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J A Cioffi K L Allen M O Lively B Kemper 《The Journal of biological chemistry》1989,264(25):15052-15058
The length of the hydrophobic core of the bovine parathyroid hormone signal peptide was modified by in vitro mutagenesis. Extension of the hydrophobic core by three amino acids at the NH2-terminal end had little effect on the proteolytic processing of the signal peptide by microsomal membranes. Deletion of 6 of the 12 amino acids in the core eliminated translocation and processing of the modified protein. Deletion of pairs of amino acids across the core resulted in position-dependent inhibition of signal activity unrelated to hydrophobicity but inversely related to the hydrophobic moments of the modified cores. Deletions in the NH2-terminal region of the core were strongly inhibitory for proteolytic processing whereas deletions in the COOH-terminal region had no effect or increased processing when assessed either co-translationally with microsomal membranes or post-translationally with purified hen oviduct signal peptidase. Deletion of cysteine 18 and alanine 19 increased processing, but deletion of cysteine alone or substitution of leucine for cysteine did not increase processing more than deletion of both residues at 18 and 19. Translations of the translocation-defective mutants with pairs of amino acids deleted in a wheat germ system were inhibited by addition of exogenous signal recognition particle suggesting that interactions of the modified signal peptides with signal recognition particle were normal. The position-dependent effects of the hydrophobic core modifications indicate that structural properties of the core in addition to hydrophobicity are important for signal activity. The parallel effects of the modifications on co-translational translocation and post-translational processing by purified signal peptidase suggest that proteins in the signal peptidase complex might be part of, or intimately associated with, membrane proteins involved in the translocation. A model is proposed in which the NH2-terminal region of the hydrophobic core binds to one subunit of the signal peptidase while the other subunit catalyzes the cleavage. 相似文献
3.
S F Nothwehr S D Hoeltzli K L Allen M O Lively J I Gordon 《The Journal of biological chemistry》1990,265(35):21797-21803
The polar, COOH-terminal c-region of signal peptides has been considered to be most important for influencing the efficiency and fidelity of signal peptidase cleavage while the hydrophobic core or h-region appears indispensable for initiating translocation. To identify structural features of residues flanking the c-region that influence the fidelity and efficiency of signal peptidase cleavage as well as co-translational translocation, we introduced six amino acid substitutions into the COOH terminus of the hydrophobic core and seven substitutions at the NH2 terminus of the mature region (the +1 position) of a model eukaryotic preprotein-human pre(delta pro)apoA-II. This preprotein contains several potential sites for signal peptidase cleavage. The functional consequences of these mutations were assayed using an in vitro co-translational translocation/processing system and by post-translational cleavage with purified, detergent-solubilized, hen oviduct signal peptidase. The efficiency of translocation could be correlated with the hydrophobic character of the residue introduced at the COOH terminus of the h-region. Some h/c boundary mutants underwent co-translational translocation across the microsomal membrane with only minimal cleavage yet they were cleaved post-translationally by hen oviduct signal peptidase more efficiently than other mutants which exhibited a high degree of coupling of co-translational translocation and cleavage. These data suggest that features at the COOH terminus of the h-domain can influence "presentation" of the cleavage site to signal peptidase. The +1 residue substitutions had minor effects on the extent of co-translational translocation and processing. However, these +1, as well as h/c boundary mutations, had dramatic effects on the site of cleavage chosen by signal peptidase, indicating that residues flanking the c-region of this prototypic eukaryotic signal peptide can affect the fidelity of its proteolytic processing. The site(s) selected by canine microsomal and purified hen oviduct signal peptidase were very similar, suggesting that "intrinsic" structural features of this prepeptide can influence the selectivity of eukaryotic signal peptidase cleavage, independent of the microsomal membrane and associated translocation apparatus. 相似文献
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Structural features in the NH2-terminal region of a model eukaryotic signal peptide influence the site of its cleavage by signal peptidase 总被引:5,自引:0,他引:5
The 20-amino acid signal peptide of human pre (delta pro)apolipoprotein A-II contains the tripartite domain structure typical of eukaryotic prepeptides, i.e. a positively charged NH2-terminal (n) region, a hydrophobic core (h) region, and a COOH-terminal polar domain (c region). This signal sequence has multiple potential sites for cotranslational processing making it an attractive model for assessing the consequences of systematic structural alterations on the site selected for signal peptidase cleavage. We previously analyzed 40 mutant derivatives of this model preprotein using an in vitro translation/canine microsome processing assay. The results showed that the position of the boundary between the h and c regions and properties of the -1 residue are critical in defining the site of cotranslational cleavage. To investigate whether structural features in the NH2-terminal region of signal peptides play a role in cleavage specificity, we have now inserted various amino acids between the positively charged n region (NH2-Met-Lys) and the h region of a "parental" pre(delta pro)apoA-II mutant that has roughly equal cleavage between Gly18 decreases and Gly20 decreases. Movement of the n/h boundary toward the NH2 terminus results in a dramatic shift in cleavage to Gly18 decreases. Replacement of the Lys2 residue with hydrophilic, negatively charged residues preserves the original sites of cleavage. Replacement with a hydrophobic residue causes cleavage to shift "upstream." Simultaneous alteration of the position of n/h and h/c boundaries has an additive effect on the site of signal peptidase cleavage. None of these mutations produced a marked decrease in the efficiency of in vitro cotranslational translocation or cleavage. However, in sequence contexts having poor signal function, introduction of hydrophobic residues between the n and h regions markedly improved the efficiency of translocation/processing. We conclude that the position of the n/h boundary as well as positioning of the h/c boundary affects the site of cleavage chosen by signal peptidase. 相似文献
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P N Herbert L L Bausserman K M Lynch A L Saritelli M A Kantor R J Nicolosi R S Shulman 《Biochemistry》1987,26(5):1457-1463
We used antisera to human A and C apolipoproteins to identify homologues of these proteins among the high-density lipoprotein apoproteins of Macaca fascicularis (cynomolgus) monkeys, and NH2-terminal analysis was used to verify the homology. The NH2-terminal sequence of the M. fascicularis apoA-I is identical with that of another Old World species, Erythrocebus patas, and differs from human apoA-I at only 4 of the first 24 residues. M. fascicularis apoA-II contains a serine for cysteine replacement at position 6 and is therefore monomeric like the apoA-II from all species below apes. Human and monkey apoA-II are not otherwise different through their first 25 residues. About 20% of M. fascicularis apoC-I aligns with human apoC-I through residue 22, and 80% lacks an NH2-terminal dipeptide. Otherwise, the monkey apoC-I differs from the human protein at only 2 of 25 positions. Two forms of M. fascicularis apoC-II were identified. ApoC-II1 is highly homologous with human apoC-II, whereas an NH2-terminal hexapeptide is absent from apoC-II2. ApoC-II2 was the predominant species, and apoC-II1 appears to represent a propeptide from which a hexapeptide prosegment is cleaved at a Gln-Asp bond. Both forms of monkey apoC-II are potent activators of lipoprotein lipase. There are two polymorphic forms of M. fascicularis apoC-III, and their electrophoretic mobilities become identical after treatment with neuraminidase. Except for a glycine for serine substitution at position 10, the first 15 NH2-terminal residues of M. fascicularis and human apoC-III are the same. 相似文献
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Simon W. Law Glenn Gray H.Bryan Brewer 《Biochemical and biophysical research communications》1983,112(1):257-264
ds-cDNA to human liver apoA-I mRNA has been cloned. Nucleic acid sequence analysis revealed that apoA-I mRNA codes for a precursor apolipoprotein, preproapoA-I, which contains 24 amino acids on the NH2-terminal end of the mature plasma apoA-I. Eighteen amino acids are contained within the hydrophobic prepeptide (Met-Lys-Ala-Ala-Val-Leu-Thr-Leu-Ala-Val-Leu-Phe-Leu-Thr-Gly-Ser-Gln-Ala) followed by a 6 amino acid propeptide (Arg-His-Phe-Top-Gln-Gln). Our results on human apoA-I are in agreement with the partial sequence of the precursor of rat apoA-I with respect to the length of the precursor sequence, location of the prepeptide, and the presence of an unusual propeptide sequence terminating in a neutral dipeptide Gln-Gln. A detailed analysis of the primary structure of normal human apoA-I mRNA will be essential to our ultimate understanding of the processing of human apoA-I and diseases characterized by molecular defects in apoA-I structure and function. 相似文献
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Signal and membrane anchor functions overlap in the type II membrane protein I gamma CAT 总被引:14,自引:7,他引:7
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I gamma CAT is a hybrid protein that inserts into the membrane of the endoplasmic reticulum as a type II membrane protein. These proteins span the membrane once and expose the NH2-terminal end on the cytoplasmic side and the COOH terminus on the exoplasmic side. I gamma CAT has a single hydrophobic segment of 30 amino acid residues that functions as a signal for membrane insertion and anchoring. The signal-anchor region in I gamma CAT was analyzed by deletion mutagenesis from its COOH-terminal end (delta C mutants). The results show that the 13 amino acid residues on the amino-terminal side of the hydrophobic segment are not sufficient for membrane insertion and translocation. Mutant proteins with at least 16 of the hydrophobic residues are inserted into the membrane, glycosylated, and partially proteolytically processed by a microsomal protease (signal peptidase). The degree of processing varies between different delta C mutants. Mutant proteins retaining 20 or more of the hydrophobic amino acid residues can span the membrane like the parent I gamma CAT protein and are not proteolytically processed. Our data suggest that in the type II membrane protein I gamma CAT, the signals for membrane insertion and anchoring are overlapping and that hydrophilic amino acid residues at the COOH-terminal end of the hydrophobic segment can influence cleavage by signal peptidase. From this and previous work, we conclude that the function of the signal-anchor sequence in I gamma CAT is determined by three segments: a positively charged NH2 terminus, a hydrophobic core of at least 16 amino acid residues, and the COOH-terminal flanking hydrophilic segment. 相似文献
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Biosynthesis of human preproapolipoprotein A-II 总被引:10,自引:0,他引:10
J I Gordon K A Budelier H F Sims C Edelstein A M Scanu A W Strauss 《The Journal of biological chemistry》1983,258(22):14054-14059
The primary translation product of human apolipoprotein A-II was purified from wheat germ and ascites cell-free lysates programmed with RNA isolated from either a hepatocellular carcinoma cell line (HepG2) or intestinal epithelium. A-II mRNA represents 0.2% of the translatable RNA in these hepatocytes and in jejunal epithelium. Plasma high density lipoprotein-associated A-II is a 77-amino acid polypeptide. The primary translation product is 100 amino acids long and contains a 23-amino acid NH2-terminal extension. Cotranslational cleavage of the cell-free product indicated that this NH2-terminal sequence consists of an 18-amino acid long signal peptide, Met-Lys-Leu-Leu-Ala-Ala-X-Val-Leu-Leu-Leu-X-X-Cys-X-Leu-X-X-, and a 5-amino acid long propeptide, Ala-Leu-Val-Arg-Arg. This functional division was confirmed by sequencing the stable intracellular form of apolipoprotein A-II isolated from HepG2 cells. Approximately 45% of the proapo-A-II is cleaved to the mature form during export from HepG2 cells. The COOH-terminal dipeptide conforms to the rule that prosegments are cleaved after paired basic residues. We have previously shown (Gordon, J. I., Sims, H. F., Lentz, S. R., Edelstein, C., Scanu, A. M., and Strauss, A. W. (1983) J. Biol. Chem. 258, 4037-4044) that proapolipoprotein A-I is not cleaved during export from these cells and contains a prosegment with a COOH-terminal Gln-Gln dipeptide. Therefore, proteolytic processing of the two principal high density lipoprotein-associated apolipoproteins proceeds along different pathways. 相似文献
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T A Rosenwasser K A Hogquist S F Nothwehr S Bradford-Goldberg P O Olins D D Chaplin J I Gordon 《The Journal of biological chemistry》1990,265(22):13066-13073
We have examined the patterns of compartmentalization of several mammalian proteins in Escherichia coli which do not have signal peptides or functional signal peptide equivalents. These proteins include (i) human proapolipoprotein A-I (proapoA-I), a 249-residue protein which contains a hexapeptide NH2-terminal prosegment plus a mature domain of 243 residues comprised of tandemly arrayed, docosapeptide repeats with predicted amphipathic alpha-helical structure; (ii) the mature apoA-I molecule without its prosegment; (iii) mouse interleukin-1 beta (IL-1 beta), a 17-kDa protein which is composed of 12 beta strands that form a tetrahedral structure; and (iv) the 31-kDa precursor of IL-1 beta, proIL-1 beta. Efficient expression of these proteins in E. coli was achieved using a plasmid that contains the nalidixic acid-inducible recA promoter and ribosome binding site from the gene 10 leader of bacteriophage T7. In induced cultures the mammalian proteins represented up to 20% of the total bacterial protein mass. Surprisingly, cell fractionation using cold (osmotic) shock indicated that proapoA-I, apoA-I, and IL-1 beta, but not its 31-kDa precursor, were segregated into the periplasmic space with high efficiency: the ratio of periplasmic space/spheroplast distribution ranged from 0.6 to 1.1 in cells harvested 60-180 min after nalidixic acid induction. Not only was this compartmentalization efficient but it was also selective: analysis of the osmotic shock fractions revealed that the periplasmic space preparations were not contaminated with cytoplasmic proteins (e.g. phosphoglycerate dehydrogenase). Sequential Edman degradation showed that these proteins had not undergone any NH2-terminal proteolytic processing. The mammalian proteins did not affect the export of a prototypic bacterial preprotein, beta-lactamase. Together the data suggest that osmotic shock fractionation of E. coli may facilitate the purification of functional foreign proteins produced in this prokaryote. They also raise the possibility that structural elements in these proteins other than conventional signal peptides may effect periplasmic targeting in E. coli. 相似文献
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Benjamin E Gewurz Hidde L Ploegh Domenico Tortorella 《The Journal of biological chemistry》2002,277(13):11306-11313
The human cytomegalovirus US2 gene product targets major histocompatibility class I molecules for degradation in a proteasome-dependent fashion. Degradation requires interaction between the endoplasmic reticulum (ER) lumenal domains of US2 and class I. While ER insertion of US2 is essential for US2 function, US2 lacks a cleavable signal peptide. Radiosequence analysis of glycosylated US2 confirms the presence of the NH(2) terminus predicted on the basis of the amino acid sequence, with no evidence for processing by signal peptidase. Despite the absence of cleavage, the US2 NH(2)-terminal segment constitutes its signal peptide and is sufficient to drive ER translocation of chimeric reporter proteins, again without further cleavage. The putative US2 signal peptide c-region is responsible for the absence of cleavage, despite the presence of a suitable -3,-1 amino acid motif for signal peptidase recognition. In addition, the US2 signal peptide affects the early processing events of the nascent polypeptide, altering the efficiency of ER insertion and subsequent N-linked glycosylation. To our knowledge, US2 is the first example of a membrane protein that does not contain a cleavable signal peptide, yet otherwise behaves like a type I membrane glycoprotein. 相似文献
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D W Andrews E Perara C Lesser V R Lingappa 《The Journal of biological chemistry》1988,263(30):15791-15798
The earliest events in protein secretion include targeting to and translocation across the endoplasmic reticulum membrane. To dissect the mechanism by which signal sequences mediate translocation in eukaryotes, we are examining the behavior of fusion proteins and deletion mutants in cell-free systems. We demonstrate that the protein domain being translocated can have profound impact on the efficiency of the translocation process. Specifically, deletions in the mature prolactin "passenger" domain, beyond the signal cleavage site, reduce the efficiency of signal function. The effect of these deletions on signal function is observed when this signal sequence is in its normal position, at the amino terminus, and when internalized by the addition of 117 amino acids of chimpanzee alpha-globin. Alterations in the interaction of the deletion mutants with the signal recognition particle and with another component of the translocation system, signal peptidase, were observed. Our results suggest that subtle changes in sequences beyond the signal cleavage site can alter the efficiency of co-translational translocation by affecting various signal-receptor interactions. 相似文献
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Sequence of the precursor to rat bone gamma-carboxyglutamic acid protein that accumulates in warfarin-treated osteosarcoma cells 总被引:5,自引:0,他引:5
A biosynthetic precursor to rat bone gamma-carboxyglutamic acid protein (BGP) was isolated from warfarin-treated ROS 17/2 osteosarcoma cells by antibody affinity chromatography followed by reverse phase high performance liquid chromatography. Thirty-two residues of its NH2-terminal sequence were determined by gas-phase protein sequence analysis. Comparison of this sequence with the known structure of rat BGP established that the intracellular precursor is a 76-residue molecule of Mr = 9120 that differs from 6000-Da bone BGP in having an NH2-terminal extension of 26 residues. This precursor appears to be generated from the primary translation product by cleavage of a hydrophobic signal peptide and is the probable substrate for gamma-carboxylation by virtue of its accumulation in the presence of warfarin. The putative targeting region for gamma-carboxylation previously identified in the leader sequences of vitamin K-dependent proteins is found in the propeptide portion of the precursor. Since the immunoreactive component secreted by warfarin-treated cells is identical in sequence to the 6000-Da BGP from bone, propeptide cleavage from the precursor is independent of gamma-carboxylation and precedes secretion of BGP from the cell. 相似文献
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The amino-terminal domain of a eukaryotic signal peptide, from bovine parathyroid hormone, was altered by in vitro mutagenesis of the cDNA. The function of "internalized" signal sequence mutants and of deletion mutants was assayed using an in vitro translation-translocation system. The addition of 11 amino acids to the NH2 terminus of the signal peptide did not prevent normal processing of the precursor protein, whereas a 23-amino acid extension blocked processing. These data suggest that the NH2-terminal sequences of internal signal peptides must be permissive of the signal function. Deletion of 6 NH2-terminal amino acids from the signal peptide had no effect on its cleavage by microsomal membranes, but removal of 10 or 13 amino acids, including all charged residues prior to the hydrophobic core, prevented processing. For both the extension and deletion mutations, processed proteins were protected from proteolytic digestion, whereas unprocessed forms were not, which indicated that the unprocessed mutant proteins were not translocated across the microsomal membrane. Translation of both the extension and deletion translocation-deficient mutants was arrested by signal recognition particle, and salt-washed microsomal membranes reversed the translational arrest. These data demonstrate that the NH2-terminal domain is not required for the interaction of signal recognition particle with the signal peptide or with signal recognition particle receptor, but is required for formation of a maximally translocation-competent complex with the microsomal membrane. 相似文献
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Secreted proteins are processed from a nascent form that contains an NH2-terminal signal peptide. During processing, the latter is cleaved by a specific NH2-terminal signal peptidase. The nascent form of phosphatidylinositol glycan (PI-G) tailed proteins contain both an NH2- and a COOH-terminal signal peptide. The two signal peptides have much in common, such as size and hydrophobicity. The COOH-terminal peptide is also cleaved during processing. We propose that the amino acid in a nascent protein that ultimately combines with the PI-G moiety be designated the omega site. Amino acids adjacent and COOH-terminal to the omega site would then be omega + 1, omega + 2, etc. In previous studies, we showed that allowable substitutions at the omega site of an engineered form of placental alkaline phosphatase (miniPLAP) are limited to 6 small amino acids. In the present study, mutations were made at the omega + 1 and omega + 2 sites. At the omega + 1 site, processing to varying degrees was observed with 8 of the 9 amino acids substituted for alanine, the normal constituent. Only the proline mutant showed no processing. By contrast, the only substituents permitted at the omega + 2 site were glycine and alanine, with only trace activity observed with serine and cysteine. Thus, just as there is a -1, -3 rule for predicting cleavage by NH2-terminal signal peptidase, there appears to be a comparable omega, omega + 2 rule for predicting cleavage/PI-G addition by COOH-terminal signal transamidase. 相似文献
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Processing of human cytomegalovirus UL37 mutant glycoproteins in the endoplasmic reticulum lumen prior to mitochondrial importation
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Mavinakere MS Williamson CD Goldmacher VS Colberg-Poley AM 《Journal of virology》2006,80(14):6771-6783
The human cytomegalovirus (HCMV) UL37 glycoprotein (gpUL37) is internally cleaved and its products divergently traffic to mitochondria or are retained in the secretory pathway. To define the requirements for gpUL37 cleavage, residues -1 and -3 of the consensus endoplasmic reticulum (ER) signal peptidase I site within exon 3 (UL37x3) were replaced by bulky tyrosines (gpUL37 cleavage site mutant I). Internal cleavage of this UL37x3 mutant was inhibited, verifying usage of the consensus site at amino acids (aa) 193/194. The full-length mitochondrial species of gpUL37 cleavage site mutant I was N glycosylated and endoglycosidase H sensitive, indicating that ER translocation and processing took place prior to its mitochondrial importation. Moreover, these results suggest that internal cleavage of gpUL37 is not necessary for its N glycosylation. Partial deletion or disruption of the UL37 hydrophobic core immediately upstream of the cleavage site resulted in decreased protein abundance, suggesting that the UL37x3 hydrophobic alpha-helix contributes to either correct folding or stability of gpUL37. Insertion of the UL37x3 hydrophobic core and cleavage site into pUL37(M), a splice variant of gpUL37 which lacks these sequences and is neither proteolytically cleaved nor N glycosylated, resulted in its internal cleavage and N glycosylation. Its NH(2)-terminal fragment, pUL37(M-NH2), was detected more abundantly in mitochondria, while its N-glycosylated C-terminal fragment, gpUL37(M-COOH), was detected predominantly in the ER in a manner analogous to that of gpUL37 cleavage products. These results indicate that UL37x3 aa 178 to 205 are prerequisite for gpUL37 internal cleavage and alter UL37 protein topology allowing N glycosylation of its C-terminal sequences. In contrast, the NH(2)-terminal UL37x1 hydrophobic leader, present in pUL37x1, pUL37(M), and gpUL37, is not cleaved from mature UL37 protein, retaining a membrane anchor for UL37 isoforms during trafficking. Taken together, these results suggest that HCMV gpUL37 undergoes sequential trafficking, during which it is ER translocated, processed, and then mitochondrially imported. 相似文献
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Proteolytic processing of human preproapolipoprotein A-I. A proposed defect in the conversion of pro A-I to A-I in Tangier's disease 总被引:14,自引:0,他引:14
J I Gordon H F Sims S R Lentz C Edelstein A M Scanu A W Strauss 《The Journal of biological chemistry》1983,258(6):4037-4044
The primary translation product of human intestinal apolipoprotein A-I mRNA was isolated from wheat germ and ascites cell-free translation systems. Comparison of its NH2-terminal sequence with that of plasma high density lipoprotein-associated A-I showed that it is initially synthesized as a preproprotein. Like rat preproapolipoprotein A-I, it contains an 18-amino acid prepeptide and a 6-amino acid propeptide. The highly unusual COOH-terminal Gln-Gln dipeptide present in the rat pro-segment is also represented at the same position in the human sequence. The functional division of the 24-amino acid NH2-terminal extention into pro- and presegments was verified by finding that the stable intracellular form of A-I in a human hepatoma cell line was the proprotein. Edman degradation of radiolabeled intracellular and extracellular A-I indicated that this apolipoprotein was secreted without proteolytic cleavage of its hexapeptide prosegment. Therefore, it appears that apolipoprotein A-I undergoes an additional proteolytic processing step before it is fully integrated into plasma high density lipoprotein. Two-dimensional gel electrophoresis of purified proapolipoprotein A-I isolated from the hepatocyte cell culture media indicated that it corresponds to isoforms 2 and 3, the basic A-I isoproteins which are the precursors of plasma A-I and the predominant plasma A-I isoforms found in patients with Tangier's disease (Zannis, V. I., Lees, A. M., Lees, R. S., and Breslow, J. L. (1982) J. Biol. Chem., 257, 4978-4986). Therefore this pathologic state probably arises from a defect in the conversion of proapolipoprotein A-I to apolipoprotein A-I. 相似文献