Biomedical subjects

G Blobel

Publications and source records attributed to G Blobel.

At least 145 records · Page 8Linked to original sources

PubMed1990

The lamin B receptor of the nuclear envelope inner membrane: a polytopic protein with eight potential transmembrane domains.

The lamin B receptor is a previously identified integral membrane protein in the nuclear envelope of turkey erythrocytes that associates with the nuclear intermediate filament protein lamin B (Worman, H. J., J. Yuan, G. Blobel, and S. D. Georgatos. 1988. Proc. Natl. Acad. Sci. USA. 85:8531-8534). In the present report, we use cell fractionation and antibodies against the lamin B receptor to localize it to an 8-M urea-extracted membrane fraction of chicken liver nuclei, supporting an inner nuclear membrane localization. We deduced the amino acid sequence of the chicken lamin B receptor from overlapping clones obtained by screening cDNA libraries with a probe generated by the polymerase chain reaction with primers based on the partial protein sequence of the isolated protein. The mature lamin B receptor has a calculated molecular mass of 73,375 D and eight segments of hydrophobic amino acids that could function as transmembrane domains as determined by hydropathy analysis. Preceding the first putative transmembrane segment is a highly charged 204-residue-long amino terminal region that contains two consensus sites for phosphorylation by protein kinase A. Since the lamin B receptor has been shown to be phosphorylated by protein kinase A in vitro and in vivo and this phosphorylation affects lamin B binding (Applebaum, J., G. Blobel, and S. D. Georgatos. 1990. J. Biol. Chem. 265:4181-4185), it is likely that this amino terminal region faces the nucleoplasm. The amino terminal region also contains three DNA-binding motifs that are found in gene regulatory proteins and histones, suggesting that the lamin B receptor may additionally play a role in gene regulation and/or chromatin organization.

Amino Acid Sequence↗

PubMed1990

The chloroplast import receptor is an integral membrane protein of chloroplast envelope contact sites.

A chloroplast import receptor from pea, previously identified by antiidiotypic antibodies was purified and its primary structure deduced from its cDNA sequence. The protein is a 36-kD integral membrane protein (p36) with eight potential transmembrane segments. Fab prepared from monospecific anti-p36 IgG inhibits the import of the ribulose-1,5-bisphosphate carboxylase small subunit precursor (pS) by interfering with pS binding at the chloroplast surface. Anti-p36 IgGs are able to immunoprecipitate a Triton X-100 soluble p36-pS complex, suggesting a direct interaction between p36 and pS. This immunoprecipitation was specific as it was abolished by a pS synthetic transit peptide, consistent with the transit sequence receptor function of p36. Immunoelectron microscopy localized p36 to regions of the outer chloroplast membrane that are in close contact with the inner chloroplast membrane. Comparison of the deduced sequence of pea p36 to that of other known proteins indicates a striking homology to a protein from spinach chloroplasts that was previously suggested to be the triose phosphate-3-phosphoglycerate-phosphate translocator (phosphate translocator) (Flügge, U. I., K. Fischer, A. Gross, W. Sebald, F. Lottspeich, and C. Eckerskorn. 1989. EMBO (Eur. Mol. Biol. Organ.) J. 8:39-46). However, incubation of Triton X-100 solubilized chloroplast envelope material with hydroxylapatite indicated that p36 was quantitatively absorbed, whereas previous reports have shown that phosphate translocator activity does not bind to hydroxylapatite (Flügge, U. I., and H. W. Heldt. 1981. Biochim. Biophys. Acta. 638:296-304. These data, in addition to the topology and import inhibition data presented in this report support the assignment of p36 as a receptor for chloroplast protein import, and argue against the assignment of the spinach homologue of this protein as the chloroplast phosphate translocator.

Amino Acid Sequence↗

PubMed1990

A nuclear localization signal binding protein in the nucleolus.

We used functional wild-type and mutant synthetic nuclear localization signal peptides of SV-40 T antigen cross-linked to human serum albumin (peptide conjugates) to assay their binding to proteins of rat liver nuclei on Western blots. Proteins of 140 and 55 kD (p140 and p55) were exclusively recognized by wild-type peptide conjugates. Free wild-type peptides competed for the wild-type peptide conjugate binding to p140 and p55 whereas free mutant peptides, which differed by a single amino acid from the wild type, competed less efficiently. The two proteins were extractable from nuclei by either low or high ionic strength buffers. We purified p140 and raised polyclonal antibodies in chicken against the protein excised from polyacrylamide gels. The anti-p140 antibodies were monospecific as judged by their reactivity with a single nuclear protein band of 140 kD on Western blots of subcellular fractions of whole cells. Indirect immunofluorescence microscopy on fixed and permeabilized Buffalo rat liver (BRL) cells with anti-p140 antibodies exhibited a distinct punctate nucleolar staining. Rhodamine-labeled wild-type peptide conjugates also bound to nucleoli in a similar pattern on fixed and permeabilized BRL cells. Based on biochemical characterization, p140 is a novel nucleolar protein. It is possible that p140 shuttles between the nucleolus and the cytoplasm and functions as a nuclear import carrier.

Amino Acid Sequence↗

PubMed1990

The 210-kD nuclear envelope polypeptide recognized by human autoantibodies in primary biliary cirrhosis is the major glycoprotein of the nuclear pore.

We have recently reported a new family of nuclear autoantibodies in a subset of patients with primary biliary cirrhosis. These antibodies bind to a nuclear envelope polypeptide(s) of approximately 200 kD, the exact identity of which was not established. In this study, we show that all of these autoantibodies are directed against a 210-kD integral membrane glycoprotein of the nuclear pore.

Autoantigens↗

PubMed1990

Identification and characterization of receptors for protein import into chloroplasts and mitochondria.

An anti-idiotypic antibody approach was used to identify chloroplast and mitochondrial protein component(s) which interact with the corresponding signal sequence. The proteins thus identified can be operationally defined as receptor(s) for import of proteins into chloroplasts and mitochondria. The import receptor(s) was found in "contact sites" between the outer and inner membrane of chloroplast envelope or of mitochondria.

Antibodies, Anti-Idiotypic↗

PubMed1989

Cyclic AMP-dependent protein kinase in canine pancreatic rough endoplasmic reticulum.

A canine pancreas homogenate was subfractionated by several differential centrifugation steps. The distribution of cAMP-dependent protein kinase in the various fractions was monitored by assaying [3H]cAMP binding and photo-cross-linking of the regulatory subunits of the enzyme (RI and RII) with radiolabeled 8-azido-cAMP. The distribution of the kinase was also compared to that of markers for the plasma membrane, the endoplasmic reticulum and the cytosol. While our results confirm previous studies suggesting the presence of cyclic AMP-dependent protein kinase in the cytosol and Golgi, a significant amount of the total [3H] cAMP binding and photolabeled R subunits (both RI and RII) were found in rough microsomes (RM). The association is relatively resistant to extraction with EDTA, low and high ionic strength solutions. These extractions unmasked several new phosphorylation substrates in the "stripped" RM that were inaccessible in the RM, possibly because they were covered by ribosomes or peripheral membrane proteins. RII with a molecular mass of 52 kDa (RII-52 kDa) was the predominant RII found in the cytosolic fraction, whereas RII-52 kDa and RII with a molecular mass of 54 kDa (RII-54 kDa) were approximately equally enriched in the RM fraction. The mobility of the RII-52 kDa-photolabeled band could be shifted to the mobility of the RII-54 kDa band by phosphorylation with purified catalytic subunit and ATP, indicating that they represent "dephospho" and "phospho" forms of RII, respectively. A more precise localization to the rough endoplasmic reticulum was accomplished by isopycnic floatation in sucrose gradients. The enzyme cobanded at the density of rough microsomes and shifted to the lower density of "stripped" microsomes after treatment with puromycin/high salt, which specifically removes ribosomes.

Animals↗

PubMed1989

A subunit of mammalian signal peptidase is homologous to yeast SEC11 protein.

Canine signal peptidase consists of a complex of five proteins (Evans, A. E., Gilmore, R., and Blobel, G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 581-585). A cDNA encoding the 21-kDa subunit of the signal peptidase complex was isolated from a liver cDNA library using an 88-base pair probe, generated by the polymerase chain reaction. The 820-base pair cDNA was sequenced and found to encode a protein of 21,585 daltons. The deduced amino acid sequence from the canine cDNA was found to be 47% identical to the yeast SEC11 protein. SEC11 has been shown to be required for signal peptide cleavage, normal rate of secretion, and cell survival in Saccharomyces cerevisiae (Böhni, P. C., Deshaies, R. J., and Schekman, R. W. (1988) J. Cell Biol. 106, 1035-1042). It is, therefore, likely that the 21-kDa subunit of signal peptidase complex is the structural and functional homologue of the yeast SEC11 gene product.

Amino Acid Sequence↗

PubMed1989

SecB functions as a cytosolic signal recognition factor for protein export in E. coli.

A purified 64 kd protein, consisting of four identical subunits of the 16 kd SecB, binds to the signal sequence of preproteins prior to their translocation across inverted vesicles (INV) derived from the E. coli plasma membrane. The purified SecB tetramer competes with canine signal recognition particle (SRP) in signal sequence binding and thus behaves as a prokaryotic equivalent of SRP. As shown by cell fractionation and immunoblot analysis with anti-SecB antibodies, SecB is a cytosolic protein. An E. coli supernatant depleted of SecB after passage through an anti-SecB Sepharose column retains full translation activity but is unable to support translocation into added INV. Translocation into INV is fully restored by readdition of purified SecB.

Bacterial Proteins↗

PubMed1989

Solubilization and characterization of yeast signal peptidase.

An efficient post-translational assay for solubilized yeast signal peptidase has been developed. The enzyme can be solubilized in nonionic detergent (0.5% Nikkol) without added salt, but salt increased the efficiency of solubilization. Radiosequencing of the cleaved substrate revealed that the enzyme removed the signal peptide. The substrate (prepro-alpha-factor) must be pretreated with sodium dodecyl sulfate to be cleaved. The enzyme displays a broad, alkaline pH optimum, retaining activity at pH 12. Moderately high temperatures (35 degrees C), excess detergent (greater than 0.5% Nikkol), or high salt (greater than 300 mM KOAc) will inactivate the enzyme. Phosphatidylcholine is necessary for optimal activity. The optimal ratio of Nikkol:lipid:sodium dodecyl sulfate is 6.4:2.2:1. The membrane association of yeast signal peptidase is resistant to carbonate extraction, indicating that it is an integral membrane protein.

Endopeptidases↗

PubMed1989

Large aqueous channels in membrane vesicles derived from the rough endoplasmic reticulum of canine pancreas or the plasma membrane of Escherichia coli.

Voltage clamp conditions were used to study the membrane permeability properties of rough microsomes (RM) derived from the rough endoplasmic reticulum of canine pancreas and inverted vesicles (InV) derived from the plasma membrane of Escherichia coli. Membrane vesicles of RM or InV were fused to a planar lipid bilayer that was formed in a hole of a partition separating two chambers. Fusion of a single RM vesicle yielded a single-step conductance increase. Some preparations yielded unitary conductances of 20, 55, 80, and 115 pS in 45 mM potassium glutamate. These channels were largely open at negative membrane potential on the cytoplasmic side of the RM membrane, mostly closed at positive voltages, permeable to amino acids, and slightly more selective for anions than cations. There was a dramatic increase in the number of open channels when 100 microM GTP was added to the cytoplasmic side of the fused RM, whereas 100 microM guanosine 5'-[gamma-thio]triphosphate caused closing of channels. ATP had no effect. A large channel of 115 pS at 45 mM potassium glutamate was also detected after the fusion of InV. As both RM and InV share the ability to translocate secretory proteins, it is possible that the 115-pS channel in both membranes represents a protein-conducting channel.

Animals↗

PubMed1989

Site-specific antibodies against the PrlA (secY) protein of Escherichia coli inhibit protein export by interfering with plasma membrane binding of preproteins.

Genetic evidence indicates that the PrlA (SecY) protein of Escherichia coli functions as a membrane integrated signal sequence receptor in protein "export"--i.e., in protein translocation across (or integration into) the plasma membrane. We have raised antibodies in rabbits against two synthetic peptides representing the hydrophilic N- or C-terminal region of PrlA. Using these antibodies as probes in cell fractionation experiments, we confirm that PrlA is an integral membrane protein of the plasma membrane of E. coli. Fab fragments prepared from each of the two antisera specifically inhibit protein export by interfering with the binding of preproteins to the plasma membrane. Inhibition of preprotein binding and export by Fab fragments was shown in a cell-free translocation system: precursors for LamB (an integral membrane protein) and for a truncated form of MalE (a periplasmic protein) were first synthesized in a membrane-depleted E. coli-derived cell-free translocation system followed by posttranslational incubation with inverted vesicles derived from the plasma membrane of E. coli. Our data thus indicate that the N and C termini of PrlA are exposed to the cytoplasm. We discuss the possibility that the transmembrane segments of PrlA could be arranged in the lipid bilayer in a cylindrical fashion, thereby delimiting a protein conducting channel, with a signal sequence binding domain represented, at least in part, by the N and C termini of PrlA. Such a channel would also contain a "stop-transfer" sequence binding domain that in response to a stop-transfer sequence would open the cylindrical channel to the lipid bilayer and permit displacement of the polypeptide from the channel to the lipid bilayer, resulting in membrane integration.

Amino Acid Sequence↗

PubMed1989

Binding of a soluble factor of Escherichia coli to preproteins does not require ATP and appears to be the first step in protein export.

We have constructed a mutant form of the maltose binding protein precursor, termed preMBP*, that, unlike its wild-type counterpart preMBP, retains translocation competence after synthesis. In a homologous Escherichia coli translation/translocation system, preMBP* was translocated either co- or posttranslationally with virtually 100% efficiency into inverted vesicles (INV) derived from the E. coli plasma membrane. Translation of preMBP* mRNA in a wheat germ system and subsequent incubation with INV yielded no translocation. However, addition of increasing amounts of an E. coli postribosomal supernatant (PRS) to the wheat germ extract stimulated preMBP* translocation with virtually 100% efficiency occurring at 100 micrograms of PRS per 50 microliters of incubation mixture. The activity in the E. coli PRS appears to be identical to the previously described "export" factor. The soluble activity can bind to preMBP* posttranslationally and in the absence of ATP. Subsequent targeting to INV and/or translocation, however, requires ATP. Binding of the soluble activity to preMBP* thus appears to be the first step in a multistep translocation reaction.

Adenosine Triphosphate↗

PubMed1989

Cytosolic factor purified from Escherichia coli is necessary and sufficient for the export of a preprotein and is a homotetramer of SecB.

We have purified to homogeneity a cytosolic factor from Escherichia coli that is required for the translocation of a preprotein into inverted vesicles of the E. coli plasma membrane. The preprotein used is a precursor of mutant maltose-binding protein. This mutant contains alterations of the carboxyl terminus. Unlike the precursor for wild-type maltose-binding protein, the mutant precursor does not acquire a protease-resistant conformation after synthesis and retains posttranslational translocation competence. The purified cytosolic factor, added posttranslationally, is necessary and sufficient to yield virtually 100% translocation of the mutant precursor into inverted vesicles. The purified factor amounts to 0.08% of the cytosolic proteins and is a 64-kDa tetramer consisting of four identical 16-kDa subunits. Amino-terminal sequence analysis revealed that it is identical to the secB gene product. The purified SecB homotetramer is part of a larger 150-kDa complex that represents the "export" factor activity. During purification, the export factor activity dissociates into a 64-kDa SecB homotetramer and unidentified component(s). For the posttranslational integration of another preprotein, the precursor for the lamB gene product, into inverted vesicles, the 64-kDa SecB homotetramer is also required but additional factor(s) makes integration more efficient.

ATP-Binding Cassette Transporters↗

PubMed1989

Nascent secretory chain binding and translocation are distinct processes: differentiation by chemical alkylation.

We have investigated the effects of chemical alkylation of microsomal membranes on nascent chain binding and translocation. Assays were conducted using either full-length or truncated preprolactin transcripts in combination with a reconstituted membrane system consisting of proteolyzed rough microsomes and the cytoplasmic domain of the signal recognition particle receptor. Treatment of rough microsomes with N-ethylmaleimide was observed to inhibit preprolactin processing at a site other than the signal recognition particle or the signal recognition particle receptor. As formation of a translocation competent junction between the ribosome/nascent chain complex and the membrane has recently been demonstrated to require GTP (Connolly, T., and R. Gilmore. J. Cell Biol. 1986. 103:2253-2261), the effects of membrane alkylation on this parameter were assessed. N-ethylmaleimide treatment did not inhibit nascent chain targeting or GTP-dependent signal sequence insertion. Translocation of the targeted and inserted nascent chain was, however, blocked. These data indicate (a) that the process of nascent chain translocation is distinct from targeting and signal sequence insertion, and (b) translocation of the peptide chain across the membrane is mediated by an N-ethylmaleimide-sensitive membrane protein component(s). To further substantiate the observation that nascent chain targeting and signal sequence insertion can be distinguished from translocation, the temperature dependencies of the two phenomena were compared. Signal sequence insertion occurred at low temperatures (4 degrees C) and was maximal between 10 and 15 degrees C. Translocation was only observed at higher temperatures and was maximal between 25 and 30 degrees C.

Alkylation↗

PubMed1989

Yeast nuclear envelope proteins cross react with an antibody against mammalian pore complex proteins.

We have used a monoclonal antibody raised against rat liver nuclear proteins to study two cross-reactive proteins in the yeast nucleus. In rat liver, this monoclonal antibody, mAb 414, binds to nuclear pore complex proteins, including one of molecular weight 62,000 (Davis, L. I., and G. Blobel. 1987. Proc. Natl. Acad. Sci. USA. 84:7552-7556). In yeast, mAb 414 cross reacts by immunoblotting with two proteins that have apparent molecular weights of 110,000 and 95,000, and are termed p110 and p95, respectively. Examination of subcellular fractions by immunoblotting shows that both p110 and p95 are located exclusively in the nuclear fraction. The mAb 414 immunoprecipitates several proteins from a crude yeast cell extract, including p110, p95, and a approximately 55-kD protein. Immunoprecipitation from subcellular fractions yields only p110 and p95 from purified nuclei, whereas the approximately 55-kD protein is immunoprecipitated from the soluble fraction. Digestion of purified nuclei with DNase to produce nuclear envelopes releases some of p110, but the majority of p110 is solubilized only after treatment of envelopes with 1 M NaCl. Immunofluorescence localization using yeast cells and isolated nuclei shows a punctate and patchy staining pattern of the nucleus. Confocal laser scanning immunofluorescence microscopy resolves the punctate and patchy staining pattern better and shows regions of fluorescence at the nuclear envelope. Postembedding immunogold electron microscopy using purified nuclei and mAb 414 shows colloidal gold decoration of the yeast nuclear envelope, but resolves pore complexes too poorly to achieve further ultrastructural localization. Immunogold labeling of nuclei followed by embedding suggests decoration of pore complexes. Thus, p110 and/or p95 are localized to the nuclear envelope in yeast, and may be components of the nuclear pore complex.

Animals↗

PubMed1989

Lamin A, lamin B, and lamin B receptor analogues in yeast.

Previous studies have shown that turkey erythrocyte lamin B is anchored to the nuclear envelope via a 58-kD integral membrane protein termed p58 or lamin B receptor (Worman H. J., J. Yuan, G. Blobel, and S. D. Georgatos. 1988. Proc. Natl. Acad. Sci. USA. 85:8531-8534). We now identify a p58 analogue in the yeast Saccharomyces cerevisiae. Turkey erythrocyte lamin B binds to yeast urea-extracted nuclear envelopes with high affinity, associating predominantly with a 58-kD polypeptide. This yeast polypeptide is recognized by polyclonal antibodies against turkey p58, partitions entirely with the nuclear fraction, remains membrane bound after urea extraction of the nuclear envelopes, and is structurally similar to turkey p58 by peptide mapping criteria. Using polyclonal antibodies against turkey erythrocyte lamins A and B, we also identify two yeast lamin forms. The yeast lamin B analogue has a molecular mass of 66 kD and is structurally related to erythrocyte lamin B. Moreover, the yeast lamin B analogue partitions exclusively with the nuclear envelope fraction, is quantitatively removed from the envelopes by urea extraction, and binds to turkey lamin A and vimentin. As many higher eukaryotic lamin B forms, the yeast analogue is chemically heterogeneous comprising two serologically related species with different charge characteristics. Antibodies against turkey lamin A detect a 74-kD yeast protein, slightly larger than the turkey lamin A. It is more abundant than the yeast lamin B analogue and partitions between a soluble cytoplasmic fraction and a nuclear envelope fraction. The yeast lamin A analogue can be extracted from the nuclear envelope by urea, shows structural similarity to turkey and rat lamin A, and binds to isolated turkey lamin B. These data indicate that analogues of typical nuclear lamina components (lamins A and B, as well as lamin B receptor) are present in yeast and behave as their vertebrate counterparts.

Animals↗

PubMed1989

Primary structure analysis of an integral membrane glycoprotein of the nuclear pore.

The complete primary structure of an integral membrane glycoprotein of the nuclear pore was deduced from the cDNA sequence. The cDNA encodes a polypeptide of 204,205 D containing a 25-residue-long signal sequence, two hydrophobic segments that could function as transmembrane segments, and 13 potential N-linked oligosaccharide addition sites. Endoglycosidase H reduces the molecular mass by approximately 9 kD suggesting that not all of these 13 sites are used. We discuss possible models for the topology of this protein in the pore membrane as well as a possible role in the formation of pores and pore complexes.

Amino Acid Sequence↗

PubMed1988

cDNA-derived primary structure of the glycoprotein component of canine microsomal signal peptidase complex.

Canine microsomal signal peptidase activity has been shown previously to co-migrate as an apparent complex of six polypeptides with molecular masses of 25, 23, 22, 21, 18, and 12 kDa. The 22- and 23-kDa species are differentially glycosylated forms of the same protein, designated SPC 22/23. The amino acid sequence of SPC 22/23 was deduced from cDNA clones. The protein is synthesized without a cleavable amino-terminal signal sequence and contains a single site for N-linked glycosylation. SPC 22/23 appears to be anchored to the rough endoplasmic reticulum membrane by a single hydrophobic segment near its amino terminus, with the remainder of the protein positioned on the lumenal side of the membrane. The amino acid sequence of SPC 22/23 shares homology with tryptic peptides derived from the hen oviduct signal peptidase glycoprotein, one of two possible proteins required for signal peptide processing in the avian system (Baker, R.K., and Lively, M.O. (1987) Biochemistry 26, 8561-8567). Therefore, the complete amino acid sequence of SPC 22/23 presented in this report corresponds to one of two possible proteins required for signal peptide processing in higher eukaryotic cells.

Amino Acid Sequence↗