Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q07644 (polypeptide)
 72,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

There are at least two different mechanisms for the transport of secretory proteins into the mammalian endoplasmic reticulum. Both mechanisms depend on the presence of a signal peptide on the respective precursor protein and involve a signal peptide receptor on the cis-side and signal peptidase on the trans-side of the membrane. Furthermore, both mechanisms involve a membrane component with a cytoplasmically exposed sulfhydryl. The decisive feature of the precursor protein with respect to which of the two mechanisms is used is the chain length of the polypeptide. The critical size seems to be around 70 amino acid residues (including the signal peptide). The one mechanism is used by precursor proteins larger than about 70 amino acid residues and involves two cytosolic ribonucleoparticles and their receptors on the microsomal surface. The other one is used by small precursor proteins and relies on the mature part within the precursor molecule and a cytosolic ATPase.
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PMID:Ribonucleoparticle-independent transport of proteins into mammalian microsomes. 209 35

Export of the outer membrane protein, OmpA, across the cytoplasmic membrane of Escherichia coli was severely inhibited by the presence of two, three, four or six additional basic residues at the N-terminus of the mature polypeptide, but not by three similarly positioned acidic residues. Because a few bacterial proteins do possess basic residues close to the leader peptidase cleavage site and because the type of inhibition described here could pose problems in the construction of hybrid secretory proteins, we also studied means of alleviating this form of export incompatibility. Inhibition was abolished when basic residues were preceded by acidic ones. Also, the processing rates of the mutants with two or six basic residues could be partially restored by increasing the length of the hydrophobic core of the signal peptide. Taking this as a precedent, it is suggested that the structure of the signal peptide is an important feature for maintenance of a reasonable rate of translocation of those exported proteins which possess basic residue(s) at the N-terminus of the mature polypeptide.
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PMID:Export incompatibility of N-terminal basic residues in a mature polypeptide of Escherichia coli can be alleviated by optimising the signal peptide. 219 18

The minimum substrate sequence recognized by signal peptidase I (SPase I or leader peptidase) was defined by measuring the kinetic parameters for a set of chemically synthesized peptides corresponding to the cleavage site of the precursor maltose binding protein (pro-MBP). The minimum sequence of a substrate hydrolyzed by SPase I at a measurable rate was the pentapeptide Ala-Leu-Ala decreases Lys-Ile. The rates of hydrolysis of this substrate, however, were several hundred-fold lower than those observed for the maturation of MBP in Escherichia coli, suggesting that in addition to these minimal sites involved in recognition, other features of pro-MBP are also needed for the optimal rate of signal peptide cleavage by SPase I. One parameter may be the length of the polypeptide chain. Studies of the synthetic peptides showed that decreasing the length of the polypeptide chain of substrates decreased the substrate efficiency measured as kcat/Km. However, in one case a decrease in the length of a peptide corresponding to -7 to +3 positions of pro-MBP to a nonapeptide (-7 to +2) increased the substrate efficiency by about 900-fold. The nonapeptide is the most efficient substrate for the enzyme in vitro so far reported. It is speculated that better peptide substrates are the ones which are able to adopt folded structures.
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PMID:Minimum substrate sequence for signal peptidase I of Escherichia coli. 224 78

The UDP-sugar hydrolase of Salmonella typhimurium has previously been reported to be located in both the inner and the outer membrane. We have cloned the gene, designated ushB, encoding this enzyme and determined its nucleotide sequence. No significant sequence homology with the periplasmic UDP-sugar hydrolase of Escherichia coli was found at either the DNA or protein level. However, a sequence is detectable, in the E. coli genome, which weakly hybridizes with a specific ushB probe. Polypeptide analysis has allowed the identification of the Salmonella hydrolase which has an Mr of 28,349 as compared to an Mr of 60,767 for the E. coli hydrolase. Most of the protein (approximately 90%) is located in the inner membrane. Two independent membrane fractionation procedures indicate that the remainder may be associated with the outer membrane. The deduced primary structure indicates the presence of an N-terminal signal peptide, although certain features of the region surrounding the putative processing site indicate that processing may be inefficient, or may not occur. Experiments with several inhibitors of signal peptidase function fail to demonstrate the appearance of a precursor form.
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PMID:Isolation, molecular characterization and expression of the ushB gene of Salmonella typhimurium which encodes a membrane-bound UDP-sugar hydrolase. 254 58

The expression of cytochrome c3 from Desulfovibrio vulgaris (Hildenborough) was examined in Escherichia coli transformed with either of two plasmids, pJ8 and pJ81. The former has an 840 bp insert of D. vulgaris DNA, containing the structural gene for cytochrome c3 (387 bp) and its promoter region. Plasmid pJ81 was generated from pJ8 by deoxyoligonucleotide-directed mutagenesis to direct the synthesis of a protein with an altered signal peptidase cleavage site [Ala(-1)----Asp(-1)]. Synthesis of the 14 kDa precursor, which was partly processed to the 12 kDa mature protein, was observed in cells of E. coli TG2(pJ8) by SDS gel electrophoresis and Western blotting. Analysis of spheroplasts revealed that the processed polypeptide was present in the periplasm while the precursor was found only in the membrane/cytoplasmic fraction. No processing was observed in E. coli TG2(pJ81) cells, due to the mutation of the signal peptide cleavage site. No insertion of haem into the E. coli product could be detected in E. coli TG2(pJ8) cells by post-electrophoretic protohaem fluorescence analysis. The sensitivity of the cytochrome c3 synthesized in E. coli TG2(pJ8) to digestion by chymotrypsin also indicated that the apoprotein was formed. The results indicate that E. coli is capable of synthesizing and exporting the cytochrome c3 polypeptide, but fails to insert the haems.
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PMID:Expression of the gene encoding cytochrome c3 from Desulfovibrio vulgaris (Hildenborough) in Escherichia coli: export and processing of the apoprotein. 256 Dec 88

Hen oviduct signal peptidase requires only two proteins for proteolysis of fully synthesized secretory precursor proteins in vitro: one with a molecular mass of 19 kilodaltons (kDa) and one which is a glycoprotein whose mass varies from 22 to 24 kDa depending on the extent of glycosylation. Purified signal peptidase has been analyzed both as part of an active catalytic unit and after electroelution of the individual proteins out of a preparative polyacrylamide gel. The multiple forms of the glycoprotein component of signal peptidase bind to concanavalin A and are shown to be derived from the same polypeptide backbone. Removal of their oligosaccharides by digestion with N-glycanase converts these proteins to a single 19.5-kDa polypeptide. The glycoproteins all exhibit very similar profiles following individual digestion with trypsin and separation of the resulting peptides by reverse-phase high-performance liquid chromatography. In addition, sequence analysis of selected peptides from corresponding regions in chromatograms representing each form of the glycoprotein reveals the same amino acid sequences. The 19-kDa signal peptidase protein does not bind concanavalin A, has a distinct tryptic peptide map from that of the glycoprotein, and appears to share no amino acid sequences in common with the glycoprotein. Its copurification on a concanavalin A-Sepharose column indicates that it must interact directly with the glycoprotein subunit.
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PMID:Purification and characterization of hen oviduct microsomal signal peptidase. 283 45

Spirochaeta aurantia DNA that coded for an antigenic determinant of the flagellin associated with the filament surface of the periplasmic flagella was isolated. When expressed in Escherichia coli, the antigenic polypeptide had an apparent molecular weight of 37,000. Sequence analysis of the antigen-encoding DNA revealed the presence of an open reading frame that determined a polypeptide with a predicted molecular weight of 31,241. This polypeptide showed a region of identity with the N-amino-terminal region of the 39,000- and 37,000-dalton flagellins of the distantly related spirochetes Treponema phagedenis and Treponema pallidum, respectively (S. J. Norris, N. W. Charon, R. G. Cook, M. D. Fuentes, and R. J. Limberger, J. Bacteriol. 170:4072-4082, 1988). The region of identity in the deduced S. aurantia polypeptide was preceded by a possible signal sequence and signal peptidase cleavage site.
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PMID:Cloning and sequence analysis of flaA, a gene encoding a Spirochaeta aurantia flagellar filament surface antigen. 292 Dec 47

The DNA sequence flanking the gene (lep) encoding signal peptidase I of Escherichia coli has been determined. The upstream flanking sequence contains a gene (lepA) that encodes a polypeptide of 598 amino acid residues and terminates 18 base pairs from the initiation codon of the lep gene. The position of the lep promoter was determined by both gene fusion with the lacZ gene as well as by S1 nuclease mapping of the lep mRNA to be 73 base pairs upstream from the initiation codon of the lepA gene. The lepA gene was cloned into a high expression vector (pIN-III), and its gene product was identified to be a protein of apparent molecular weight of 76,000. This gene product was preferentially localized in the cytoplasmic membrane and periplasmic fractions upon subcellular fractionation. The DNA sequence immediately downstream of the lep gene contains features consistent with a rho factor-independent transcriptional termination site, indicating that the lep operon encodes only two proteins (lepA and lep).
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PMID:Characterization of the lep operon of Escherichia coli. Identification of the promoter and the gene upstream of the signal peptidase I gene. 298 48

To investigate putative sorting domains in precursors to polypeptide hormones, we have constructed fusion proteins between the amino terminus of preproinsulin (ppI) and the bacterial cytoplasmic enzyme chloramphenicol acetyltransferase (CAT). Our aim is to identify sequences in ppI, other than the signal peptide, that are necessary to mediate the intracellular sorting and secretion of the bacterial enzyme. Here we describe the in vitro translation of mRNAs encoding two chimeric molecules containing 71 and 38 residues, respectively, of the ppI NH2 terminus fused to the complete CAT sequence. The ppI signal peptide and 14 residues of the B-chain were sufficient to direct the translocation and segregation of CAT into microsomal membrane vesicles. Furthermore, the CAT enzyme underwent N-linked glycosylation, presumably at a single cryptic site, with an efficiency that was comparable to that of native glycoproteins synthesized in vitro. Partial amino-terminal sequencing demonstrated that the downstream sequences in the fusion proteins did not alter the specificity of signal peptidase, hence cleavage of the ppI signal peptide occurred at precisely the same site as in the native precursor. This is in contrast to results found in prokaryotic systems. These data demonstrate that the first 38 residues of ppI encode all the information necessary for binding to the endoplasmic reticulum membrane, translocation, and proteolytic (signal sequence) processing.
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PMID:The NH2 terminus of preproinsulin directs the translocation and glycosylation of a bacterial cytoplasmic protein by mammalian microsomal membranes. 302 97

Cytochrome P450b is an integral membrane protein of the rat hepatocyte endoplasmic reticulum (ER) which is cotranslationally inserted into the membrane but remains largely exposed on its cytoplasmic surface. The extreme hydrophobicity of the amino-terminal portion of P450b suggests that it not only serves to initiate the cotranslational insertion of the nascent polypeptide but that it also halts translocation of downstream portions into the lumen of the ER and anchors the mature protein in the membrane. In an in vitro system, we studied the cotranslational insertion into ER membranes of the normal P450b polypeptide and of various deletion variants and chimeric proteins that contain portion of P450b linked to segments of pregrowth hormone or bovine opsin. The results directly established that the amino-terminal 20 residues of P450b function as a combined insertion-halt-transfer signal. Evidence was also obtained that suggests that during the early stages of insertion, this signal enters the membrane in a loop configuration since, when the amino-terminal hydrophobic segment was placed immediately before a signal peptide cleavage site, cleavage by the luminally located signal peptidase took place. After entering the membrane, the P450b signal, however, appeared to be capable of reorienting within the membrane since a bovine opsin peptide segment linked to the amino terminus of the signal became translocated into the microsomal lumen. It was also found that, in addition to the amino-terminal combined insertion-halt-transfer signal, only one other segment within the P450b polypeptide, located between residues 167 and 185, could serve as a halt-transfer signal and membrane-anchoring domain. This segment was shown to prevent translocation of downstream sequences when the amino-terminal combined signal was replaced by the conventional cleavable insertion signal of a secretory protein.
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PMID:Signals for the incorporation and orientation of cytochrome P450 in the endoplasmic reticulum membrane. 304 40


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