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P Walter

Publications and source records attributed to P Walter.

At least 253 records · Page 14Linked to original sources
PubMed1989

Deletion analysis in the amino-terminal extension of methionyl-tRNA synthetase from Saccharomyces cerevisiae shows that a small region is important for the activity and stability of the enzyme.

MESI, the structural gene for methionyl-tRNA synthetase from Saccharomyces cerevisiae encodes an amino-terminal extension of 193 amino acids, based on the comparison of the encoded protein with the Escherichia coli methionyl-tRNA synthetase. We examined the contribution of this polypeptide region to the activity of the enzyme by creating several internal deletions in MESI which preserve the correct reading frame. The results show that 185 amino acids are dispensable for activity and stability. Removal of the next 5 residues affects the activity of the enzyme. The effect is more pronounced on the tRNA aminoacylation step than on the adenylate formation step. The Km for ATP and methionine are unaltered indicating that the global structure of the enzyme is maintained. The Km for tRNA increased slightly by a factor of 3 which indicates that the positioning of the tRNA on the surface of the molecule is not affected. There is, however, a great effect on the Vmax of the enzyme. Examination of the three-dimension structure of the homologous E. coli methionyl-tRNA synthetase indicates that the amino acid region preceding the mononucleotide-binding fold does not participate directly in the catalytic cleft. It could, however, act at a distance by propagating a mutational alteration to the catalytic residues.

Amino Acid Sequence↗
PubMed1989

Model for signal sequence recognition from amino-acid sequence of 54K subunit of signal recognition particle.

Protein targeting to the endoplasmic reticulum in mammalian cells is catalysed by signal recognition particle (SRP). Cross-linking experiments have shown that the subunit of relative molecular mass 54,000 (Mr 54K; SRP54) interacts directly with signal sequences as they emerge from the ribosome. Here we present the sequence of a complementary DNA clone of SRP54 which predicts a protein that contains a putative GTP-binding domain and an unusually methionine-rich domain. The properties of this latter domain suggest that it contains the signal sequence binding site. A previously uncharacterized Escherichia coli protein has strong homology to both domains. Closely homologous GTP-binding domains are also found in the alpha-subunit of the SRP receptor (SR alpha, docking protein) in the endoplasmic reticulum membrane and in a second E. coli protein, ftsY, which resembles SR alpha. Recent work has shown that SR alpha is a GTP-binding protein and that GTP is required for the release of SRP from the signal sequence and the ribosome on targeting to the endoplasmic reticulum membrane. We propose that SRP54 and SR alpha use GTP in sequential steps of the targeting reaction and that essential features of such a pathway are conserved from bacteria to mammals.

Amino Acid Sequence↗
PubMed1989

Binding of ADP to rat liver cytosolic proteins and its influence on the ratio of free ATP/free ADP.

In a cytosolic extract from rat liver, the number and the concentration of ADP-binding sites as well as their dissociation constants were determined by using the rate-of-dialysis technique. Interfering cytosolic adenylate kinase was extracted from the cytosol by affinity chromatography on Ap5A-agarose, and remaining traces of enzyme activity were inhibited with (+)-catechin. Binding of ADP to cytosolic proteins was increased by poly(ethylene glycol) and decreased by EDTA. The effect of 0.1 mM-EDTA could be reversed by addition of equimolar concentrations of Mn2+ or Mg2+. In presence of 5% poly(ethylene glycol), added to increase local protein concentration, two binding sites for ADP were observed, with KD values of 1.9 microM (site I) and 10.8 microM (site II). The concentration of these binding sites, when extrapolated to cellular protein concentrations, were 30 microM (site I) and 114 microM (site II). It is concluded that a minimum of about 50% of total cytosolic ADP is bound to proteins, and that the ratio of free ATP/free ADP is at least twice that of total ATP/total ADP.

Adenosine Diphosphate↗
PubMed1989

Molecular sorting in the secretory pathway.

Proteins can be secreted from animal cells by either a constitutive or a regulated pathway; those destined for regulated secretion are actively sorted into dense-core secretory granules. Although sorting is generally assumed to be accomplished by specific carriers, the nature of these carriers remains elusive. In this study, peptide hormones were used as affinity ligands to purify a set of 25-kilodalton proteins from canine pancreatic tissue. Their ligand specificities and patterns of expression have the characteristics of sorting carriers.

Animals↗
PubMed1989

Isolation of a cDNA clone of the 14-kDa subunit of the signal recognition particle by cross-hybridization of differently primed polymerase chain reactions.

Using an enhancement of the polymerase chain reaction (PCR) technique, we have isolated a complementary DNA encoding SRP14 (14-kDa subunit), one of six proteins contained in the signal recognition particle (SRP). Several pools of degenerate oligonucleotides encoding different peptide sequences of SRP14 were used to generate amplified DNA by the PCR. A cross-hybridization procedure was developed to identify the authentic SRP14 cDNA clone among the amplified DNA products obtained by PCR. The basis of this approach is the assumption that a partial cDNA of SRP14 should be the only DNA product common to two amplification reactions primed with different degenerate oligonucleotide mixtures. The partial canine cDNA of SRP14 identified by this procedure served as a probe to isolate a complete cDNA clone of SRP14 from a mouse embryonic cDNA library in lambda phage gt10.

Amino Acid Sequence↗
PubMed1989

Evidence for a two-step mechanism involved in assembly of functional signal recognition particle receptor.

The signal recognition particle (SRP) and SRP receptor act sequentially to target nascent secretory proteins to the membrane of the ER. The SRP receptor consists of two subunits, SR alpha and SR beta, both tightly associated with the ER membrane. To examine the biogenesis of the SRP receptor we have developed a cell-free assay system that reconstitutes SR alpha membrane assembly and permits both anchoring and functional properties to be assayed independently. Our experiments reveal a mechanism involving at least two distinct steps, targeting to the ER and anchoring of the targeted molecule on the cytoplasmic face of the membrane. Both steps can be reconstituted in vitro to restore translocation activity to ER microsomes inactivated by alkylation with N-ethyl-maleimide. The characteristics elucidated for this pathway distinguish it from SRP-dependent targeting of secretory proteins, SRP-independent ER translocation of proteins such as prepromellitin, and direct insertion mechanisms of the type exemplified by cytochrome b5.

Endoplasmic Reticulum↗
PubMed1989

Protein translocation across the endoplasmic reticulum membrane: identification by photocross-linking of a 39-kD integral membrane glycoprotein as part of a putative translocation tunnel.

The molecular environment of secretory proteins during translocation across the ER membrane was examined by photocross-linking. Nascent preprolactin chains of various lengths, synthesized by in vitro translation of truncated messenger RNAs in the presence of N epsilon-(5-azido-2-nitrobenzoyl)-Lys-tRNA, signal recognition particle, and microsomal membranes, were used to position photoreactive probes at various locations within the membrane. Upon photolysis, each nascent chain species was cross-linked to an integral membrane glycoprotein with a deduced mass of 39 kD (mp39) via photoreactive lysines located in either the signal sequence or the mature prolactin sequence. Thus, different portions of the nascent preprolactin chain are in close proximity to the same membrane protein during the course of translocation, and mp39 therefore appears to be part of the translocon, the specific site of protein translocation across the ER membrane. The similarity of the molecular and cross-linking properties of mp39 and the glyco-protein previously identified as a signal sequence receptor (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature [Lond.]. 328: 830-833) suggests that these two proteins may be identical. Our data indicate, however, that mp39 does not (or not only) function as a signal sequence receptor, but rather may be part of a putative translocation tunnel.

Cross-Linking Reagents↗
PubMed1989

Signal recognition particle mediates a transient elongation arrest of preprolactin in reticulocyte lysate.

Signal recognition particle (SRP) is a ribonucleoprotein that functions in the targeting of ribosomes synthesizing presecretory proteins to the ER. SRP binds to the signal sequence as it emerges from the ribosome, and in wheat germ extracts, arrests further elongation. The translation arrest is released when SRP interacts with its receptor on the ER membrane. We show that the delay of elongation mediated by SRP is not unique to wheat germ translation extracts. Addition of mammalian SRP to reticulocyte lysates resulted in a delay of preprolactin synthesis due to increased ribosome pausing at specific sites on preprolactin mRNA. Addition of canine pancreatic microsomal membranes to reticulocyte lysates resulted in an acceleration of preprolactin synthesis, suggesting that the endogenous SRP present in the reticulocyte lysate also delays synthesis of secretory proteins.

Animals↗
PubMed1989

Saccharomyces cerevisiae and Schizosaccharomyces pombe contain a homologue to the 54-kD subunit of the signal recognition particle that in S. cerevisiae is essential for growth.

We have isolated and sequenced genes from Saccharomyces cerevisiae (SRP54SC) and Schizosaccharomyces pombe (SRP54sp) encoding proteins homologous to both the 54-kD protein subunit (SRP54mam) of the mammalian signal recognition particle (SRP) and the product of a gene of unknown function in Escherichia coli, ffh (Römisch, K., J. Webb, J. Herz, S. Prehn, R. Frank, M. Vingron, and B. Dobberstein. 1989. Nature (Lond.). 340:478-482; Bernstein H. D., M. A. Poritz, K. Strub, P. J. Hoben, S. Brenner, P. Walter. 1989. Nature (Lond.). 340:482-486). To accomplish this we took advantage of short stretches of conserved sequence between ffh and SRP54mam and used the polymerase chain reaction (PCR) to amplify fragments of the homologous yeast genes. The DNA sequences predict proteins for SRP54sc and SRP54sp that are 47% and 52% identical to SRP54mam, respectively. Like SRP54mam and ffh, both predicted yeast proteins contain a GTP binding consensus sequence in their NH2-terminal half (G-domain), and methionine-rich sequences in their COOH-terminal half (M-domain). In contrast to SRP54mam and ffh the yeast proteins contain additional Met-rich sequences inserted at the COOH-terminal portion of the M-domain. SRP54sp contains a 480-nucleotide intron located 78 nucleotides from the 5' end of the open reading frame. Although the function of the yeast homologues is unknown, gene disruption experiments in S. cerevisiae show that the gene is essential for growth. The identification of SRP54sc and SRP54sp provides the first evidence for SRP related proteins in yeast.

Amino Acid Sequence↗
PubMed1989

Cassette mutagenic analysis of the yeast invertase signal peptide: effects on protein translocation.

The coding sequence of the SUC2 locus was placed under the control of the constitutive ADH1 promoter and transcription terminator in a centromere-based yeast plasmid vector from which invertase is expressed in a Suc- strain of Saccharomyces cerevisiae. Mutants in the signal peptide sequence were produced by replacing this region of the gene with synthetic oligonucleotide cassettes containing mixtures of nucleotides at several positions. The mutants could be divided into three classes on the basis of the ability to secrete invertase. Class I mutants produced secreted invertase but in reduced amount. The class II mutant, 4-55B, also exhibited reduced a level of invertase, but a significant fraction of the enzyme was intracellular. Class III mutants were partially defective in translocation from the cytoplasm to the endoplasmic reticulum and produced enzymatically active, unglycosylated preinvertase in the cytoplasm. Class III mutant preinvertases were also defective in translocation across canine pancreas microsomes. These results suggested that the reduced level of invertase resulted from proteolytic degradation of inefficiently transported intermediates. Comparison of the sequences of the mutant signal peptides indicated that amino acids at the extreme amino terminus and adjacent to the cleavage site play a crucial role in the secretory process when combined with a mutation within the hydrophobic core.

Amino Acid Sequence↗
PubMed1989

The use of gene-fusions to determine membrane protein topology in Saccharomyces cerevisiae.

We have used protein-fusions to study in Saccharomyces cerevisiae the topology and integration of arginine permease. Since this membrane protein does not contain a cleavable signal sequence, we sought to identify the first internal signal by, initially, fusing the cytoplasmic enzyme, galactokinase, to various positions along the amino-terminal region, and then measuring in vitro the translocation of galactokinase across the membrane of the endoplasmic reticulum. Five fusion proteins were examined that contained, progressively, zero to four hydrophobic segments. The galactokinase moiety of fusion 5, but not fusions 1-4, was translocated. Fusion 4 differed from 5 by only the fourth hydrophobic segment, indicated that this region contains the first internal signal. From this we conclude that hydrophobic segment IV spans the membrane, and that the hydrophilic domain amino-terminal to it lies on the cytoplasmic side of the membrane. If any of the first three segments actually span the membrane, then they probably integrate by a mechanism that differs from segment IV.

Amino Acid Transport Systems↗
PubMed1989

Angiogenesis and hemodynamics of microvasculature of transplanted islets of Langerhans.

Transplantation of isolated islets of Langerhans is frequently followed by early loss of islet function. Because whether this is caused by insufficient vascularization or graft rejection is unknown, angiogenesis and microvascularization of islet grafts were studied in vivo by means of intravital microscopy. After transplantation of syngeneic islets in hamster dorsal skin-fold chambers, 97% (n = 66) of the islets exhibited the first signs of angiogenesis at days 2-4, characterized by sinusoidal sacculations and capillary sprouts. After 10 days, angiogenesis was completed, consisting of a microvascular network similar to those of islets in situ: arterial supply, afferent and efferent capillary loops, and venular drainage. Functional density of microvessels was 700.1 +/- 127.0 cm-1, and erythrocyte velocity was 0.58 +/- 0.35 mm/s. Intracellular insulin was demonstrated immunohistochemically. Electron-microscopic studies revealed normal fine structure of the capillary wall. The model allows in vivo analysis of microvascular phenomena occurring in host-vs.-graft reaction after allogeneic and xenogeneic islet transplantation. Furthermore, it may be used to quantitatively assess immunosuppressive regimens.

Animals↗
PubMed1989

[The significance of vascular surgery treatment of complications following percutaneous transluminal angioplasty of the lower extremities].

In 147 patients suffering from arterial occlusive disease of the lower extremities 162 PTAs of the iliac, femoral and popliteal arteries were performed. Thirteen complications occurred in 12 patients, while surgical intervention was necessary in seven cases (4.3%). Hematoma at the site of the arterial puncture were the most frequent complications, requiring acute surgical intervention (n = 3). In two patients, acute vascular occlusion after PTA required bypass operation and thrombendarteriectomy, respectively. In one patient, presenting with an iliac stenosis, PTA was performed using a transaxillar approach and was complicated by an axillar hematoma due to the ruptur of the arteria subscapularis. Furthermore, one patient with an extended hematoma of the abdominal wall required surgical intervention. The results presented indicate that PTA of the lower extremities for treatment of arterial occlusive disease is a procedure with low risk. Complications, which require acute operative therapy, can be treated mostly successful by small surgical interventions.

Angioplasty, Balloon↗
PubMed1988

Isolation and characterization of a cDNA clone encoding the 19 kDa protein of signal recognition particle (SRP): expression and binding to 7SL RNA.

Signal recognition particle (SRP) consists of a 7SL RNA molecule and 6 protein subunits. We have isolated and characterized cDNA clones from human liver which encode the 19kDa protein subunit (SRP19). This subunit binds to the RNA directly and mediates binding of a second polypeptide, the 54kDa subunit which is involved in signal sequence recognition. Amino acid sequences deduced from the human cDNA sequence were identical to amino acid sequences of tryptic peptides from canine pancreatic SRP19. In vitro transcription and translation of the human cDNA resulted in a protein product the same size as canine SRP19 which could be immunoprecipitated by an antiserum raised against canine SRP19. SRP19 synthesized in a cell-free system specifically bound to 7SL RNA. The sequence of SRP19 is discussed with respect to its binding to 7SL RNA.

Amino Acid Sequence↗