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P Walter

Publications and source records attributed to P Walter.

At least 271 records · Page 15Linked to original sources
PubMed1988

Heavy-chain binding protein recognizes aberrant polypeptides translocated in vitro.

Immunoglobulin heavy-chain binding protein (BiP, GRP-78) associates tightly in the endoplasmic reticulum (ER) with newly synthesized proteins that are incompletely assembled, have mutant structures, or are incorrectly glycosylated. The function of BiP has been suggested to be to prevent secretion of incorrectly folded or incompletely assembled protein, to promote folding or assembly of proteins, or to solubilize protein aggregates within the ER lumen. Here we examine the interaction of BiP with newly synthesized polypeptides in an in vitro protein translation-translocation system. We find that BiP forms tight complexes with nonglycosylated yeast invertase and incorrectly disulphide-bonded prolactin, but does not associate detectably with either glycosylated invertase or correctly disulphide-bonded prolactin. Moreover, BiP associates detectably only with completed chains of prolactin, not with chains undergoing synthesis. We conclude that BiP recognizes and binds with high affinity in vitro to aberrantly folded or aberrantly glycosylated polypeptides, but not to all nascent chains as they are folding.

Carrier Proteins↗
PubMed1988

Each of the activities of signal recognition particle (SRP) is contained within a distinct domain: analysis of biochemical mutants of SRP.

Signal recognition particle (SRP), a small ribonucleoprotein required for targeting secretory proteins to the ER, has three known functions: signal recognition, elongation arrest, and translocation promotion. Because SRP is inactivated by the sulfhydryl alkylating reagent N-ethylmaleimide (NEM), we have attempted to establish structure-function relationships within SRP by assembling particles in which a single protein is modified. Alkylation of the 68/72 kd protein of SRP yields a particle that arrests elongation but fails to promote translocation and no longer interacts with SRP receptor. Alkylation of the 54 kd protein yields a particle that fails to recognize signal sequences. This approach has allowed us to map activities to specific protein domains on SRP, and should be generally useful for analyzing other ribonucleoproteins.

Animals↗
PubMed1988

The yeast VAS1 gene encodes both mitochondrial and cytoplasmic valyl-tRNA synthetases.

S1 mapping on the VAS1 structural gene indicates the existence of two classes of transcripts initiating at distinct in-frame translation start codons. The longer class of VAS1 transcripts initiates upstream of both ATG codons located 138 base pairs away and the shorter class downstream of the first ATG. A mutation that destroys the first AUG on the long message results in respiratory deficiency but does not affect viability. Mutation of the ATG at position 139 leads to lethality because the initiating methionine codon of the essential cytoplasmic valyl-tRNA synthetase has been destroyed. N-terminal protein sequence data further confirm translation initiation at ATG-139 for the cytoplasmic valyl-tRNA synthetase. From these results, we conclude that the VAS1 single gene encodes both mitochondrial and cytoplasmic valyl-tRNA synthetases. The presequence of the mitochondrial valyl-tRNA synthetase shows amino acid composition but not the amphiphilic character of imported mitochondrial proteins. From mutagenesis of the ATG-139 we conclude that the presequence specifically targets the cytoplasmically synthesized mitochondrial valyl-tRNA synthetase to the mitochondrial outer membrane and prevents binding of the enzyme core to cytoplasmic tRNAVal.

Amino Acid Sequence↗
PubMed1988

The affinity of signal recognition particle for presecretory proteins is dependent on nascent chain length.

We have developed an assay in which incomplete preprolactin chains of varying lengths are targeted to the endoplasmic reticulum (ER) membrane in an elongation independent manner. The reaction had the same molecular requirements as nascent chain translocation across the ER membrane, namely, it was signal recognition particle (SRP) dependent, and required the nascent chain to be present as peptidyl tRNA (i.e. most likely ribosome associated) and to have its signal sequence exposed outside the ribosome. We found that the efficiency of the targeting reaction dropped dramatically as the chains grew longer than 140 amino acids in length, which probably reflected a decrease in affinity of the nascent chain-ribosome complex for SRP. Thus at physiological SRP concentrations (10 nM) there appears a sharp cut-off point in the ability of these chains to be targeted, while at high SRP concentrations (270 nM) all chains could be targeted. In kinetic experiments, high concentrations of SRP were found to change the time in elongation after which translocation of the nascent polypeptide could no longer occur.

Animals↗
PubMed1988

Ribosome pausing and stacking during translation of a eukaryotic mRNA.

We have devised a sensitive assay to determine the distribution of translating ribosomes on a mRNA. Using this assay to monitor ribosome transit on bovine preprolactin mRNA, we have detected four major positions of ribosome pausing in both wheat-germ and rabbit reticulocyte extracts. Two of these rate-limiting steps represent initiation and termination. One pause occurs after approximately 75 amino acids have been polymerized; signal recognition particle arrests preprolactin synthesis at this position. The other internal pause occurs at 160 amino acids. In these latter two cases ribosomes stop at a GGC glycine codon; however, two other GGC codons are translated without apparent pausing. Surprisingly, we find that up to nine ribosomes are tightly stacked behind each pausing ribosome, such that the ribosome centers are only 27-29 nucleotides apart. The assay should prove useful for probing mechanisms of translational regulation.

Animals↗
PubMed1988

Small ribonucleoproteins in Schizosaccharomyces pombe and Yarrowia lipolytica homologous to signal recognition particle.

We have partially purified ribonucleoproteins (RNPs) from Schizosaccharomyces pombe and Yarrowia lipolytica with properties resembling those of mammalian signal recognition particle (SRP). In both species of yeast we have identified a single major RNA species in the size range of SRP RNA (256 nucleotides in S. pombe and 270 nucleotides in Y. lipolytica) present in postribosomal salt extracts of the cytoplasm. The RNPs containing these RNAs sediment in sucrose gradients at 11 S and 10 S for S. pombe and Y. lipolytica, respectively. Analysis of genomic clones of these RNAs has revealed that (i) they are encoded by single copy genes; (ii) they share two short conserved sequences that match the A and B boxes defined for polymerase III promoters; (iii) they can be folded into secondary structures that closely match that defined by phylogenetic analysis of higher eukaryotic SRP RNAs; and (iv) they show primary sequence conservation in short regions predicted to be single stranded. Both of the yeast RNAs bind under stringent conditions to canine SRP proteins. Most importantly, RNase protection of the S. pombe RNA by the individual canine SRP proteins, p19 and p68/72, shows that the proteins recognize homologous elements of the mammalian and yeast RNA. Taken together these data suggest strongly that we have identified yeast SRP homologues.

Animals↗
PubMed1988

Binding sites of the 19-kDa and 68/72-kDa signal recognition particle (SRP) proteins on SRP RNA as determined in protein-RNA "footprinting".

We have used the nuclease alpha-sarcin to map the binding sites of the 19-kDa and the 68/72-kDa proteins of signal recognition particle (SRP) on SRP RNA. We found that the regions of protection to nuclease afforded by the two proteins were distinct. p19 protected primarily the two tips in the RNA secondary structure. p68/72 protected a large region extending across the center of the particle and altered the nuclease pattern in the regions that p19 would bind, suggesting that these two proteins may be in close proximity in the particle. The protection afforded by the two proteins in combination was equal to the sum of the individual protections. We have not observed cooperativity in the binding of these two proteins as assessed by the protection assay; nor do we have any evidence that the structure becomes more compact as it assembles. The map derived from this "footprint" analysis places the signal recognition domain (p54 bound to the RNA via the 19-kDa protein) and the elongation arrest domain (associated with the Alu end of the particle) on opposite ends of the particle. Thus, it is possible that SRP recognizes signals by the direct interaction of p54 with the signal sequence at the nascent chain exit site and simultaneously blocks elongation by the entrance of p9/14 into the aminoacyl tRNA site 16 nm away.

Animals↗
PubMed1988

Full-length prepro-alpha-factor can be translocated across the mammalian microsomal membrane only if translation has not terminated.

We have previously shown that fully synthesized prepro-alpha-factor (pp alpha F), the precursor for the yeast pheromone alpha-factor, can be translocated posttranslationally across yeast rough microsomal (RM) membranes from a soluble, ribosome-free pool. We show here that this is not the case for translocation of pp alpha F across mammalian RM. Rather we found that a small amount of translocation of full-length pp alpha F is observed, but is solely due to polypeptide chains that were still ribosome bound and covalently attached to tRNA, i.e., not terminated. In addition, both signal recognition particle (SRP) and SRP receptor are required, i.e., the same targeting machinery that is normally responsible for the coupling between protein synthesis and translocation. Thus, the molecular requirements for targeting are distinct from posttranslational translocation across yeast RM. As termination is generally regarded as part of translation, the translocation of full-length pp alpha F across mammalian RM does not occur "posttranslationally," albeit independent of elongation. Most other proteins for which posttranslational translocation across mammalian RM was previously claimed fall into the same category in that ribosome attachment as peptidyl-tRNA is required. To clearly separate these two distinct processes, we suggest that the term posttranslational be reserved for those processes that occur in the complete absence of the translational machinery. We propose the term "ribosome-coupled translocation" for the events described here.

Animals↗
PubMed1988

Prepro-carboxypeptidase Y and a truncated form of pre-invertase, but not full-length pre-invertase, can be posttranslationally translocated across microsomal vesicle membranes from Saccharomyces cerevisiae.

We have determined that prepro-carboxypeptidase Y and a truncated form of pre-invertase can be translocated across the yeast microsomal membrane post-translationally in a homologous in vitro system. The yeast secretory protein prepro-alpha-factor which was previously shown to be an efficient posttranslational translocation substrate is therefore not unique in this regard, but rather the yeast ER protein translocation machinery is generally capable of accepting substrates from a ribosome-free, soluble pool. However, within our detection limits, full-length pre-invertase could not be translocated posttranslationally, but was translocated co-translationally. This indicates that not every fully synthesized pre-protein can use this pathway, presumably because normal or aberrant folding characteristics can interfere with translocation competence.

Carboxypeptidases↗
PubMed1988

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.

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.

Cytoplasm↗
PubMed1988

Application of radioimmunoassay to monitor treatment of human cerebral gliomas with bleomycin entrapped within liposomes.

A radioimmunoassay was assessed for its suitability for monitoring the blood and urinary concentrations of bleomycin entrapped within large unilamellar liposomes in patients receiving therapeutic doses of the drug. Bleomycin entrapped within liposomes was administered by direct intracerebral injection to three patients with grade III to IV cerebral gliomas, twice weekly for up to six weeks. No clinically important toxic effects were observed which could have been attributed to these preparations, although all three patients showed progressive deterioration in their clinical condition. This type of treatment may be more beneficial to patients at an earlier stage of their disease.

Adolescent↗
PubMed1988

[Neonatal carcinoma. Review of the literature apropos of a case].

A cortical adrenal carcinoma arising during the neonatal period and associated with virilization recurred after an initial excision and is in complete remission after a follow-up period of 10 years. The authors have performed a review of the medical literature which has yielded 56 neonatal carcinomas including this one. More than half of these cases concerned the thyroid gland (15 cases) and the adrenal cortex (14 cases). These were followed by malignant melanomas (12 cases), carcinomas of the parotid gland (6 cases), oropharyngeal region (3 cases), adrenal medulla (2 cases) and stomach, liver, breast and cutaneous sweat gland (one case each). Six tumors were associated with congenital malformations which included Beckwith-Wiedeman's syndrome; 4 tumors arose from preexisting tissue lesions; in one case, oral contraceptives were taken during pregnancy. The main treatment of neonatal carcinomas is surgery. Local recurrences (7 cases) do not markedly affect the outcome, whereas metastases (12 cases) have a very unfavorable prognosis. Superficial tumors are diagnosed earlier and more frequently cured. Out of 44 cases for which the outcome is known, only 18 children survived.

Abnormalities, Multiple↗
PubMed1987

Wild type and mutant signal peptides of Escherichia coli outer membrane lipoprotein interact with equal efficiency with mammalian signal recognition particle.

The signal peptide of the outer membrane lipoprotein (OMLP) of Escherichia coli was shown to be capable of promoting protein translocation across mammalian microsomal membranes in vitro. We assayed translocation of a fusion protein containing the OMLP signal peptide and nine amino acids of OMLP fused in frame to beta-lactamase. The efficiency with which the mammalian translocation machinery recognizes and accepts the OMLP signal peptide as substrate is indistinguishable from that of mammalian secretory proteins. Upon translocation mammalian signal peptidase processes the pre-OMLP-beta-lactamase protein at different sites than are utilized in vivo by E. coli OMLP signal peptidase (signal peptidase II) but that can be predicted as mammalian signal peptidase cleavage sites. Mutants in the OMLP signal peptide were tested for their ability to promote translocation of the fusion protein in this assay system. It has been shown previously that mutants in the positively charged amino acids at the amino terminus of the signal peptide severely delay the translocation of OMLP in vivo in E. coli. However, these mutants had no detectable effect either on signal recognition by mammalian signal recognition particle or on the efficiency of translocation itself.

Amino Acid Sequence↗
PubMed1987

Mathematical modeling of the effects of the signal recognition particle on translation and translocation of proteins across the endoplasmic reticulum membrane.

The kinetics of the signal recognition particle(SRP)-mediated process of protein translocation across the endoplasmic reticulum membrane was studied by mathematical modeling and complementary experiments. The following results were obtained. (1) A model according to which SRP directs the ribosome, rather than the mRNA, to the membrane is supported by experiments designed to discriminate between the two alternatives. (2) This model describes both steady-state and synchronized translation experiments and makes a number of predictions. (3) The interaction between a nascent protein and SRP may be described by two parameters: (i) a binding constant which can be attributed to the structure of the signal peptide, and (ii) the size of the "SRP-window", i.e. the distance between the first and the last site on the polypeptide chain that can interact with SRP. For preprolactin a binding constant of 1 to 2.5 nmol-1l was estimated. Modeling of the synchronized synthesis of ovalbumin indicates that it has a much weaker binding constant than preprolactin (approximately 0.25 nmol-1l) although we cannot exclude the possibility that the SRP-window may be also smaller. (4) A better understanding of the molecular effects of SRP on translation and translocation through the rough endoplasmic reticulum membrane has been achieved. Inhibition of the steady-state rate of translation by SRP requires a stoichiometric interaction of SRP with ribosomes carrying nascent polypeptide chains and will occur only when ribosomes are piled up back to the initiation site. Translocation, on the other hand, requires only the catalytic action of SRP and is determined by the local concentration of protein-synthesizing ribosomes accumulated at the site(s) of SRP interaction. As a consequence, translational inhibition by SRP may sometimes fail to occur, depending either on the type of protein or on experimental conditions, such as a high mRNA concentration, even if translocation can be demonstrated. (5) A rough extrapolation to the conditions in vivo indicates that all synthesized polypeptide chains destined for translocation across or integration into the endoplasmic reticulum membrane are indeed quantitatively translocated and that no translational inhibition occurs.

Endoplasmic Reticulum↗