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At least 73 records · Page 4Linked to original sources
PubMed1991

Effects of protein size on the rate of import of the precursors of aldehyde dehydrogenase and ornithine transcarbamylase into rat liver mitochondria.

It is known that a signal peptide is required for the import of a protein into mitochondrial matrix. It is also known that a signal peptide can be attached to any protein and allow it to be imported. We recently reported that the rate of import of rat liver mitochondrial aldehyde dehydrogenase precursor was slower than that of ornithine transcarbamylase precursor (Wang TTY, Farrés J, and Weiner H. Arch Biochem Biophys 272, 440-449, 1989). It was not known if the difference in the rate of import was related to the fact that the mature portion of aldehyde dehydrogenase is larger (500 amino acids compared with 322 amino acids) or because the signal peptides were different. We further showed that treatment of the mitochondria with alcohols caused an inhibition of the import of the precursor of aldehyde dehydrogenase but not that of ornithine transcarbamylase. In the present study we constructed chimeric proteins that contained the signal peptide from one precursor protein and the mature portion from the other. We found that the rate of import was related to the overall size of the precursor protein. Consistent with this observation was finding that a truncated aldehyde dehydrogenase precursor, which contained 317 amino acids, was imported more rapidly than was the authentic precursor. Consistent with this finding was the fact that butanol caused the inhibition of only the large precursor proteins. Thus, it appears that size of the protein being imported is a major determinant of the rate at which a precursor protein is imported into mitochondria.

Aldehyde Dehydrogenase↗
PubMed1990

Cloning and characterization of the human colipase cDNA.

Pancreatic lipase hydrolyzes dietary triglycerides to monoglycerides and fatty acids. In the presence of bile salts, the activity of pancreatic lipase is markedly decreased. The activity can be restored by the addition of colipase, a low molecular weight protein secreted by the pancreas. The action of pancreatic lipase in the gut lumen is dependent upon its interaction with colipase. As a first step in elucidating the molecular events governing the interaction of lipase and colipase with each other and with fatty acids, a cDNA encoding human colipase was isolated from a lambda gt11 cDNA library with a rabbit polyclonal anti-human colipase antibody. The full-length 525 bp cDNA contained an open reading frame encoding 112 amino acids, including a 17 amino acid signal peptide. The predicted protein sequence contains 100% of the published protein sequence for human colipase determined by chemical methods, but predicts the presence of five additional NH2-terminal amino acids and four additional COOH-terminal amino acids. Comparison of the predicted protein sequence with the known sequences of colipase from other species reveals regions of extensive identity. In vitro translation of mRNA transcribed from the cDNA gave a protein of the expected molecular size that was processed by pancreatic microsomal membranes. Sequence analysis of the in vitro translation product after processing demonstrated signal peptide cleavage and the presence of a human procolipase, as exists in the pig and horse colipases. DNA blot analysis was consistent with the presence of a single gene for colipase. RNA blot analysis demonstrated tissue-specific expression of colipase mRNA in the pancreas. Thus, we report, for the first time, a cDNA for colipase.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

Processing of two protein precursors yields four mature guinea pig seminal vesicle secretory proteins.

The translation of the two most abundant guinea pig seminal vesicle epithelium mRNAs (1800 nucleotides and 950 nucleotides) and the subsequent processing of their protein products were studied in an effort to elucidate the mechanism by which the four mature guinea pig seminal vesicle epithelium (GPSVE) secretory proteins are produced. The primary translation products of the 1800 nt and 950 nt mRNAs are two secretory protein precursors of 45 kDa and 20 kDa, respectively. Removal of signal peptides from these two precursors produces proteins of 43 kDa and 18.5 kDa, which are recognized by polyclonal antisera directed against the four mature secretory proteins. The existence of further processing intermediates in the production of the secretory proteins is suggested by the appearance of other immunoreactive polypeptides following incubation of GPSVE in nutrient medium containing [3H] leucine. Immunological and pulse-chase analysis strongly suggests that the 43-kDa protein gives rise to SVP-1, -3, and -4 and that SVP-2 is derived from the 18.5-kDa protein.

Animals↗
PubMed1990

Identification and sequencing of the spheroidin gene of Choristoneura biennis entomopoxvirus.

Entomopoxviruses are a class of insect viruses whose virions are embedded in cytoplasmic occlusion bodies. The major component of these protective complexes is a protein called spheroidin. An open reading frame encoding the spheroidin gene of Choristoneura biennis entomopoxvirus has been identified and sequenced in our laboratory. This protein coding region is 1023 nucleotides long and specifies a polypeptide of 38,500 Da. Spheroidin was purified by SDS polyacrylamide gel electrophoresis, electroeluted, and its amino terminus sequence was determined on a gas phase sequencer. We observed that the first 20 N-terminal amino acids were absent in the mature processed form of the spheroidin molecule. Examination of these 20 residues revealed their hydrophobic nature and close resemblance to the consensus signal peptide sequence which is commonly found on membrane proteins. The DNA sequence of the spheroidin gene predicted a processed polypeptide with a molecular weight of 36 kDa. However, spheroidin was observed to aggregate in complexes composed of 50-kDa monomers. Intermolecular disulfide bonds were shown to play major roles in the formation and structure of these viral occlusion bodies. The difference in molecular weight between the predicted protein and its counterpart in infected cells is likely due to post-translational modifications. Indeed, two potential asparagine-linked glycosylation sites are present on the spheroidin molecule. The 5' flanking regions of the spheroidin gene and the vaccinia major core protein precursor gene P4b were shown to share substantial homology.

Amino Acid Sequence↗
PubMed1988

Efficient production of native, biologically active human cystatin C by Escherichia coli.

A cDNA encoding the mature human cysteine proteinase inhibitor cystatin C was fused to the coding sequence for the Escherichia coli outer membrane protein A signal peptide, and the recombinant gene was expressed in E. coli under the control of the lambda PR promoter, an optimized Shine-Dalgarno sequence and the lambda cI 857 repressor. When induced at 42 degrees C, such cells expressed large amounts of recombinant cystatin C. The recombinant protein was isolated in high yield and characterized. All physicochemical properties investigated, including the positions of disulfide bonds, indicated that the E. coli derived cystatin C was identical to cystatin C isolated from human biological fluids, except that the proline residue in position three was not hydroxylated. The recombinant protein displayed full biological activity against papain, cathepsin B and dipeptidyl peptidase I.

Amino Acid Sequence↗
PubMed1991

Intramolecular chaperone: the role of the pro-peptide in protein folding.

Subtilisin, an alkaline serine protease, is produced in the bacterium as pre-pro-subtilisin; the pre-peptide of 29 amino acid residues is the signal peptide essential for the secretion of prosubtilisin from the cytoplasm into the culture medium. On the other hand, the pro-peptide of 77 residues covalently linked to the amino terminal end of the subtilisin intramolecularly guides the folding of subtilisin into the active enzyme. Importantly, the pro-peptide is not required for the enzymatic activity and is removed intramolecularly by autoprocessing upon the completion of the protein folding. In this review, I will first summarize all the data concerning the functions of the subtilisin pro-peptide. On the basis of these results, I shall discuss a new general concept, an intramolecular chaperone to explain the essential role of the pro-peptide in protein folding.

Amino Acid Sequence↗
PubMed1990

Sequence analysis, tissue distribution and regulation by cell depolarization, and second messengers of bovine secretogranin II (chromogranin C) mRNA.

Secretogranin II is a very acidic, tyrosine-sulfated protein found in secretory granules of cells belonging to the diffuse neuroendocrine system. It gained more general importance recently as a universal immunohistochemical marker for endocrine neoplasms. Sequence information was obtained from secretogranin II isolated from bovine anterior pituitaries, allowing the isolation of cDNA clones and deduction of its primary structure. Bovine secretogranin II is a 586-amino acid protein of 67,455 Da which is preceded by a signal peptide of 27 residues and contains 9 pairs of basic amino acids in its sequence which are used as potential cleavage sites for generation of physiologically active peptides. Moderately abundant mRNA levels were found in adrenal medulla, pituitary, hippocampus, and caudate. Secretogranin II message was absent from parathyroid gland, adrenal cortex, kidney, liver, and spleen. Depolarization of isolated chromaffin cells by various secretagogues significantly up-regulated secretogranin II mRNA levels by mechanisms distinct from those established for chromogranins and neuropeptides, components maintained along with secretogranin II in neuroendocrine storage vesicles.

Adrenal Medulla↗
PubMed1987

Nucleotide sequence of the gene from the cyanobacterium Anacystis nidulans R2 encoding the Mn-stabilizing protein involved in photosystem II water oxidation.

The gene for the Mn-stabilizing protein (MSP; the so-called extrinsic 33-kDa protein) that is involved in photosystem II water oxidation was cloned and sequenced from the genome of the cyanobacterium Anacystis nidulans R2. The gene (here designated woxA) was shown to be present in a single copy. The deduced amino acid sequence indicated that the translation product consisted of 277 amino acid residues with a Mr of 29,306. The comparison of the sequence with that of mature MSP from spinach chloroplasts suggested that the translation product is a precursor whose amino-terminal 28 amino acid residues represent the signal peptide for the protein to cross the thylakoid membrane into the lumen. The length of the putative signal peptide was less than half that of the transit peptide for thylakoid-lumenal proteins of higher plants, whereas the structural profile of the putative signal peptide was similar to that of the carboxyl-terminal portion of the higher plant transit peptides. The amino acid sequence of the mature A. nidulans R2 MSP showed rather low homology (48-49%) to higher plant MSPs, but the conserved amino acid residues appeared to be clustered. Five clusters were tentatively assigned, in which the homology values were in a range of 66-70%. Domains essential for the functioning of MSP are expected to be situated in these clusters. It is of note that the two cysteine residues in MSP were conserved, and the disulfide linkage between them may play an important role in maintaining the tertiary structure of MSP.

Amino Acid Sequence↗
PubMed1990

Expression and secretion of Mirabilis antiviral protein in Escherichia coli and its inhibition of in vitro eukaryotic and prokaryotic protein synthesis.

Mirabilis antiviral protein (MAP), a ribosome-inactivating protein, exhibits inhibitory effects on both plant virus infection and protein synthesis. To study these functions by site-specific mutagenesis, the total synthetic gene of MAP was constructed and expressed in Escherichia coli. However, the growth of the host was inhibited by the products, and the yield of MAP was very low. To improve the system for expressing MAP, an expression vector, pSH7, was constructed. This vector is based on the high copy number plasmid pUC19 and includes PL promoter and temperature-sensitive cI857 repressor. The plasmid also contains the ompA signal sequence and the total synthetic MAP gene. The MAP gene was expressed and its product was secreted into the culture medium after E. coli transformants were cultivated at 30 degrees C and the temperature was raised to 42 degrees C. The secreted MAP was then purified and characterized. This protein was identical to native MAP as determined by its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the amino acid sequence at the NH2 terminus, and its inhibitory effect on in vitro protein synthesis. MAP was found to inhibit the in vitro protein synthesis of rabbit reticulocyte and wheat germ. It further showed an IC50 concentration of approximately 200 nM in an E. coli in vitro translation system in contrast to ricin A-chain, a well known ribosome-inactivating protein.

Amino Acid Sequence↗
PubMed1985

Elongation arrest is not a prerequisite for secretory protein translocation across the microsomal membrane.

Signal recognition particle (SRP) is a ribonucleoprotein consisting of six distinct polypeptides and one molecule of small cytoplasmic 7SL RNA. It was previously shown to promote the co-translational translocation of secretory proteins across the endoplasmic reticulum by (a) arresting the elongation of the presecretory nascent chain at a specific point, and (b) interacting with the SRP receptor, an integral membrane protein of the endoplasmic reticulum which is active in releasing the elongation arrest. Recently a procedure was designed by which the particle could be disassembled into its protein and RNA components. We have further separated the SRP proteins into four homogeneous fractions. When recombined with each other and with 7SL RNA, they formed fully active SRP. Particles missing specific proteins were assembled in the hope that some of these would retain some functional activity. SRP(-9/14), the particle lacking the 9-kD and 14-kD polypeptides, was fully active in promoting translocation, but was completely inactive in elongation arrest. This implied that elongation arrest is not a prerequisite for protein translocation. SRP receptor was required for SRP(-9/14)-mediated translocation to occur, and thus must play some role in the translocation process in addition to releasing the elongation arrest.

Animals↗
PubMedDate not supplied

The role of the mature part of secretory proteins in translocation across the plasma membrane and in regulation of their synthesis in Escherichia coli.

Presently available data are reviewed which concern the role of the mature parts of secretory precursor proteins in translocation across the plasma membrane of Escherichia coli. The following conclusions can be drawn; i) signals, acting in a positive fashion and required for translocation do not appear to exist in the mature polypeptides; ii) a number of features have been identified which either affect the efficiency of translocation or cause export incompatibility. These are: alpha) protein folding prior to translocation; beta) restrictions regarding the structure of N-terminus; gamma) presence of lipophilic anchors; delta) too low a size of the precursor. Efficiency of translocation is also enhanced by binding of chaperonins (SecB, trigger factor, GroEL) to precursors. Binding sites for chaperonins appear to exist within the mature parts of the precursors but the nature of these sites has remained rather mysterious. Mutant periplasmic proteins with a block in release from the plasma membrane have been described, the mechanism of this block is not known. The mature parts of secretory proteins can also be involved in the regulation of their synthesis. It appears that exported proteins are already recognized as such before they are channelled into the export pathway and that their synthesis can be feed-back inhibited at the translational level.

Bacterial Proteins↗
PubMed1990

Mitochondrial precursor protein. Effects of 70-kilodalton heat shock protein on polypeptide folding, aggregation, and import competence.

A hybrid precursor protein constructed by fusing the mitochondrial matrix-targeting signal of rat preornithine carbamyl transferase to murine cytosolic dihydrofolate reductase (designated pO-DHFR) was expressed in Escherichia coli. Following purification under denaturing conditions, pO-DHFR was capable of membrane translocation when diluted directly into import medium containing purified mitochondria but lacking cytosolic extracts. This import competence was lost with time, however, when the precursor was diluted and preincubated in medium lacking mitochondria, unless cytosolic proteins (provided by rabbit reticulocyte lysate) were present. Identical results were obtained for purified precursor made by in vitro translation. The ability of the cytosolic proteins to maintain the purified precursor in an import-competent state was sensitive to protease, N-ethylmaleimide (NEM), and was heat labile. Further, this activity appeared to be signal sequence dependent. ATP was not required for the maintenance of pO-DHFR competence, nor did purified 70-kDa heat shock protein (the constitutive form of Hsp70) substitute for this activity. Interestingly, however, purified Hsp70 prevented aggregation of the precursor in an ATP-dependent manner and, as well, retarded the apparent rate and extent of pO-DHFR folding. Partial purification of reticulocyte lysate proteins indicated that competence activity resides within a large mass protein fraction (200-250 kDa) that contains Hsp70. Sucrose density gradient analysis revealed that pO-DHFR reversibly interacts with components of this fraction. Pretreatment of the fraction with NEM, however, significantly stabilized the subsequent formation of a complex with the precursor. The results indicate that Hsp70 can retard precursor polypeptide folding and prevent precursor aggregation; however, by itself, Hsp70 cannot confer import competence to pO-DHFR. Maintenance of import competence correlates with interactions between the precursor and an NEM-sensitive cytosolic protein fraction. Efficient dissociation of the precursor from this complex appears to require a reactive thiol moiety on the cytosolic protein(s).

Adenosine Triphosphate↗
PubMed1986

Characterization of secretory protein translocation: ribosome-membrane interaction in endoplasmic reticulum.

Secretory proteins are synthesized on ribosomes bound to the membrane of the endoplasmic reticulum (ER). After the selection of polysomes synthesizing secretory proteins and their direction to the membrane of the ER via signal recognition particle (SRP) and docking protein respectively, the polysomes become bound to the ER membrane via an unknown, protein-mediated mechanism. To identify proteins involved in protein translocation, beyond the (SRP-docking protein-mediated) recognition step, controlled proteolysis was used to functionally inactivate rough microsomes that had previously been depleted of docking protein. As the membranes were treated with increasing levels of protease, they lost their ability to be functionally reconstituted with the active cytoplasmic fragment of docking protein (DPf). This functional inactivation did not correlate with a loss of either signal peptidase activity, nor with the ability of the DPf to reassociate with the membrane. It did correlate, however, with a loss of the ability of the microsomes to bind ribosomes. Ribophorins are putative ribosome-binding proteins. Immunoblots developed with monoclonal antibodies against canine ribophorins I and II demonstrated that no correlation exists between the protease-induced inability to bind ribosomes and the integrity of the ribophorins. Ribophorin I was 85% resistant and ribophorin II 100% resistant to the levels of protease needed to totally eliminate ribosome binding. Moreover, no direct association was found between ribophorins and ribosomes; upon detergent solubilization at low salt concentrations, ribophorins could be sedimented in the presence or absence of ribosomes. Finally, the alkylating agent N-ethylmaleimide was shown to be capable of inhibiting translocation (beyond the SRP-docking protein-mediated recognition step), but had no affect on the ability of ribosomes to bind to ER membranes. We conclude that potentially two additional proteinaceous components, as yet unidentified, are involved in protein translocation. One is protease sensitive and possibly involved in ribosome binding, the other is N-ethylmaleimide sensitive and of unknown function.

Binding Sites↗
PubMed1992

Targeting of proteins into the peroxisomal matrix.

During the last few years much has been learned regarding signals that target proteins into peroxisomes. The emphasis in the near future will undoubtedly shift towards the elucidation of the mechanism of import. The use of mammalian and yeast cells deficient in peroxisome assembly and/or import (Zoeller & Raetz, 1986; Erdmann et al., 1989; Cregg et al., 1990; Morand et al., 1990; Tsukamoto, Yokota & Fujiki, 1990) should provide a handle on the genes (Erdmann et al., 1991; Tsukamoto et al., 1991) involved in these processes. This will have to be coupled with further development of in vitro systems which will permit the dissection of the steps in the translocation of proteins into peroxisomes. Though some progress has been made in the development of such assays (Imanaka et al., 1987; Small et al., 1987, 1988; Miyazawa et al., 1989), the fragility of peroxisomes and the absence of biochemical hallmarks of import (such as protein modifications or proteolytic processing) have hindered progress. Since peroxisomes exist in the form of a reticulum in mammalian cells (Gorgas, 1984), all peroxisome purification schemes (from mammalian cells at least) must undoubtedly rupture the peroxisomes, which then reseal to form vesicular structures. Additionally, the reliance on the latency of catalase alone as a major criterion for the integrity of peroxisomes ignores the fact that many other matrix proteins leak out of peroxisomes at vastly different rates during purification of the organelles (Thompson & Krisans, 1990). In view of these problems, the development of peroxisomal transport assays with semi-intact cells would also constitute an important advance.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1985

GTP-binding membrane protein of Escherichia coli with sequence homology to initiation factor 2 and elongation factors Tu and G.

The amino acid sequence of LepA protein, which has been shown to be cotranscribed with signal peptidase I in Escherichia coli, was compared with greater than 2000 known protein sequences. It was revealed that, of the 598 amino acid residues contained in LepA, an amino-terminal domain of 112 residues is homologous to a domain of similar size found in initiation factor 2, elongation factor Tu, and elongation factor G (IF2, EF-Tu, and EF-G), factors required for translation in E. coli. In this domain, 46 and 34 residues align perfectly with the corresponding regions of EF-G and EF-Tu, respectively. If functionally conserved residues within this domain (19 for EF-G and 17 for EF-Tu) are included, the overall resemblance is 58% and 46%, respectively, for EF-G and EF-Tu. A similar domain exists internally in IF2, where there is 42% overall resemblance with the domain of LepA. Immediately adjacent to this region is a small sequence of limited similarity that exists not only in EF-G, EF-Tu, and IF2 but also in the protooncogene c-Ha-ras-1 (from human bladder) and other GTP-binding proteins. Given these homologies, GTP-photoaffinity labeling and subcellular fractionation experiments were undertaken, and it was found that LepA is indeed a membrane-bound GTP-binding protein.

Amino Acid Sequence↗
PubMed1989

A membrane component of the endoplasmic reticulum that may be essential for protein translocation.

We have purified a glycosylated, membrane-spanning protein of relative molecular mass approximately 34,000 (Mr approximately 34 K) from canine microsomes that appears to be essential for protein translocation across the endoplasmic reticulum (ER) as shown by the inhibitory action of antibodies directed against it and of monovalent Fab-fragments produced from them. The ER membrane contains at least as many molecules of the 34 K membrane protein as bound ribosomes. The protein can be detected immunologically in tissues of various organisms, indicating an universal function.

Animals↗
PubMed1992

Replication control of plasmid R1: RepA synthesis is regulated by CopA RNA through inhibition of leader peptide translation.

The replication frequency of plasmid R1 is post-transcriptionally controlled by an antisense RNA, CopA, that binds to the leader region in the RepA mRNA, CopT, and ultimately inhibits the synthesis of the replication initiator protein RepA. We present results demonstrating that CopA controls RepA synthesis indirectly. A reading frame for a 24 amino acid leader peptide (Tap, translational activator peptide) is located in the region between the copA and repA genes. A translational fusion between the tap and lacZ genes was used to demonstrate that tap is translated and controlled by CopA. Stop codons (UAA, UAG and UGA) introduced at three different positions within the tap gene led to a severe decrease in repA expression. Specific suppression of the stop codons reversed the effect. This indicates that tap translation is required for RepA synthesis. Phylogenetic comparisons between IncFII-like plasmids, together with previous in vitro and in vivo results (Ohman and Wagner, 1989, 1991), suggest that a stable RNA stem-loop structure sequesters the repA ribosome binding site irrespective of CopA-CopT duplex formation. The results presented here show that ribosomes translating the tap reading frame have to terminate close to the start codon of repA to permit reinitiation (direct translational coupling), and that transient disruption of the inhibitory RNA stem-loop is insufficient for activation of repA translation. The possibility that direct translational coupling is required because of a suboptimal repA RBS cannot be excluded.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗