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Biomedical subjects

G Blobel

Publications and source records attributed to G Blobel.

At least 127 records · Page 7Linked to original sources
PubMed1991

Biochemical fractionation and assembly of the membrane components that mediate nascent chain targeting and translocation.

Fractionation of a microsomal detergent extract with ammonium sulfate allows separation of the signal recognition particle receptor (SR alpha), which is required for targeting of the nascent chain, from other microsomal proteins, such as signal peptidase, whose activity is displayed during subsequent translocation. The reconstituted SR alpha-enriched fraction is functional in assays of precursor targeting and elongation arrest release but lacks translocation activity. This defect can be complemented by addition, prior to reconstitution, of a separate protein subfraction. In addition, protein components necessary for translocation can be reversibly depleted from the complementary fraction, under conditions where precursor targeting is retained, by sulfhydryl-directed chromatography. Thus, precursor binding and translocation can be biochemically uncoupled, indicating that they are sequential reactions mediated by distinct components.

Animals↗
PubMed1991

Binding of lamin A to polynucleosomes.

Morphological observations suggest a close association between heterochromatin and the nuclear lamina. To investigate the molecular aspects of this association, we have established a simple sedimentation assay employing purified lamin proteins, or their 125I-labeled derivatives, and polynucleosomal particles isolated from avian erythrocytes. We report here that purified, unlabeled lamin A and 125I-lamin A, but not 125I-lamin B or 125I-bovine serum albumin, bind to polynucleosomes in a saturable and specific fashion. The specific binding of 125I-lamin A is of high affinity (Kd = approximately 1 x 10(-9) M) and is distinctly temperature-dependent. This interaction is not affected by exogenous polyionic agents such as polylysine and DNA, but it can be abolished by protease digestion of the polynucleosomes. These data suggest that nuclear lamin A maintains a direct association with a proteinaceous constituent of interphase chromatin.

Adenosine Triphosphate↗
PubMed1991

A protein-conducting channel in the endoplasmic reticulum.

The existence of a protein-conducting channel in the endoplasmic reticulum membrane was demonstrated by electrophysiological techniques. Pancreatic rough microsome (RM) vesicles were fused to one side (cis) of a planar lipid bilayer separating two aqueous compartments of 50 mM salt. This exposed the cytoplasmic surface of the RMs, with its attached ribosomes, to the cis chamber. Addition of 100 microM puromycin to the cis side caused a large increase in membrane conductance, presumably the result of puromycin-induced clearance of nascent protein chains from the lumen of protein-conducting channels. When puromycin was added at low concentrations (0.33 microM), single channels of 220 pS were observed. These closed when the salt concentration was raised to levels at which ribosomes detach from the membrane (150-400 mM), indicating that the attached ribosome keeps the channel in an open conformation. A mechanism for a complete cycle of opening and closing of the protein-conducting channel is suggested.

Edetic Acid↗
PubMed1991

Signal peptide analogs derived from two chloroplast precursors interact with the signal recognition system of the chloroplast envelope.

We have used synthetic peptides representing segments of the signal sequences of preferredoxin (pFd) and the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase (pS) to study interactions with the signal sequence recognition system at the chloroplast surface. Peptides representing the COOH-terminal 30 amino acids of the pFd and pS signal peptides were able to completely and reversibly inhibit the import of their homologous precursors into isolated chloroplasts at a 2.5 microM concentration. Import was blocked at the level of precursor binding to the chloroplast. This inhibition of precursor binding and import was not due to disruption of chloroplast integrity as incubation of isolated chloroplasts with the peptides did not cause measurable perturbation of the envelope membranes. The peptides also were able to block the import of the heterologous precursor protein, suggesting that pS and pFd share a common signal sequence recognition system. Visualization of the bound peptides at the chloroplast surface by indirect immunofluorescence microscopy using antipeptide antibodies gave a marked punctate staining pattern. This pattern is consistent with the localization of chloroplast import receptor(s) at contact zones between the inner and outer envelope membranes.

Amino Acid Sequence↗
PubMed1991

cDNA cloning and sequencing of human fibrillarin, a conserved nucleolar protein recognized by autoimmune antisera.

We have isolated a 1.1-kilobase cDNA clone that encodes human fibrillarin by screening a hepatoma library in parallel with DNA probes derived from the fibrillarin genes of Saccharomyces cerevisiae (NOP1) and Xenopus laevis. RNA blot analysis indicates that the corresponding mRNA is approximately 1300 nucleotides in length. Human fibrillarin expressed in vitro migrates on SDS gels as a 36-kDa protein that is specifically immunoprecipitated by antisera from humans with scleroderma autoimmune disease. Human fibrillarin contains an amino-terminal repetitive domain approximately 75-80 amino acids in length that is rich in glycine and arginine residues and is similar to amino-terminal domains in the yeast and Xenopus fibrillarins. The occurrence of a putative RNA-binding domain and an RNP consensus sequence within the protein is consistent with the association of fibrillarin with small nucleolar RNAs. Protein sequence alignments show that 67% of amino acids from human fibrillarin are identical to those in yeast fibrillarin and that 81% are identical to those in Xenopus fibrillarin. This identity suggests the evolutionary conservation of an important function early in the pathway for ribosome biosynthesis.

Amino Acid Sequence↗
PubMed1991

Yeast signal peptidase contains a glycoprotein and the Sec11 gene product.

Partially purified yeast microsomal signal peptidase appears to be a complex of four polypeptides of 13, 18, 20, and 25 kDa. The 18-kDa chain is the product of the Sec11 gene, which is necessary for signal peptidase activity. The 25-kDa subunit is a glycoprotein that binds Con A. Two related methods for purification of the enzyme are presented; the first includes removal of peripheral membrane proteins from microsomes by alkali extraction, solubilization of the enzyme by nonionic detergent and high salt, and four different chromatographic procedures. An alternative method was developed based on lectin-affinity chromatography.

Chromatography↗
PubMed1991

Network antibodies identify nuclear lamin B as a physiological attachment site for peripherin intermediate filaments.

We studied the molecular associations between peripherin (a neuronal, type III intermediate filament subunit) and nuclear lamins. We show here that isolated peripherin binds selectively to mammalian lamin B under in vitro conditions. We further demonstrate that a synthetic peptide, representing the proximal part of peripherin's tail domain (P1), also associates with mammalian lamin B in a saturable, cooperative, and specific fashion. Laboratory animals immunized with P1 spontaneously develop idiotypic and anti-idiotypic antibodies recognizing peripherin and lamin B, respectively. These data provide essentially in vivo evidence that lamin B represents a constitutive nuclear "receptor" site for the tail domains of peripherin intermediate filaments.

Amino Acid Sequence↗
PubMed1990

Identification of a receptor for protein import into mitochondria.

Anti-idiotypic antibodies, prepared using a chemically synthesized signal peptide of a mitochondrial precursor protein, recognized a mitochondrial integral membrane protein (p32). Fab fragments derived from both anti-idiotypic antibodies and monospecific antibodies against purified p32 inhibited protein import into mitochondria. Moreover, anti-p32 antibodies specifically immunoprecipitated a precursor-p32 complex after detergent solubilization of mitochondria. Immunoelectron microscopy and subfractionation of mitochondria indicate that p32 is located in contact sites between the outer and inner mitochondrial membranes.

Antibodies, Anti-Idiotypic↗
PubMed1990

Isolation and characterization of the gene for a yeast mitochondrial import receptor.

We have previously identified an integral membrane protein (p32) from Saccharomyces cerevisiae as a receptor for protein import into mitochondria, and have localized it to the mitochondrial outer membrane at contact sites. Here we report isolation of the corresponding mitochondrial import receptor gene, termed MIR1. The deduced amino-acid sequence of p32 shows roughly 40% identity with proteins of bovine heart and rat liver that have been suggested to be mitochondrial phosphate carriers. Haploid cells carrying a disrupted MIR1 allele were unable to grow on a non-fermentable carbon source but grew in media containing glucose, indicating that the MIR1 protein is essential for mitochondrial function. Compared with wild type, amounts of some mitochondrial proteins were markedly reduced in cells containing a disrupted MIR1 allele, whereas levels of others were unchanged. This indicates that yeast contains more than one pathway for protein import into mitochondria.

Amino Acid Sequence↗
PubMed1990

Identification and characterization of autoantibodies against the nuclear envelope lamin B receptor from patients with primary biliary cirrhosis.

We have identified autoantibodies from two patients with primary biliary cirrhosis (PBC) that recognize the nuclear envelope of mammalian cells on indirect immunofluorescence microscopy. These antibodies bind to a 58-kD integral membrane protein (p58) of the turkey erythrocyte nuclear envelope, which has been previously identified as a membrane receptor for lamin B (Worman, H. J., J. Yuan, G. Blobel, and S. D. Georgatos. 1988. Proc. Natl. Acad. Sci. USA. 85:8531). The antibodies also bind to a 61-kD integral membrane protein (p61) of the rat liver nuclear envelope. Affinity-purified antibodies eluted from turkey p58 bind to rat p61, showing that the two proteins share an epitope(s) and that p61 is likely the rat liver lamin B receptor. In human nuclear envelopes, the antigen recognized has an apparent molecular mass close to that of avian protein. These findings, along with the previous discovery of autoantibodies against an integral membrane glycoprotein (gp210) of the nuclear pore membrane in patients with PBC, suggest that antibodies against integral membrane proteins of the nuclear envelope are characteristic of a subset of patients with PBC.

Animals↗
PubMed1990

Two subunits of the canine signal peptidase complex are homologous to yeast SEC11 protein.

Canine microsomal signal peptidase activity was previously isolated as a complex of five subunits (25, 22/23, 21, 18, and 12 kDa). Two of the signal peptidase complex (SPC) subunits (23/23 and 21 kDa) have been cloned and sequenced. One of these, the 21-kDa subunit, was observed to be a mammalian homolog of SEC11 protein (Sec11p) (Greenburg, G., Shelness, G. S., and Blobel, G. (1989) J. Biol. Chem. 264, 15762-15765) a gene product essential for signal peptide processing and cell growth in yeast (Böhni, P.C., Deshqies, R.J., and Schekman, R.W. (1988) J. Cell Biol. 106, 1035-1042). cDNA clones for the 18-kDa SPC subunit have now been characterized and found to encode a second SEC11p homolog. Both the 18- and 21-kDa canine SPC subunits are integral membrane proteins by virtue of their resistance to alkaline extraction. Upon detergent solubilization, both proteins are found in a complex with the 22/23 kDa SPC subunit, the only SPC subunit containing N-linked oligosaccharide. No steady-state pool of canine Sec11p-like monomers is detected in microsomal membranes. Alkaline extraction of microsomes prior to solubilization or solubilization at alkaline pH causes partial dissociation of the SPC. The Sec11p-like subunits displaced from the complex under these conditions demonstrate no signal peptide processing activity by themselves. The existence of homologous subunits is common to a number of known protein complexes and provides further evidence that the association between SPC proteins observed in vitro may be physiologically relevant to the mechanism of signal peptide processing and perhaps protein translocation.

Amino Acid Sequence↗
PubMed1990

In vivo phosphorylation of the lamin B receptor. Binding of lamin B to its nuclear membrane receptor is affected by phosphorylation.

Previous studies have shown that the nuclear envelope of avian erythrocytes contains a 58-kDa integral membrane protein (p58) which serves as a receptor for the karyoskeletal protein lamin B (Worman, J. H., Yuan, J., Blobel, G., and Georgatos, S. D. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8531-8534). We now demonstrate that p58 is phosphorylated in vivo at serine residues and that its phosphorylation is stimulated by isoproterenol in a dose-dependent fashion. We further show that dephosphorylation of p58 reduces significantly its binding to lamin B. These data suggest that phosphorylation may constitute one of the major mechanisms regulating the lamina-nuclear membrane interactions.

Animals↗
PubMed1990

Isolation and sequencing of NOP1. A yeast gene encoding a nucleolar protein homologous to a human autoimmune antigen.

We have identified the gene for the yeast nucleolar protein p38 and deduced the primary structure of p38 from its sequence. We propose the name NOP1 (nucleolar protein 1) for this gene. NOP1 encodes a 327 amino acid protein of 34,470 daltons and is flanked by potential promoter and polyadenylation sequences. Blot analyses indicate that the mRNA transcribed from NOP1 is approximately 1.3 kilobases in size and that there is one NOP1 gene per haploid genome. The amino-terminal sequence of p38 is homologous with the 31 known amino-terminal residues of the autoimmune antigen fibrillarin, confirming the previously observed similarity between p38 and this mammalian nucleolar protein. Consistent with this, p38 cross-reacts with serum from a patient with the autoimmune disease scleroderma. A putative nuclear localization signal can be identified in p38. Interestingly, a repetitive amino acid sequence motif begins near the amino terminus of p38. This motif is approximately 80 residues long, is rich in glycine and arginine, and shows striking sequence homology to mammalian nucleolins and certain nucleic acid binding proteins.

Amino Acid Sequence↗
PubMed1990

Assembly of translocation-competent proteoliposomes from detergent-solubilized rough microsomes.

Canine pancreas rough microsomes were solubilized in a high salt buffer containing sodium cholate, a detergent extract prepared by high speed centrifugation, and vesicles were reconstituted from the extract by a detergent dialysis procedure. The reconstituted vesicles, lacking resident lumenal proteins, translocated in vitro-synthesized preprolactin in a cotranslational, SRP-dependent manner. The translocated precursor was processed to mature prolactin and protected from digestion by exogenous protease. Vesicles were also reconstituted from detergent extracts depleted of glycoproteins by chromatography on concanavalin-Sepharose. The depleted vesicles, containing a full complement of SRP receptor but deficient in the glycoprotein subunit of signal peptidase, the ribophorins, and other glycoprotein components, were functional in the targeting and binding of nascent preprolactin but exhibited a greatly diminished capacity for translocation.

Animals↗
PubMed1990

Reconstitution of protein translocation from detergent-solubilized Escherichia coli inverted vesicles: PrlA protein-deficient vesicles efficiently translocate precursor proteins.

Proteoliposomes were reconstituted by detergent dialysis of a sodium cholate extract of inverted vesicles derived from Escherichia coli plasma membrane. The translocation of precursor proteins into reconstituted vesicles occurred at high efficiency and was SecB dependent. The protein composition of the reconstituted vesicles differed markedly from that of native vesicles. Immunoblot analysis of the sodium cholate extract and of the reconstituted vesicles indicated that PrlA (SecY) protein remained largely unsolubilized under the described conditions and was virtually absent from the reconstituted vesicles, suggesting that PrlA may not be required for in vitro translocation.

ATP-Binding Cassette Transporters↗