scholarly journals An ATP-binding membrane protein is required for protein translocation across the endoplasmic reticulum membrane.

1991 ◽  
Vol 2 (10) ◽  
pp. 851-859 ◽  
Author(s):  
D L Zimmerman ◽  
P Walter
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Microsomal Membrane ◽  
Atp Binding ◽  
Endoplasmic Reticulum Membrane ◽  
Receptor Complex

The role of nucleotides in providing energy for polypeptide transfer across the endoplasmic reticulum (ER) membrane is still unknown. To address this question, we treated ER-derived mammalian microsomal vesicles with a photoactivatable analogue of ATP, 8-N3ATP. This treatment resulted in a progressive inhibition of translocation activity. Approximately 20 microsomal membrane proteins were labeled by [alpha 32P]8-N3ATP. Two of these were identified as proteins with putative roles in translocation, alpha signal sequence receptor (SSR), the 35-kDa subunit of the signal sequence receptor complex, and ER-p180, a putative ribosome receptor. We found that there was a positive correlation between inactivation of translocation activity and photolabeling of alpha SSR. In contrast, our data demonstrate that the ATP-binding domain of ER-p180 is dispensable for translocation activity and does not contribute to the observed 8-N3ATP sensitivity of the microsomal vesicles.

scholarly journals Targeting of the hepatitis B virus precore protein to the endoplasmic reticulum membrane: after signal peptide cleavage translocation can be aborted and the product released into the cytoplasm.

1988 ◽  
Vol 106 (4) ◽  
pp. 1093-1104 ◽  
Author(s):  
P D Garcia ◽  
J H Ou ◽  
W J Rutter ◽  
P Walter
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Core Protein ◽  
Signal Sequence ◽  
Signal Peptidase ◽  
Amino Terminus ◽  
Endoplasmic Reticulum Membrane ◽  
Nucleic Acid Binding ◽  
Precore Region ◽  
B Virus

The major hepatitis B virus (HBV) core protein is a viral structural protein involved in nucleic acid binding. Its coding sequence contains an extension of 29 codons (the "precore" region) at the amino terminus of the protein which is present in a fraction of the viral transcripts. This region is evolutionarily conserved among mammalian and avian HBVs, suggesting it has functional importance, although at least for duck HBV it has been shown to be nonessential for replication of infectious virions. Using in vitro assays for protein translocation across the endoplasmic reticulum membrane, we found that the precore region of the HBV genome encodes a signal sequence. This signal sequence was recognized by signal recognition particle, which targeted the nascent precore protein to the endoplasmic reticulum membrane with efficiencies comparable to those of other mammalian secretory proteins. A 19-amino acid signal peptide was removed by signal peptidase on the lumenal side of the microsomal membrane, generating a protein similar to the HBV major core protein, but containing 10 additional amino acids from the precore region at its amino terminus. Surprisingly, we found that 70-80% of this signal peptidase-cleaved product was localized on the cytoplasmic side of the microsomal vesicles and was not associated with the membranes. We conclude that translocation was aborted by an unknown mechanism, then the protein disengaged from the translocation machinery and was released back into the cytoplasm. Thus, a cytoplasmically disposed protein was created whose amino terminus resulted from signal peptidase cleavage. The remaining 20-30% appeared to be completely translocated into the lumen of the microsomes. A deletion mutant lacking the carboxy-terminal nucleic acid binding domain of the precore protein was similarly partitioned between the lumen of the microsomes and the cytoplasmic compartment, indicating that this highly charged domain is not responsible for the aborted translocation. We discuss the implications of our findings for the protein translocation process and suggest a possible role in the virus life cycle.

scholarly journals Structural and functional characterization of Sec66p, a new subunit of the polypeptide translocation apparatus in the yeast endoplasmic reticulum.

1993 ◽  
Vol 4 (9) ◽  
pp. 931-939 ◽  
Author(s):  
D Feldheim ◽  
K Yoshimura ◽  
A Admon ◽  
R Schekman
Keyword(s):  
Endoplasmic Reticulum ◽  
Signal Sequence ◽  
Functional Characterization ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Endoplasmic Reticulum Membrane ◽  
Restrictive Temperature ◽  
Permissive Temperature

SEC66 encodes the 31.5-kDa glycoprotein of the Sec63p complex, an integral endoplasmic reticulum membrane protein complex required for translocation of presecretory proteins in Saccharomyces cerevisiae. DNA sequence analysis of SEC66 predicts a 23-kDa protein with no obvious NH2-terminal signal sequence but with one domain of sufficient length and hydrophobicity to span a lipid bilayer. Antibodies directed against a recombinant form of Sec66p were used to confirm the membrane location of Sec66p and that Sec66p is a glycoprotein of 31.5 kDa. A null mutation in SEC66 renders yeast cells temperature sensitive for growth. sec66 cells accumulate some secretory precursors at a permissive temperature and a variety of precursors at the restrictive temperature. sec66 cells show defects in Sec63p complex formation. Because sec66 cells affect the translocation of some, but not all secretory precursor polypeptides, the role of Sec66p may be to interact with the signal peptide of presecretory proteins.

Sec62p, A Component of the Endoplasmic Reticulum Protein Translocation Machinery, Contains Multiple Binding Sites for the Sec-Complex

2000 ◽  
Vol 11 (11) ◽  
pp. 3859-3871 ◽  
Author(s):  
Sandra Wittke ◽  
Martin Dünnwald ◽  
Nils Johnsson
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Endoplasmic Reticulum Membrane ◽  
Sequence Recognition ◽  
Multiple Binding Sites ◽  
Oligomeric Protein ◽  
Multiple Binding ◽  
Endoplasmic Reticulum Protein

SEC62 encodes an essential component of the Sec-complex that is responsible for posttranslational protein translocation across the membrane of the endoplasmic reticulum in Saccharomyces cerevisiae. The specific role of Sec62p in translocation was not known and difficult to identify because it is part of an oligomeric protein complex in the endoplasmic reticulum membrane. An in vivo competition assay allowed us to characterize and dissect physical and functional interactions between Sec62p and components of the Sec-complex. We could show that Sec62p binds via its cytosolic N- and C-terminal domains to the Sec-complex. The N-terminal domain, which harbors the major interaction site, binds directly to the last 14 residues of Sec63p. The C-terminal binding site of Sec62p is less important for complex stability, but adjoins the region in Sec62p that might be involved in signal sequence recognition.

scholarly journals Protein translocation mutants defective in the insertion of integral membrane proteins into the endoplasmic reticulum.

1992 ◽  
Vol 3 (2) ◽  
pp. 129-142 ◽  
Author(s):  
C J Stirling ◽  
J Rothblatt ◽  
M Hosobuchi ◽  
R Deshaies ◽  
R Schekman
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Protein Translocation ◽  
Secretory Protein ◽  
Polyclonal Antiserum ◽  
Secretory Proteins ◽  
Temperature Sensitive ◽  
Endoplasmic Reticulum Membrane ◽  
Membrane Spanning ◽  
Coa Reductase

Yeast mutants defective in the translocation of soluble secretory proteins into the lumen of the endoplasmic reticulum (sec61, sec62, sec63) are not impaired in the assembly and glycosylation of the type II membrane protein dipeptidylaminopeptidase B (DPAPB) or of a chimeric membrane protein consisting of the multiple membrane-spanning domain of yeast hydroxymethylglutaryl CoA reductase (HMG1) fused to yeast histidinol dehydrogenase (HIS4C). This chimera is assembled in wild-type or mutant cells such that the His4c protein is oriented to the ER lumen and thus is not available for conversion of cytosolic histidinol to histidine. Cells harboring the chimera have been used to select new translocation defective sec mutants. Temperature-sensitive lethal mutations defining two complementation groups have been isolated: a new allele of sec61 and a single isolate of a new gene sec65. The new isolates are defective in the assembly of DPAPB, as well as the secretory protein alpha-factor precursor. Thus, the chimeric membrane protein allows the selection of more restrictive sec mutations rather than defining genes that are required only for membrane protein assembly. The SEC61 gene was cloned, sequenced, and used to raise polyclonal antiserum that detected the Sec61 protein. The gene encodes a 53-kDa protein with five to eight potential membrane-spanning domains, and Sec61p antiserum detects an integral protein localized to the endoplasmic reticulum membrane. Sec61p appears to play a crucial role in the insertion of secretory and membrane polypeptides into the endoplasmic reticulum.

The protein translocation machinery of the endoplasmic reticulum

1982 ◽  
Vol 300 (1099) ◽  
pp. 225-228 ◽  
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Rough Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Integral Membrane Proteins ◽  
Multiple Copies ◽  
And Function ◽  
Lysosomal Proteins

The rough endoplasmic reticulum (r.e.r.) has been postulated to possess a single translation-coupled translocation system (in multiple copies) that effects signal sequence-mediated translocation of all secretory and lysosomal proteins and integration of all integral membrane proteins whose port of entry is the rough endoplasmic reticulum (G. Blobel 1980 Proc. natn. Acad. Sci. U.S.A. 77, 1496—1500). Two proteins have been isolated that are components of the r.e.r. translocation system. Their properties and function in protein translocation across and integration into membranes are discussed.

scholarly journals Sls1p Stimulates Sec63p-Mediated Activation of Kar2p in a Conformation-Dependent Manner in the Yeast Endoplasmic Reticulum

2000 ◽  
Vol 20 (18) ◽  
pp. 6923-6934 ◽  
Author(s):  
Mehdi Kabani ◽  
Jean-Marie Beckerich ◽  
Claude Gaillardin
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Synthetic Lethality ◽  
In Vitro Assays ◽  
Endoplasmic Reticulum Membrane ◽  
Dependent Manner ◽  
Dose Dependent

ABSTRACT We previously characterized the SLS1 gene in the yeastYarrowia lipolytica and showed that it interacts physically with YlKar2p to promote translocation across the endoplasmic-reticulum membrane (A. Boisramé, M. Kabani, J. M. Beckerich, E. Hartmann, and C. Gaillardin, J. Biol. Chem. 273:30903–30908, 1998). A Y. lipolytica Kar2p mutant was isolated that restored interaction with an Sls1p mutant, suggesting that the interaction with Sls1p could be nucleotide and/or conformation dependent. This result was used as a working hypothesis for more accurate investigations in Saccharomyces cerevisiae. We show by two-hybrid an in vitro assays that the S. cerevisiae homologue of Sls1p interacts with ScKar2p. Using dominant lethal mutants of ScKar2p, we were able to show that ScSls1p preferentially interacts with the ADP-bound conformation of the molecular chaperone. Synthetic lethality was observed between ΔScsls1 and translocation-deficientkar2 or sec63-1 mutants, providing in vivo evidence for a role of ScSls1p in protein translocation. Synthetic lethality was also observed with ER-associated degradation and folding-deficient kar2 mutants, strongly suggesting that Sls1p functions are not restricted to the translocation process. We show that Sls1p stimulates in a dose-dependent manner the binding ofScKar2p on the lumenal J domain of Sec63p fused to glutathione S-transferase. Moreover, Sls1p is shown to promote the Sec63p-mediated activation of Kar2p's ATPase activity. Our data strongly suggest that Sls1p could be the first GrpE-like protein described in the endoplasmic reticulum.

Expression of Viral Membrane Proteins from Cloned cDNA by Microinjection into Eucaryotic Cell Nuclei

1982 ◽  
Vol 40 ◽  
pp. 26-29
Author(s):  
H. Garoff ◽  
Cl. Kondor-Koch
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Signal Sequence ◽  
Endoplasmic Reticulum Membrane ◽  
In Vitro Mutagenesis ◽  
Cell Nuclei ◽  
Eucaryotic Cell ◽  
Cloned Cdna ◽  
The Relationship

The relationship between a protein's structure and its function can be explored in detail by in vitro mutagenesis of a cloned DNA molecule encoding the protein and expression of its mutagenized form in a eucaryotic cell. This will be a useful approach to study the assembly of spanning viral or plasma membrane proteins into the endoplasmic reticulum membrane and their transport to the cell surface. Questions that could be answered using this technique include:Is the signal sequence alone sufficient for translocation of a polypeptide chain across the endoplasmic reticulum membrane? Is a cytoplasmic protein (e.g. a viral capsid protein) translocated when linked to a signal sequence?

scholarly journals Translocation of proteins across the endoplasmic reticulum. II. Signal recognition protein (SRP) mediates the selective binding to microsomal membranes of in-vitro-assembled polysomes synthesizing secretory protein.

1981 ◽  
Vol 91 (2) ◽  
pp. 551-556 ◽  
Author(s):  
P Walter ◽  
G Blobel
Keyword(s):  
Endoplasmic Reticulum ◽  
Signal Sequence ◽  
Preceding Paper ◽  
Secretory Protein ◽  
Microsomal Membrane ◽  
Endoplasmic Reticulum Membrane ◽  
Signal Recognition ◽  
Lipid Signal ◽  
Recognition Protein

Translocation-competent microsomal membrane vesicles of dog pancreas were shown to selectively bind nascent, in vitro assembled polysomes synthesizing secretory protein (bovine prolactin) but not those synthesizing cytoplasmic protein (alpha and beta chain of rabbit globin). This selective polysome binding capacity was abolished when the microsomal vesicles were salt-extracted but was restored by an 11S protein (SRP, Signal Recognition Protein) previously purified from the salt-extract of microsomal vesicles (Walter and Blobel, 1980. Proc. Natl. Acad. Sci. U. S. A. 77:7112-7116). SRP-dependent polysome recognition and binding to the microsomal membrane was shown to be a prerequisite for chain translocation. Modification of SRP by N-ethyl maleimide abolished its ability to mediate nascent polysome binding to the microsomal vesicles. Likewise, polysome binding to the microsomal membrane was largely abolished when beta-hydroxy leucine, a Leu analogue, was incorporated into nascent secretory polypeptides. The data in this and the preceding paper provide conclusive experimental evidence that chain translocation across the endoplasmic reticulum membrane is a receptor-mediated event and thus rule out proposals that chain translocation occurs spontaneously and without the mediation by proteins. Moreover, our data here demonstrate conclusively that the initial events that lead to translocation and provide for its specificity are protein-protein (signal sequence plus ribosome with SRP) and not protein-lipid (signal sequence with lipid bilayer) interactions.

scholarly journals Topology and functional domains of Sec63p, an endoplasmic reticulum membrane protein required for secretory protein translocation.

1992 ◽  
Vol 12 (7) ◽  
pp. 3288-3296 ◽  
Author(s):  
D Feldheim ◽  
J Rothblatt ◽  
R Schekman
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Protein Translocation ◽  
Protein A ◽  
Integral Membrane Protein ◽  
Secretory Protein ◽  
Carboxyl Terminus ◽  
Endoplasmic Reticulum Membrane ◽  
Cell Fractionation ◽  
Network Cell

SEC63 encodes a protein required for secretory protein translocation into the endoplasmic reticulum (ER) of Saccharomyces cerevisiae (J. A. Rothblatt, R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman, J. Cell Biol. 109:2641-2652, 1989). Antibody directed against a recombinant form of the protein detects a 73-kDa polypeptide which, by immunofluorescence microscopy, is localized to the nuclear envelope-ER network. Cell fractionation and protease protection experiments confirm the prediction that Sec63p is an integral membrane protein. A series of SEC63-SUC2 fusion genes was created to assess the topology of Sec63p within the ER membrane. The largest hybrid proteins are unglycosylated, suggesting that the carboxyl terminus of Sec63p faces the cytosol. Invertase fusion to a loop in Sec63p that is flanked by two putative transmembrane domains produces an extensively glycosylated hybrid protein. This loop, which is homologous to the amino terminus of the Escherichia coli heat shock protein, DnaJ, is likely to face the ER lumen. By analogy to the interaction of the DnaJ and Hsp70-like DnaK proteins in E. coli, the DnaJ loop of Sec63p may recruit luminal Hsp70 (BiP/GRP78/Kar2p) to the translocation apparatus. Mutations in two highly conserved positions of the DnaJ loop and short deletions of the carboxyl terminus inactivate Sec63p activity. Sec63p associates with several other proteins, including Sec61p, a 31.5-kDa glycoprotein, and a 23-kDa protein, and together with these proteins may constitute part of the polypeptide translocation apparatus. A nonfunctional DnaJ domain mutant allele does not interfere with the formation of the Sec63p/Sec61p/gp31.5/p23 complex.

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