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B Dobberstein

Publications and source records attributed to B Dobberstein.

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

Common principles of protein translocation across membranes.

Most major systems that transport proteins across a membrane share the following features: an amino-terminal transient signal sequence on the transported protein, a targeting system on the cis side of the membrane, a hetero-oligomeric transmembrane channel that is gated both across and within the plane of the membrane, a peripherally attached protein translocation motor that is powered by the hydrolysis of nucleoside triphosphate, and a protein folding system on the trans side of the membrane. These transport systems are divided into two families: export systems that export proteins out of the cytosol, and import systems that transport proteins into cytosol-like compartments.

Amino Acid Sequence↗
PubMed1995

A complex of the signal sequence binding protein and the SRP RNA promotes translocation of nascent proteins.

Translocation of proteins across the endoplasmic reticulum membrane is initiated by the signal recognition particle (SRP), a cytoplasmic ribonucleoprotein complex consisting of a 7S RNA and six polypeptides. To investigate the functions of the SRP components, we have tested the activities of several SRP subparticles. We show that the SRP GTPase (SRP54) alone binds a signal sequence and discriminates it from a non-signal sequence. Although SRP54 alone is unable to promote translocation, SRP54 in a complex with SRP RNA is both necessary and sufficient to promote translocation of an elongation-arrested nascent protein in a GTP-regulated manner. For co-translational translocation, additional SRP components are required. We discuss the implications of our results for the function of the Escherichia coli SRP which is homologous to the SRP54/SRP-RNA complex.

Animals↗
PubMed1995

Interference of distinct invariant chain regions with superantigen contact area and antigenic peptide binding groove of HLA-DR.

In the endoplasmic reticulum, MHC class II alpha beta dimers associate with the trimeric invariant chain (li), generating a nine-subunit (alpha beta li)3 complex. In the presence of li, the peptide binding groove is blocked, so that loading with self or antigenic peptides can only occur after proteolytic removal of li in specialized post-Golgi compartments. The class II-associated invariant chain peptide region of li (about residues 81-104) is known to mediate binding to class II molecules and blockade of the groove, but this does not exclude additional contact sites for li. Using a set of overlapping li peptides and recombinant soluble li, we demonstrate here that a large segment of li encompassing approximately residues 71 to 128 interacts with HLA-DR molecules. The N- and C-terminal regions of this li segment appear to bind outside the peptide groove to the contact area for the staphylococcal superantigen Staphylococcus aureus enterotoxin B on the alpha 1 domain. The core region of this segment (residues 95-108) prevents binding of antigenic peptides, probably by interaction with the peptide groove. Occupation of the groove with antigenic peptides abolishes binding not only of the core region, but also that of those li peptides that bind outside the groove. These findings suggest the existence of distinct conformational states of class II molecules, with li binding preferentially to one form.

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

The protein-conducting channel in the membrane of the endoplasmic reticulum is open laterally toward the lipid bilayer.

Lipids and proteins were found to contact a nascent type II membrane protein, as well as a nascent secretory protein, during their insertion into the membrane of the endoplasmic reticulum. This suggests that the protein-conducting channel is open laterally toward the lipid bilayer during an early stage of protein insertion. Contact to lipids was confined to the hydrophobic core region of the respective signal or signal anchor sequence. Thus, the nascent polypeptide is positioned in the translocation complex such that the signal or signal anchor sequence faces the lipid bilayer, whereas the hydrophilic, translocating portion is in proteinaceous environment.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1994

Formation of SRP-like particle induces a conformational change in E. coli 4.5S RNA.

E. coli P48 protein is homologous to the SRP54 component of the eukaryotic signal recognition particle. In vivo, P48 is associated with 4.5S RNA which shares a homology with eukaryotic SRP RNA. To study the interaction between P48 and 4.5S RNA in vitro, we used 4.5S RNA with fluorescein coupled to the 3'-terminal ribose. Upon binding of P48, the fluorescent 4.5S RNA shows a substantial decrease in fluorescence. Fluorescence quenching as well as anisotropy measurements reveal that the effect is not due to a direct interaction of P48 with the dye. This suggests that the binding of P48 induces a conformational change in 4.5S RNA which affects the structure at the 3' end of the RNA. From equilibrium titrations with fluorescent 4.5S RNA, a dissociation constant of 0.15 microns is obtained for the RNA.protein complex. The formation of the complex is not affected by GTP binding to or hydrolysis by P48.

Bacterial Proteins↗
PubMed1994

An alternative protein targeting pathway in Escherichia coli: studies on the role of FtsY.

In Escherichia coli, a signal recognition particle (SRP) has been identified which binds specifically to the signal sequence of presecretory proteins and which appears to be essential for efficient translocation of a subset of proteins. In this study we have investigated the function of E. coli FtsY which shares sequence similarity with the alpha-subunit of the eukaryotic SRP receptor ('docking protein') in the membrane of the endoplasmic reticulum. A strain was constructed which allows the conditional expression of FtsY. Depletion of FtsY is shown to cause the accumulation of the precursor form of beta-lactamase, OmpF and ribose binding protein in vivo, whereas the processing of various other presecretory proteins is unaffected. Furthermore, FtsY-depleted inverted cytoplasmic membrane vesicles are shown to be defective in the translocation of pre-beta-lactamase using an in vitro import assay. Subcellular localization studies revealed that FtsY is located in part at the cytoplasmic membrane with which it seems peripherally associated. These observations suggest that FtsY is the functional E. coli homolog of the mammalian SRP receptor.

Bacterial Outer Membrane Proteins↗
PubMed1994

Isolation and characterization of the intracellular MHC class II compartment.

An intracellular compartment has been isolated to which MHC class II molecules are transported on their way to the plasma membrane. They arrive with an associated invariant chain which is then proteolytically processed while MHC class II molecules acquire antigenic peptide. These loaded class II molecules then leave the compartment devoid of invariant chain and bound for the plasma membrane. This compartment represents a new stage in the endocytic/lysosomal pathway.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1994

Mammalian and Escherichia coli signal recognition particles.

Recent evidence from both biochemical and genetic studies indicates that protein targeting to the prokaryotic cytoplasmic membrane and the eukaryotic endoplasmic reticulum membrane may have more in common than previously thought. A ribonucleoprotein particle was identified in Escherichia coli that consists of at least one protein (P48 or Ffh) and one RNA molecule (4.5S RNA), both of which exhibit strong sequence similarity with constituents of the mammalian signal recognition particle (SRP). Like the mammalian SRP, the E. coli SRP binds specifically to the signal sequence of presecretory proteins. Depletion of either P48 or 4.5S RNA affects translation and results in the accumulation of precursors of several secreted proteins. This review discusses the recent studies and speculates on the position of the SRP in the complex network of protein interactions involved in translation and membrane targeting in E. coli.

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

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↗
PubMed1993

Assembly of the 68- and 72-kD proteins of signal recognition particle with 7S RNA.

Signal recognition particle (SRP), the cytoplasmic ribonucleoprotein particle that mediates the targeting of proteins to the ER, consists of a 7S RNA and six different proteins. The 68- (SRP68) and 72- (SRP72) kD proteins of SRP are bound to the 7S RNA of SRP as a heterodimeric complex (SRP68/72). Here we describe the primary structure of SRP72 and the assembly of SRP68, SRP72 and 7S RNA into a ribonucleoprotein particle. The amino acid sequence deduced from the cDNA of SRP72 reveals a basic protein of 671 amino acids which shares no sequence similarity with any protein in the sequence data libraries. Assembly of SRP72 into a ribonucleoprotein particle required the presence of 7S RNA and SRP68. In contrast, SRP68 alone specifically bound to 7S RNA. SRP68 contacts the 7S RNA via its NH2-terminal half while COOH-terminal portions of SRP68 and SRP72 are in contact with each other in SRP. SRP68 thus serves as a link between 7S RNA and SRP72. As a large NH2-terminal domain of SRP72 is exposed on SRP it may be a site of contact to other molecules involved in the SRP cycle between the ribosome and the ER membrane.

Amino Acid Sequence↗
PubMed1993

The MHC class II-associated invariant chain contains two endosomal targeting signals within its cytoplasmic tail.

The oligomeric complex formed by major histocompatibility complex (MHC) class II alpha and beta chains and invariant chain (Ii) assembles in the endoplasmic reticulum and is then transported via the Golgi complex to compartments of the endocytic pathway. When Ii alone is expressed in CV1 cells it is sorted to endosomes. The Ii cytoplasmic tail has been found to be essential for targeting to these compartments. In order to characterize further the signals responsible for endosomal targeting, we have deleted various segments of the cytoplasmic tail. The Ii mutants were transiently expressed and the cellular location of the proteins was analyzed biochemically and morphologically. The cytoplasmic tail of Ii was found to contain two endosomal targeting sequences within its cytoplasmic tail; one targeting sequence was present within amino acid residues 12-29 and deletion of this segment revealed the presence of a second endosomal targeting sequence, located within the first 11 amino acid residues. The presence of a leucine-isoleucine pair at positions 7 and 8 within this sequence was found to be essential for endosomal targeting. In addition, the presence of this L-I motif lead to accumulation of Ii molecules in large endosomal vacuoles containing lysosomal marker proteins. Both wild type Ii and Ii mutant molecules containing only one endosomal targeting sequence were rapidly internalized from the plasma membrane. When the Ii cytoplasmic tail was fused to the membrane-spanning region of neuraminidase, a resident plasma membrane protein, the resulting chimera (INA) was found in endocytic compartments containing lysosomal marker proteins. Thus the cytoplasmic tail of Ii is sufficient for targeting to the endocytic/lysosomal pathway.

Amino Acid Sequence↗
PubMed1992

Signal-sequence recognition by an Escherichia coli ribonucleoprotein complex.

Hydrophobic signal-sequences direct the transfer of secretory proteins across the inner membrane of prokaryotes and the endoplasmic reticulum membranes of eukaryotes. In mammalian cells, signal-sequences are recognized by the 54K protein (M(r) 54,000) of the signal recognition particle (SRP) which is believed to hold the nascent chain in a translocation-competent conformation until it contacts the endoplasmic reticulum membrane. The SRP consists of a 7S RNA and six different polypeptides. The 7S RNA and the 54K signal-sequence-binding protein (SRP54) of mammalian SRP exhibit strong sequence similarity to the 4.5S RNA and P48 protein (Ffh) of Escherichia coli which form a ribonucleoprotein particle. Depletion of 4.5S RNA or overproduction of P48 causes the accumulation of the beta-lactamase precursor, although not of other secretory proteins. Whether 4.5S RNA and P48 are part of an SRP-like complex with a role in protein export is controversial. Here we show that the P48/4.5S RNA ribonucleoprotein complex interacts specifically with the signal sequence of a nascent secretory protein and therefore is a signal recognition particle.

Bacterial Proteins↗