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G Blobel

Publications and source records attributed to G Blobel.

At least 91 records · Page 5Linked to original sources
PubMed1995

Nup120p: a yeast nucleoporin required for NPC distribution and mRNA transport.

To extend our understanding of the mechanism by which the nuclear pore complex (NPC) mediates macromolecular transport across the nuclear envelope we have focused on defining the composition and molecular organization of the yeast NPC. Peptide sequence analysis of a polypeptide with a M(r) of approximately 100,000 present in a highly enriched yeast NPC fraction identified a novel yeast nucleoporin we term Nup120p. Nup120p corresponds to the open reading frame (ORF) YKL057c identified by the yeast genome sequencing project. The ORF predicts a protein with a calculated molecular mass of 120.5 kD containing two leucine zipper motifs, a short coiled-coil region and limited primary sequence similarity to Nup133p. Nup120p was localized to the NPC using a protein A-tagged chimera in situ by indirect immunofluorescence microscopy. Deletion of the NUP120 gene caused clustering of NPCs at one side of the nuclear envelope, moderate nucleolar fragmentation and slower cell growth. Transfer of nup120 delta cells to 37 degrees C resulted in the nuclear accumulation of poly(A)+ mRNA, extensive fragmentation of the nucleolus, spindle defects, and cell death.

Amino Acid Sequence↗
PubMed1994

Isolation of components of the chloroplast protein import machinery.

Components of the protein import machinery of the chloroplast were isolated by a procedure in which the import machinery was engaged in vitro with a tagged import substrate under conditions that yielded largely chloroplast envelope-bound import intermediates. Subsequent detergent solubilization of envelope membranes showed that six envelope polypeptides copurified specifically and, apparently, stoichiometrically with the import intermediates. Four of these polypeptides are components of the outer membrane import machinery and are associated with early import intermediates. Two of these polypeptides have been characterized. One is a homolog of the heat shock protein hsp70; the other one is a channel-protein candidate.

Adenosine Triphosphate↗
PubMed1994

Identification of two GTP-binding proteins in the chloroplast protein import machinery.

Two of four proteins that associated with translocation intermediates during protein import across the outer chloroplast envelope membrane were identified as guanosine triphosphate (GTP)-binding proteins. Both proteins are integral membrane proteins of the outer chloroplast membrane, and both are partially exposed on the chloroplast surface where they were accessible to thermolysin digestion. Engagement of the outer membrane's import machinery by an import substrate was inhibited by slowly hydrolyzable or non-hydrolyzable GTP analogs. Thus, these GTP-binding proteins may function in protein import into chloroplasts.

Adenosine Triphosphate↗
PubMed1994

cDNA-derived primary structure of the 25-kDa subunit of canine microsomal signal peptidase complex.

We purified the 25-kDa subunit of the pentameric signal peptidase complex (SPC 25) from canine pancreas microsomes and utilized amino acid sequence data from tryptic fragments for its molecular cloning and sequencing. Its primary structure (calculated molecular mass of 25,831 Da) shows the presence of two hydrophobic domains of which one is likely to serve as a transmembrane segment. Consistent with being the only SPC subunit with at least one intrachain disulfide bond, SPC 25 contains 4 Cys residues. SPC 25 does not contain a cleavable signal peptide, and it is proposed that the transmembrane segment doubles as signal sequence. The primary structure of SPC 25 is unrelated to any of the other three SPC subunits (SPC 22/23, SPC 21, and SPC 18) that have so far been molecularly cloned and sequenced. Neither is it similar to any protein in the data bases, except that it is 95% identical to an open reading frame of an incomplete and randomly isolated human cDNA clone.

Amino Acid Sequence↗
PubMed1994

Colocalization of vertebrate lamin B and lamin B receptor (LBR) in nuclear envelopes and in LBR-induced membrane stacks of the yeast Saccharomyces cerevisiae.

We have expressed human lamin B and the chicken lamin B receptor (LBR), either separately or together, in yeast and have monitored the subcellular location of the expressed proteins by immunofluorescence microscopy, immunoelectron microscopy, and cell fractionation. At the light microscopic level, the heterologous lamin B localized to the yeast nuclear rim and at electron microscopic resolution was found subjacent to the yeast inner nuclear membrane. These data indicate that vertebrate lamin B was correctly targeted in yeast. Expression of the heterologous LBR, either alone or together with the heterologous lamin B, resulted in the formation of membrane stacks primarily adjacent to the nuclear envelope, but also projecting from the nuclear envelope into the cytoplasm or under the plasma membrane. Double immunoelectron microscopy showed colocalization of the heterologous lamin B and LBR in the yeast nuclear envelope and in the LBR-induced membrane stacks. Cell fractionation showed the presence of the heterologous lamin B and LBR in a subnuclear fraction enriched in nuclear envelopes. The heterologous lamin B was extracted at 8 M urea, but not at 4 M urea, thus behaving as a peripheral membrane protein and indistinguishable from assembled lamins. The heterologous LBR was not extracted by 8 M urea, indicating that it was integrated into the membrane. The observed colocalization and cofractionation are consistent with previously reported in vitro binding data and suggest that heterologous lamin B and LBR interact with each other when coexpressed in yeast.

Animals↗
PubMed1994

Purification of a Ran-interacting protein that is required for protein import into the nucleus.

Previously we reported the isolation of two cytosolic fractions (A and B) from Xenopus ovary that are required sequentially to support protein import into the nuclei of digitonin-permeabilized cells. Fraction A is required for recognition of the nuclear localization sequence and targeting to the nuclear envelope, whereas fraction B is required for the subsequent translocation of the bound substrate into the nucleus. The first protein required for fraction B activity to be purified was the small GTPase Ran (ras-related nuclear protein). Here we report the purification of the second (and final) protein required for fraction B activity. By SDS/PAGE, the purified protein appeared as a single band with an apparent molecular mass of 10 kDa, but the native protein fractionated upon gel filtration chromatography with an apparent size of 30 kDa. Peptide sequence analysis indicated that the purified protein was highly homologous to a previously identified human protein of unknown function called placental protein 15 (pp15) and to the predicted protein product of a yeast open reading frame from Saccharomyces cerevisiae.

Amino Acid Sequence↗
PubMed1994

Nup107 is a novel nuclear pore complex protein that contains a leucine zipper.

We have previously described procedures for the isolation of potential nuclear pore complex proteins (nucleoporins or Nups) from rat liver nuclear envelopes and their subsequent subfractionation into those binding to wheat germ agglutinin (WGA) and those that do not. One of these non-WGA-reactive proteins, termed Nup155, was previously molecularly cloned and sequenced and by immunoelectron microscopy shown to be a bona fide nucleoporin. Here we have characterized a second protein of the non-WGA-reactive type and show that it is a Nup as well. Molecular cloning and sequencing revealed that the protein has a calculated molecular mass of 107.2 kDa. It is, therefore, termed Nup107 (for nucleoporin of 107 kDa). Like other Nups that have so far been analyzed, Nup107 contains an abundance of kinase consensus sites. However, the primary structure of Nup107 is unique and not similar to other proteins in the data banks. Its most striking feature is a leucine zipper in its carboxyl-terminal region.

Amino Acid Sequence↗
PubMed1994

The human CAN protein, a putative oncogene product associated with myeloid leukemogenesis, is a nuclear pore complex protein that faces the cytoplasm.

We have carried out partial amino acid sequence analysis of a putative nuclear pore complex protein (nucleoporin) of rat that reacts with wheat germ agglutinin and with the polyspecific monoclonal antibody 414. Surprisingly, these partial amino acid sequence data revealed a high degree of similarity with the human CAN protein, the complete cDNA-derived primary structure of which was reported by Von Lindern et al. [Von Lindern, M., Fornerod, M., van Baal, S., Jaegle, M., de Wit, T., Buijs, A. & Grosveld, G. (1992) Mol. Cell. Biol. 12, 1687-1697]. The CAN protein has been proposed to be a putative oncogene product associated with myeloid leukemogenesis. Its subcellular localization was not established. To confirm that the putative rat nucleoporin is indeed a homolog of the human CAN protein and to determine its subcellular localization, we expressed a 39-kDa internal segment of the 213,790-Da human CAN protein in Escherichia coli and raised monospecific antibodies, which reacted with the putative rat nucleoporin. Immunofluorescence microscopy of HeLa cells gave a punctate nuclear surface staining pattern characteristic of nucleoporins, and immunoelectron microscopy yielded specific decoration of the cytoplasmic side of the nuclear pore complex. This suggests that the protein is part of the short fibers that emanate from the cytoplasmic aspect of the nuclear pore complex. In agreement with previously proposed nomenclature for nucleoporins, we propose the alternative term nup214 (nucleoporin of 214 kDa) for the CAN protein.

Amino Acid Sequence↗
PubMed1994

A G protein involved in nucleocytoplasmic transport: the role of Ran.

Ran is the only known member of the Ras superfamily of small GTP-binding proteins to be localized primarily inside the nucleus. Recently, Ran was unexpectedly identified as one of the soluble factors required for nuclear import. As this protein has also been implicated in RNA export, nuclear import and export may be more closely related than previously thought, with Ran playing a key role in each.

Animals↗
PubMed1994

POM152 is an integral protein of the pore membrane domain of the yeast nuclear envelope.

We have identified a concanavalin A-reactive glycoprotein of 150 kD that coenriches with isolated yeast nuclear pore complexes. Molecular cloning and sequencing of this protein revealed a single canonical transmembrane segment. Epitope tagging and localization by both immunofluorescence and immunoelectron microscopy confirmed that it is a pore membrane protein. The protein was termed POM152 (for pore membrane protein of 152 kD) on the basis of its location and cDNA-deduced molecular mass. POM152 is likely to be a type II membrane protein with its NH2-terminal region (175 residues) and its COOH-terminal region (1,142 residues) positioned on the pore side and cisternal side of the pore membrane, respectively. The proposed cisternally exposed domain contains eight repetitive motifs of approximately 24 residues. Surprisingly, POM152 deletion mutants were viable and their growth rate was indistinguishable from that of wild-type cells at temperatures between 17 and 37 degrees C. However, overproduction of POM152 inhibited cell growth. When expressed in mouse 3T3 cells, POM152 was found to be localized to the pore membrane, suggesting a conserved sorting pathway between yeast and mammals.

3T3 Cells↗
PubMed1994

NUP145 encodes a novel yeast glycine-leucine-phenylalanine-glycine (GLFG) nucleoporin required for nuclear envelope structure.

We have isolated and characterized the gene encoding a fourth yeast glycine-leucine-phenylalanine-glycine (GLFG) repeat nucleoporin with a calculated molecular mass of 145.3 kD, and therefore termed NUP145. The amino-terminal half of Nup145p is similar to two previously identified GLFG nucleoporins, Nup116p and Nup100p (Wente, S. R., M. P. Rout, and G. Blobel. 1992. J. Cell Biol. 119:705-723). A deletion/disruption in the amino-terminal half of NUP145 (nup145 delta N) had only a slight effect on cell growth at temperatures between 17 and 37 degrees C. However, immunofluorescence microscopy of nup145 delta N cells with antinucleoporin antibodies showed that the characteristic punctate nuclear staining normally seen in wild-type yeast cells was reduced, with the majority of the signal located in one or two intense spots at the nuclear periphery. Thin section electron microscopy analysis revealed the presence of what appeared to be successive herniations of the nuclear envelope forming grape-like structures at primarily one site on the nup145 delta N nuclei. These successive herniations contained numerous NPC-like structures, correlating to the limited bright patches of anti-nucleoporin immunofluorescence signal. In some cases the successive herniations were small. Occasionally, however, multi-lobulated nuclei were seen. We suggest that the ultrastructural phenotype of nup145 delta N cells is due to a defective interaction of nup145 delta N NPCs with the surrounding pore membrane domain of the nuclear envelope. We have also analyzed the synthetic lethal phenotypes among GLFG nucleoporin mutant alleles, and found that strains harboring nup116 and either nup100 or nup145 mutations were not viable. This, in combination with the morphological analysis, may reflect overlapping yet distinct roles for these three GLFG nucleoporins in NPC-nuclear envelope interactions.

Amino Acid Sequence↗
PubMed1994

NAP57, a mammalian nucleolar protein with a putative homolog in yeast and bacteria.

We report the identification and molecular characterization of a novel nucleolar protein of rat liver. As shown by coimmunoprecipitation this protein is associated with a previously identified nucleolar protein, Nopp140, in an apparently stoichiometric complex and has therefore been termed NAP57 (Nopp140-associated protein of 57 kD). Immunofluorescence and immunogold electron microscopy with NAP57 specific antibodies show colocalization with Nopp140 to the dense fibrillar component of the nucleolus, to coiled bodies, and to the nucleoplasm. Immunogold staining in the nucleoplasm is occasionally seen in the form of curvilinear tracks between the nucleolus and the nuclear envelope, similar to those previously reported for Nopp140. These data suggest that Nopp140 and NAP57 are indeed associated with each other in these nuclear structures. The cDNA deduced primary structure of NAP57 shows a protein of a calculated molecular mass of 52,070 that contains a putative nuclear localization signal near its amino and carboxy terminus and a hydrophobic amino acid repeat motif extending across 84 residues. Like Nopp140, NAP57 lacks any of the known consensus sequences for RNA binding which are characteristic for many nucleolar proteins. Data bank searches revealed that NAP57 is a highly conserved protein. A putative yeast (S. cerevisiae) homolog is 71% identical. Most strikingly, there also appears to be a smaller prokaryotic (E. coli and B. subtilis) homolog that is nearly 50% identical to NAP57. This indicates that NAP57 and its putative homologs might serve a highly conserved function in both pro- and eukaryotes such as chaperoning of ribosomal proteins and/or of preribosome assembly.

Amino Acid Sequence↗
PubMed1993

The GTP-binding protein Ran/TC4 is required for protein import into the nucleus.

Two cytosolic fractions (A and B) from Xenopus oocytes are sufficient to support protein import into the nuclei of digitonin-permeabilized cells. Fraction A recognizes the nuclear localization sequence (NLS) and binds the import substrate to the nuclear envelope, whereas fraction B mediates the subsequent passage of the bound substrate into the nucleus. Here we report that two interacting components are required for full fraction-B activity, purify one of these components to homogeneity, and show that it is the highly abundant GTP-binding protein Ran (Ras-related nuclear protein)/TC4.

Amino Acid Sequence↗
PubMed1993

SecA protein is required for translocation of a model precursor protein into inverted vesicles of Escherichia coli plasma membrane.

We have investigated whether the SecA protein is required for in vitro translocation of a model presecretory protein into inverted vesicles (INV) of the Escherichia coli plasma membrane. Contrary to previous reports, we found that urea-extracted INV that contained only the membrane-integral form of SecA were fully translocation active. Proteoliposomes that were reconstituted from a detergent extract of INV did contain a full complement of membrane-integral SecA but < 1% of SecY. These proteoliposomes were fully translocation active. However, immunodepletion of > 90% of the SecA from the detergent extract yielded proteoliposomes that were translocation inactive. Addition of purified SecA to the SecA-depleted proteoliposomes restored translocation. The amounts of SecA required to saturate translocation activity were equivalent to those present as membrane-integral SecA in INV. These data indicate that SecA is necessary for protein translocation, and reinforce our previous conclusion that SecY is not required. Contrary to previous reports, we find that membrane-integral SecA is not irreversibly inactivated by 6 M urea and that membrane-integral SecA and SecY do not form a stoichiometric protein complex in the membrane.

Adenosine Triphosphatases↗
PubMed1993

Molecular and biochemical characterization of a recombinant human PKC-delta family member.

Two cDNA clones coding for the human protein kinase C-delta (PKC-delta) were fortuitously isolated during the process of screening a human library for a cDNA clone of an unrelated protein, the nucleolar protein fibrillarin. The two human homologues have about 88% nucleotide sequence identity to the rat and mouse PKC-delta cDNA clones. A comparison of the predicted amino acid sequences of the two human PKC-delta clones with the rat and mouse homologues indicated a greater degree of sequence divergence (89-90% homology) compared to the high degree of sequence conservation observed with other human PKC family members and their mammalian counterparts. Expression of the clones in the baculovirus insect-cell expression system indicated that both proteins exhibited phorbol ester binding activity, and were dependent upon phosphatidylserine and diacylglycerol for maximal activation. Further characterization of the properties of the human PKC-delta revealed substrate and lipid dependencies distinct from other members of the protein kinase C family; including PKC-deltas isolated from other species. The dissimilarities in the predicted amino acid sequences between the human and other mammalian species could account in part for some of these observed biochemical differences.

Amino Acid Sequence↗
PubMed1993

Lumenal proteins of the mammalian endoplasmic reticulum are required to complete protein translocation.

The role of the lumenal contents (reticuloplasm) of the endoplasmic reticulum (ER) in protein translocation was determined by in vitro analysis. Depletion of the reticuloplasm from mammalian rough microsomes revealed two distinct stages of the translocation reaction. The first stage, translocation up to and including signal peptide cleavage, was insensitive to the loss of the reticuloplasm, whereas the second stage, net transfer of the nascent chain into the ER lumen, was reticuloplasm dependent. In reticuloplasm-depleted membranes, signal-cleaved and glycosylated translocation intermediates were observed to transit free from the translocation channel to the cis, or cytoplasmic, side of the membrane. This translocation defect was complemented by reconstitution of lumenal proteins into depleted membranes. We propose that lumenal proteins are necessary for unidirectional protein translocation in mammalian ER.

Animals↗