Search PubMed⌕ Search

Biomedical subjects

G Blobel

Publications and source records attributed to G Blobel.

At least 163 records · Page 9Linked to original sources
PubMed1988

Identification of a G protein in rough endoplasmic reticulum of canine pancreas.

Employing [32P]ADP-ribosylation by pertussis toxin we have identified a G protein that is located in the rough endoplasmic reticulum of canine pancreas and therefore termed it GRER. Identification of GRER is based on the following data. A 41-kDa polypeptide was the only polypeptide that was [32P]ADP-ribosylated by pertussis toxin in pancreas rough microsomes. Guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) and 1 mM ATP, 6 mM MgCl2, 10 mM NaF (AMF) inhibited ADP-ribosylation of this polypeptide. The [32P]ADP-ribosylated 41-kDa polypeptide was immunoprecipitated by antisera which specifically recognized the C-terminal residues of the alpha subunits of Gi and transducin, indicating that the 41-kDa polypeptide is immunologically related to the alpha subunits of heterotrimeric G proteins. Treatment with GTP gamma S resulted in a reduction in the sedimentation rate of the [32P]ADP-ribosylated, detergent-solubilized GRER. It also induced the release of the [32P]ADP-ribosylated 41-kDa polypeptide from rough microsomes in the absence of detergent, unlike ADP-ribosylated alpha subunits of plasma membrane-associated G proteins. These data are consistent with an oligomeric nature of GRER. The codistribution of GRER with an endoplasmic reticulum marker protein during subcellular fractionation and the lack of plasma membrane contamination of the rough microsomal fraction, combined with the isodensity of GRER with rough microsomes as well as the isodensity of GRER with "stripped" microsomes after extraction of rough microsomes with EDTA and 0.5 M KCl, localized GRER to the rough endoplasmic reticulum. Preliminary experiments suggest that GRER appears not to be involved in translocation of proteins across the rough endoplasmic reticulum membrane.

Adenosine Diphosphate Ribose↗
PubMed1988

Prepro-alpha-factor has a cleavable signal sequence.

MAT alpha Saccharomyces cerevisiae secrete a small peptide mating pheromone termed alpha-factor. Its precursor, prepro-alpha-factor, is translocated into the endoplasmic reticulum and glycosylated at three sites. The glycosylated form is the major product in a yeast in vitro translation/translocation system. However, there is another translocated, nonglycosylated product that contains a previously unidentified modification. Contrary to previous results suggesting that the signal sequence of prepro-alpha-factor is not cleaved, amino-terminal radiosequencing has identified this product as prepro-alpha-factor without its signal sequence, that is, pro-alpha-factor. The translocated, glycosylated proteins are also processed by signal peptidase. Moreover, we have found that both purified eukaryotic and prokaryotic signal peptidase can process prepro-alpha-factor. Experiments using a yeast secretory mutant (sec 18) blocked in transport from the endoplasmic reticulum to the Golgi indicate that the protein is also cleaved in vivo. Finally, characterization of the Asn-linked oligosaccharide present on pro-alpha-factor in the yeast in vitro system by use of specific glucosidase and mannosidase inhibitors indicates that they have had the three terminal glucoses and probably one mannose removed. Therefore they most likely consist of Man8GlcNAc2 structures, identical to those found in the endoplasmic reticulum in vivo.

Amino Acid Sequence↗
PubMed1988

70K heat shock related proteins stimulate protein translocation into microsomes.

A yeast cytosol is shown to contain two distinct activities that stimulate protein translocation across microsomal membranes. One activity was purified. It consists of two constitutively expressed 70K heat shock related proteins that increase the rate of translocation. Possible mechanisms of action of these proteins are discussed.

Adenosine Triphosphate↗
PubMed1988

Heterotypic and homotypic associations between the nuclear lamins: site-specificity and control by phosphorylation.

Using purified components in affinity chromatography and blot binding assays, we have found that rat liver lamins A, B, and C can associate in homotypic and heterotypic fashions. Heterotypic A-B and C-B complexes are unusually stable and involve the common amino-terminal domain of lamins A and C, but not their helical "rod" domain. A synthetic peptide, comprising the first 32 amino acid residues of lamins A and C, is able to fully compete with the intact molecules for binding to lamin B. Conversely, heterotypic A-C associations and homotypic A-A and C-C interactions appear significantly weaker than A/C-B binding and do not involve the lamin A and C amino-terminal domain. Homotypic B-B complexes are not formed to any considerable extent unless isolated lamin B subunits are "superphosphorylated" in vitro with protein kinase A. However, when lamins A and C are similarly modified, no changes in their binding specificity can be detected. These data suggest that the nuclear lamina, unlike other multicomponent intermediate filaments, constitutes a nonobligatory heteropolymer. They also indicate that cAMP-dependent phosphorylation of interphase lamin B could cause remodeling of the lamina and establishment of homopolymeric domains.

Adenosine Triphosphate↗
PubMed1988

A lamin B receptor in the nuclear envelope.

Using a solution binding assay, we show that purified 125I-labeled lamin B binds in a saturable and specific fashion to lamin-depleted avian erythrocyte nuclear membranes with a Kd of approximately 0.2 microM. This binding is significantly greater than the binding of 125I-labeled lamin A and is competitively inhibited by unlabeled ligand. We demonstrate that a 58-kDa integral membrane protein (p58) is a lamin B receptor by virtue of its abundance in the nuclear envelope and association with 125I-labeled lamin B in ligand blotting assays. Specific antibodies raised against p58 recognize one protein in isolated nuclei and partially block 125I-labeled lamin B binding to lamin-depleted nuclear membranes. Cell fractionation and indirect immunofluorescence microscopy show that p58 is located in the periphery of the nucleus. This protein may serve as a membrane attachment site for the nuclear lamina by acting as a specific receptor for lamin B.

Animals↗
PubMed1988

Identification and characterization of a yeast nucleolar protein that is similar to a rat liver nucleolar protein.

We have produced monoclonal antibodies against purified nuclei from the yeast Saccharomyces cerevisiae and have characterized three different antibodies that recognize a protein with an apparent molecular weight of 38,000, termed p38. Subcellular fractionation shows that virtually all of p38 occurs in the nuclear fraction. High concentrations of salt (1 M) or urea (6 M) effectively solubilize p38 from a nuclear envelope fraction prepared by digestion of nuclei with DNase. Indirect immunofluorescence demonstrates a crescent shaped distribution of p38 at the inner periphery of the nucleus, with p38 extending between dividing pairs of cells during (closed) mitosis. Postembedding immunogold electron microscopy shows decoration of the densely stained "crescent" region of the yeast nucleus, confirming the localization of p38 to the nucleolus. One of the monoclonals, D77, cross reacts on immunoblots with a single protein of molecular weight 37,000 from purified rat liver nuclei. Indirect immunofluorescence localizes this protein to the nucleolus, and shows that it is dispersed throughout the cell during mitosis. The yeast and rat liver nucleolar proteins behave similarly when electrophoresed in two dimensions, and appear to have basic pI values. Analysis of immunological cross-reactivity using D77, and antibodies specific for nucleolar proteins from other sources, suggests that the rat liver protein is fibrillarin, and demonstrates that p38 shares epitopes with fibrillarin, as well as with other vertebrate nucleolar proteins.

Animals↗
PubMed1988

70-kD heat shock-related protein is one of at least two distinct cytosolic factors stimulating protein import into mitochondria.

We have developed an in vitro system in which the posttranslational import of Put2 (delta-pyrroline-5-carboxylate dehydrogenase), into yeast mitochondria is dependent on the addition of yeast postribosomal supernatant (PRS). When mRNA for a nuclear-encoded yeast mitochondrial matrix protein, Put2, was translated in a wheat germ cell-free system, import into posttranslationally added yeast mitochondria was negligible. However, when a yeast PRS was added, significant import was observed. The import stimulating activity of the yeast PRS was shown to consist of at least two distinct factors. One of these is the recently purified 70-kD heat shock-related protein Ssalp/Ssa2p, two proteins that are 98% homologous. The other factor is an N-ethylmaleimide-sensitive protein(s). Both factors act synergistically.

Biological Transport↗
PubMed1987

Lamin B autoantibodies in sera of certain patients with systemic lupus erythematosus.

Sera from four patients with systemic lupus erythematosus containing antibodies that yield nuclear rim staining of HEp-2 cells by indirect immunofluorescence were identified and characterized. Each serum contained autoantibodies reacting strongly with lamin B on western blots. One of the four sera displayed weaker reactivity with lamins A and C, while the other three displayed only minimal reactivity with lamins A and C. Titers of antilamin antibodies ranged from 1:1,250 to 1:36,250. Two of the sera also reacted at a dilution of 1:20 with cytoplasmic filaments of PTK-2 cells, suggesting that a small fraction of the autoantibodies in these sera may bind to alpha-helical domains of the lamins that are homologous to those of intermediate filaments. The majority of the antilamin antibodies in these patients' sera are specific for portions of the lamin B molecule that are not homologous to lamins A and C, however. The findings suggest that autoantibodies to the nuclear lamina may, in some instances, be responsible for a rim pattern in the fluorescent antinuclear antibody assay. In addition, autoantibodies to the nuclear lamina in sera of certain patients with systemic lupus erythematosus may be useful for defining the molecular structure and biological functions of lamin B, as well as for studying mechanisms of autoimmunity.

Antibody Specificity↗
PubMed1987

Protein import into chloroplasts requires a chloroplast ATPase.

We have transcribed mRNA from a cDNA clone coding for pea ribulose-1,5-bisphosphate carboxylase, translated the mRNA in a wheat germ cell-free system, and studied the energy requirement for posttranslational import of the [35S]methionine-labeled protein into the stroma of pea chloroplasts. We found that import depends on ATP hydrolysis within the stroma. Import is not inhibited when H+, K+, Na+, or divalent cation gradients across the chloroplast membranes are dissipated by ionophores, as long as exogenously added ATP is also present during the import reaction. Our data suggest that protein import into the chloroplast stroma requires a chloroplast ATPase that does not function to generate a membrane potential for driving the import reaction but that exerts its effect in another, yet-to-be-determined, mode. We have carried out a preliminary characterization of this ATPase regarding its nucleotide specificity and the effects of various ATPase inhibitors.

Adenosine Triphosphatases↗
PubMed1987

Soluble factors stimulating secretory protein translocation in bacteria and yeast can substitute for each other.

mRNA for prepro-alpha-factor (pp alpha), a yeast secretory glycoprotein, was translated in a wheat germ cell-free system that was posttranslationally supplemented either with inverted vesicles from the plasma membrane of Escherichia coli (INV) or with microsomes from Saccharomyces cerevisiae. A postribosomal supernatant (PRS) from E. coli was found to stimulate translocation of pp alpha across the INV membrane. A yeast PRS could substitute for its E. coli counterpart. Likewise, an E. coli PRS could substitute for a yeast PRS and stimulate translocation of pp alpha across yeast microsomal membranes.

Bacterial Proteins↗
PubMed1987

Multiple topogenic sequences in bovine opsin.

Bovine opsin, a polytopic integral membrane protein, contains seven transmembrane segments connecting eight hydrophilic domains alternating on each side of the membrane. To localize topogenic sequences that might specify the distinct topology of opsin in the membrane, we constructed various opsin mutants, each containing only one transmembrane segment. Messenger RNAs transcribed from these mutants were translated in a cell-free system supplemented with microsomal membranes. Among six of the seven transmembrane segments of opsin that were analyzed, five were able to function as signal sequences and also expressed stop-transfer sequences of variable strength. By the criteria of extractability at pH 11 and protease sensitivity, the presence of a signal sequence in combination with a "strong" stop-transfer sequence yielded integration into the lipid bilayer of the majority of chains. However, in combination with a "weak" stop-transfer sequence, we observed integration into the lipid bilayer of only some chains, with the others either completely translocated across the membrane or retained in a water-accessible space in the membrane.

Amino Acid Sequence↗
PubMed1987

Binding of two desmin derivatives to the plasma membrane and the nuclear envelope of avian erythrocytes: evidence for a conserved site-specificity in intermediate filament-membrane interactions.

Using solution binding assays, we found that a 45-kDa fragment of desmin, lacking 67 residues from the N terminus, could specifically associate with avian erythrocyte nuclear envelopes but not with plasma membranes from the same cells. It was also observed that a 50-kDa desmin peptide, missing 27 C-terminal residues, retained the ability to bind to both membrane preparations. Displacement experiments with an excess of purified vimentin suggested that the two desmin derivatives were interacting with a previously identified vimentin receptor at the nuclear envelope, the protein lamin B [Georgatos, S. & Blobel, G. (1987) J. Cell Biol. 105, 117-127]. Additional analysis by affinity chromatography confirmed this conclusion. Employing an overlay assay, we demonstrated that the 50-kDa fragment, but not the 45-kDa desmin peptide, was capable of interacting with the plasma membrane polypeptide ankyrin (a known vimentin attachment site), as was intact vimentin. Conversely, the nuclear envelope protein lamin B was recognized by both fragments but not by a chymotryptic peptide composed solely of the helical rod domain of desmin. These data imply that the lamin B-binding site on desmin resides within the 21 residues following its helical rod domain, whereas the ankyrin-associating region is localized within its N-terminal head domain, exactly as in the case of vimentin.

Animals↗
PubMed1987

Nuclear pore complex contains a family of glycoproteins that includes p62: glycosylation through a previously unidentified cellular pathway.

Using a monoclonal antibody (mAb 414), we previously identified a protein of 62 kDa (p62) that was localized to the nuclear pore complex by immunoelectron microscopy. We also showed that p62 binds specifically to wheat germ agglutinin. Therefore, we proposed that this nuclear pore complex protein might be a member of a recently characterized family of glycoproteins that are labeled by in vitro galactosylation of rat liver nuclei and contain O-linked monosaccharidic GlcNAc residues. In support of this, we now show that incubation with N-acetylglucosaminidase reduces the molecular mass of p62 by approximately 3 kDa because of the removal of terminal GlcNAc residues. Moreover, p62 can be galactosylated in vitro by using UDP-[3H]galactose and galactosyltransferase. We also show that most of the GlcNAc residues are added within 5 min of synthesis, when p62 is soluble and cytosolic. Thus, the addition of GlcNAc is carried out in the cytoplasm and is clearly distinct from the N- and O-linked glycosylation pathways of the endoplasmic reticulum and Golgi complex. Using another mAb with a broad specificity for nuclear GlcNAc-containing proteins, we show by immunofluorescence and protein blotting of subnuclear fractions that some of these proteins are in the interior of the nucleus, and others are most likely located in the pore complex.

Animals↗
PubMed1987

Two distinct attachment sites for vimentin along the plasma membrane and the nuclear envelope in avian erythrocytes: a basis for a vectorial assembly of intermediate filaments.

In vitro binding studies with isolated bovine lens vimentin and avian erythrocyte membranes reveal the existence of two functionally distinct sets of intermediate filament attachment sites. One population of such receptors is located along the nuclear envelope and comprises polypeptides recognizing the carboxy-terminal tail domain of vimentin. Vimentin associates with these nuclear surface receptors in a cooperative manner and forms extensive 10-nm filaments in a concentration-dependent fashion. Conversely, the plasma membrane contains binding sites that interact in a noncooperative, saturable fashion with vimentin, recognizing its amino-terminal head domain. The functional dichotomy of the vimentin-binding sites under in vitro conditions may reflect a vectorial assembly process whereby 10-nm filaments, although structurally apolar, acquire polar features brought about by the differential attachment to specific receptors arranged along the plasma membrane and the nuclear envelope.

Animals↗
PubMed1987

Lamin B constitutes an intermediate filament attachment site at the nuclear envelope.

We found that urea extraction of turkey erythrocyte nuclear envelopes abolished their ability to bind exogenous 125I-vimentin, while, at the same time, it removed the nuclear lamins from the membranes. After purification of the lamins from such urea extracts, a specific binding between isolated vimentin and lamin B, or a lamin A + B hetero-oligomer, was detected by affinity chromatography. Similar analysis revealed that the 6.6-kD vimentin tail piece was involved in this interaction. By other approaches (quantitative immunoprecipitation, rate zonal sedimentation, turbidometric assays) a substoichiometric lamin B-vimentin binding was determined under in vitro conditions. It was also observed that anti-lamin B antibodies but not other sera (anti-lamin A, anti-ankyrin, preimmune) were able to block 70% of the binding of 125I-vimentin to native, vimentin-depleted, nuclear envelopes. These data, which were confirmed by using rat liver nuclear lamins, indicate that intermediate filaments may be anchored directly to the nuclear lamina, providing a continuous network connecting the plasma membrane skeleton with the karyoskeleton of eukaryotic cells.

Animals↗
PubMed1986

Identification and characterization of a nuclear pore complex protein.

We describe studies using a monoclonal antibody that recognizes a 62 kd protein (p62) of rat liver nuclei. This protein remains associated with the nuclear pore complex-lamina fraction resulting from treatment of nuclei with DNAase, RNAase, and nonionic detergent. Immunofluorescence revealed a strikingly punctate pattern of nuclear rim staining. By immunoferritin microscopy, p62 was specifically localized to the pore complex. Thus, pore complexes can be resolved by fluorescence light microscopy. Pulse chase analysis of labeled tissue culture cells showed that p62 is synthesized as a soluble cytoplasmic precursor of 61 kd, which is incorporated into the nuclear fraction with an unusually long t1/2 of about 6 hr. Incorporation is followed by modification that may involve addition of N-acetylglucosamine residues.

Acetylglucosamine↗