TRAM is needed. Features that distinguish TRAM-dependent and -independent signal sequences include the length of their charged, NH2-terminal region and the structure of their hydrophobic core. In cases where TRAM is required for translocation, it is not needed for the initial interaction of the ribosome/nascent chain complex with the ER membrane but for a subsequent step inside the membrane in which the nascent chain is inserted into the translocation site in a protease-resistant manner. Thus, TRAM functions in a signal sequence-dependent manner at a critical, early phase of the translocation process.

Amino Acid Sequence↗

PubMed1996

Protein transport across the eukaryotic endoplasmic reticulum and bacterial inner membranes.

Protein transport across the endoplasmic reticulum membrane can occur by two pathways, a co- and a post-translational one. In both cases, polypeptides are first targeted to translocation sites in the membrane by virtue of their signal sequences and then transported across or inserted into the phospholipid bilayer, most likely through a protein-conducting channel. Key components of the translocation apparatus have now been identified and the translocation pathways seem likely to be related to each other but mechanistically distinct. Protein transport across the bacterial inner membrane is both similar to and different from the process in eukaryotes. Other pathways of protein translocation exist that bypass the ones involving classical signal sequences.

Bacteria↗

PubMed1995

A hydrophobic region of ricin A chain which may have a role in membrane translocation can function as an efficient noncleaved signal peptide.

Ricin A chain is a polypeptide of 267 amino acids containing a hydrophobic region near its carboxyl-terminus (residues 245-256) which has been implicated in the membrane translocation step necessary for this catalytically active toxin to reach its intracellular substrate. DNA fusions were constructed that encoded hybrid proteins consisting of carboxyl-terminal residues 233-267 or residues 238-267 of ricin A chain preceding mouse dihydrofolate reductase. When in vitro transcripts prepared from these constructs were translated in cell-free systems, the ricin A chain-derived sequences functioned as efficient signal peptides which directed dihydrofolate reductase into microsomes or into proteoliposomes containing microsomal membrane components.

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

The Sec61 complex is essential for the insertion of proteins into the membrane of the endoplasmic reticulum.

Cross-linking studies have implicated Sec61 alpha as the principal component adjacent to newly synthesised membrane proteins during insertion into the endoplasmic reticulum. Using proteoliposomes which have been reconstituted from purified components of the endoplasmic reticulum [Görlich, D and Rapoport, T.A., Cell 75 (1993) 615-630] we have found that the Sec61 complex, consisting of three subunits, is essential for the insertion of single-spanning membrane proteins. This is true for signal-anchor proteins of both orientations, and for proteins with a cleavable signal sequence. These results support the view that Sec61 alpha is a major component of the ER translocation site and promotes both the insertion of membrane proteins and the translocation of secretory proteins.

Endoplasmic Reticulum↗

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↗

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↗