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1.
Expression of membrane-associated and secreted variants of gp160 of human immunodeficiency virus type 1 in vitro and in continuous cell lines. 总被引:19,自引:16,他引:3
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The gene encoding the 856-amino-acid envelope glycoprotein, gp160, of human immunodeficiency virus type 1 was mutagenized and transfected into Chinese hamster ovary cells. Continuous cell lines that constitutively produced gp160 variants were isolated and used to study the biosynthesis and orientation of gp160 in cellular membranes. In vivo studies of gp160 variants failed to reveal domains upstream of amino acid residue 665 that could serve as stop transfer sequences. Analyses of gp160 variants expressed in vitro in a translation-coupled translocation system were consistent with the in vivo studies and provided evidence that gp160 is a simple bitopic membrane protein. A model for the orientation and function of gp160 in cellular membranes is presented. The cell lines described provide a convenient source of the gp120-gp41 complex. 相似文献
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Protein sorting between mitochondrial membranes specified by position of the stop-transfer domain 总被引:3,自引:2,他引:1
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Recently, we fused a matrix-targeting signal to a large fragment of vesicular stomatitis virus G protein, which contains near its COOH-terminus a well-characterized endoplasmic reticulum (ER) stop-transfer sequence; the hybrid G protein was sorted to the inner mitochondrial membrane (Nguyen, M., and G. C. Shore. 1987. J. Biol. Chem. 262:3929-3931). Here, we show that the 19 amino acid G stop-transfer domain functions in an identical fashion when inserted toward the COOH-terminus of an otherwise normal matrix precursor protein, pre-ornithine carbamyl transferase; after import, the mutant protein was found anchored in the inner membrane via the stop-transfer sequence, with its NH2 terminus facing the matrix and its short COOH-terminal tail located in the intermembrane space. However, when the G stop-transfer sequence was placed near the NH2 terminus, the protein was inserted into the outer membrane, in the reverse orientation (NH2 terminus facing out, with a large COOH-terminal fragment located in the intermembrane space). These observations for mitochondrial topogenesis can be explained by a simple extension of existing models for ER sorting. 相似文献
4.
Nucleotide sequence of dcrA, a Desulfovibrio vulgaris Hildenborough chemoreceptor gene, and its expression in Escherichia coli.
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The amino acid sequence of DcrA (Mr = 73,000), deduced from the nucleotide sequence of the dcrA gene from the anaerobic, sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough, indicates a structure similar to the methyl-accepting chemotaxis proteins from Escherichia coli, including a periplasmic NH2-terminal domain (Mr = 20,700) separated from the cytoplasmic COOH-terminal domain (Mr = 50,300) by a hydrophobic, membrane-spanning sequence of 20 amino acid residues. The sequence homology of DcrA and these methyl-accepting chemotaxis proteins is limited to the COOH-terminal domain. Analysis of dcrA-lacZ fusions in E. coli by Western blotting (immunoblotting) and activity measurements indicated a low-level synthesis of a membrane-bound fusion protein of the expected size (Mr = approximately 137,000). Expression of the dcrA gene under the control of the Desulfovibrio cytochrome c3 gene promoter and ribosome binding site allowed the identification of both full-length DcrA and its NH2-terminal domain in E. coli maxicells. 相似文献
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A positively charged region is a determinant of the orientation of cytoplasmic membrane proteins in Escherichia coli 总被引:6,自引:0,他引:6
Basic amino acid residues were introduced into an extracellular (periplasmic) domain, preceding a membrane-spanning hydrophobic domain, of SecY, an integral cytoplasmic membrane protein. The localization of the domain was monitored as to the alkaline phosphatase activity of TnPhoA fused adjacent to the domain. The alkaline phosphatase activity of such Escherichia coli cells drastically decreased when positive charges were introduced, indicating that on the introduction the SecY domain showed a change in localization from the periplasm to the cytoplasm. In another experiment, positive charges were introduced to the same periplasmic domain of another SecY-PhoA fusion protein, in which PhoA is fused to the cytoplasmic domain of SecY following the particular hydrophobic domain. The alkaline phosphatase activity increased drastically when positive charges were introduced, indicating that the SecY domain fused to PhoA showed a change in localization from the cytoplasm to the periplasm. In both experiments, the removal of a large amino-terminal portion of the SecY domain did not alter the effect of the positive charge introduction. Changes in localization of SecY domains thus demonstrated were also supported by a protease accessibility test on spheroplasts. It is proposed that a positively charged region adjacent to a membrane-embedded hydrophobic region tends to be stabilized on the cytoplasmic surface of the membrane, which in turn endows the hydrophobic region with the ability to act as a stop-transfer sequence or a signal sequence and consequently determines the orientation of the hydrophobic region in the membrane. 相似文献
6.
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
7.
A non-hydrophobic sequence that contributes to the biogenesis of a transmembrane protein is termed a stop-transfer effector (STE). To examine the mechanism of STE-mediated stop-transfer, a series of fusion proteins were constructed containing variants of a putative STE from murine IgM fused to an otherwise translocated hydrophobic sequence. Unexpectedly, the fraction of molecules adopting transmembrane topology was insensitive to many amino acid substitutions within the STE sequence but varied directly with the number of negative charges. Furthermore, when present at the amino terminus of a reporter, mutants were observed that adopted type I (amino terminus lumenal) and type II (amino terminus cytoplasmic) transmembrane topologies, demonstrating that the STE sequence can be located at either side of the endoplasmic reticulum membrane. Our results suggest that recognition of a broad structural feature formed primarily by negatively charged residues within the STE halts translocation and triggers membrane integration, even when the negative charges end up on the cytoplasmic side of the membrane. Since functional STE sequences photocross-link to two membrane proteins not previously identified at the translocon, these unique proteins are presumably involved in recognizing STE sequences and/or facilitating STE function. 相似文献
8.
Molecular dissection of the NH2-terminal signal/anchor sequence of rat dipeptidyl peptidase IV 总被引:7,自引:3,他引:4
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Dipeptidyl peptidase IV (DPPIV) is a membrane glycoprotein with a type II orientation in the plasma membrane. As shown in a cell-free translation system, the amino-terminal 34 amino acids of rat DPPIV are involved in translocating nascent polypeptide across the membrane of microsomes and in anchoring the translocated polypeptide in the microsomal membrane. The amino-terminal sequence performing this dual function is composed of: a central hydrophobic core of 22 amino acid residues; 6 amino-terminal residues preceding the hydrophobic core (MKTPWK); and 6 residues following the hydrophobic core. The six residues preceding the hydrophobic core are exposed on the outside (cytoplasmic side) of the microsomal membrane. Site-directed mutagenesis studies show that deletion of this cytoplasmic domain, excluding the amino-terminal initiating methionine, does not affect translocation of nascent DPPIV polypeptide, but does affect significantly anchoring of the translocated polypeptide in the microsomal membrane. In contrast, changing the two cytoplasmic Lys to Glu residues or shortening of the hydrophobic core from 22 to 15 residues or converting the last 11e of the shortened hydrophobic core into Ala affects neither translocation across nor anchoring of the DPPIV polypeptide in the microsomal membrane. These and other structural features of the DPPIV amino-terminal signal-anchor sequences are discussed along with other types of sequences for their role in targeting nascent polypeptides to the RER. 相似文献
9.
Analysis of progressive deletions of the transmembrane and cytoplasmic domains of influenza hemagglutinin 总被引:31,自引:12,他引:19
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Site-directed oligonucleotide mutagenesis has been used to introduce chain termination codons into the cloned DNA sequences encoding the carboxy-terminal transmembrane (27 amino acids) and cytoplasmic (10 amino acids) domains of influenza virus hemagglutinin (HA). Four mutant genes were constructed which express truncated forms of HA that lack the cytoplasmic domain and terminate at amino acids 9, 14, 17, or 27 of the wild-type hydrophobic domain. Analysis of the biosynthesis and intracellular transport of these mutants shows that the cytoplasmic tail is not needed for the efficient transport of HA to the cell surface; the stop-transfer sequences are located in the hydrophobic domain; 17 hydrophobic amino acids are sufficient to anchor HA stably in the membrane; and mutant proteins with truncated hydrophobic domains show drastic alterations in transport, membrane association, and stability. 相似文献
10.
Intracellular sorting and targeting of melanosomal membrane proteins: identification of signals for sorting of the human brown locus protein, gp75 总被引:13,自引:1,他引:12
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《The Journal of cell biology》1995,130(4):807-820
The structural and functional integrity of cytoplasmic organelles is maintained by intracellular mechanisms that sort and target newly synthesized proteins to their appropriate cellular locations. In melanocytic cells, melanin pigment is synthesized in specialized organelles, melanosomes. A family of melanocyte-specific proteins, known as tyrosinase-related proteins that regulate melanin pigment synthesis, is localized to the melanosomal membrane. The human brown locus protein, tyrosinase-related protein-1 or gp75, is the most abundant glycoprotein in melanocytic cells, and is a prototype for melanosomal membrane proteins. To investigate the signals that allow intracellular retention and sorting of glycoprotein (gp)75, we constructed protein chimeras containing the amino-terminal extracellular domain of the T lymphocyte surface protein CD8, and transmembrane and cytoplasmic domains of gp75. In fibroblast transfectants, chimeric CD8 molecules containing the 36-amino acid cytoplasmic domain of gp75 were retained in cytoplasmic organelles. Signals in the gp75 cytoplasmic tail alone, were sufficient for intracellular retention and targeting of the chimeric proteins to the endosomal/lysosomal compartment. Analysis of subcellular localization of carboxy-terminal deletion mutants of gp75 and the CD8/gp75 chimeras showed that deletion of up amino acids from the gp75 carboxyl terminus did not affect intracellular retention and sorting, whereas both gp75 and CD8/gp75 mutants lacking the carboxyl-terminal 27 amino acids were transported to the cell surface. This region contains the amino acid sequence, asn-gln-pro-leu-leu-thr, and this hexapeptide is conserved among other melanosomal proteins. Further evidence showed that this hexapeptide sequence is necessary for intracellular sorting of gp75 in melanocytic cells, and suggested that a signal for sorting melanosomal proteins along the endosomal/lysosomal pathway lies within this sequence. These data provide evidence for common signals for intracellular sorting of melanosomal and lysosomal proteins, and support the notion that lysosomes and melanosomes share a common endosomal pathway of biogenesis. 相似文献
11.
N-terminal basic amino acids are not required for translocation and processing of preproparathyroid hormone 总被引:1,自引:0,他引:1
An N-terminal deletion mutant of preproparathyroid hormone that contains a single basic amino acid, lysine, in the N-terminal domain of the signal peptide is translocated across the endoplasmic reticulum membrane similarly to intact preproparathyroid hormone. To examine the function of charged residues preceeding the hydrophobic core, the lysine was replaced by an uncharged (methionine) or negatively charged (glutamic acid) amino acid. The translocational activity of the mutant signal peptides was assayed in a reticulocyte lysate system containing chicken oviduct microsomal membranes. Altering the net charge of the N-terminal domain did not abolish signal sequence activity, although the efficiency of translocation was decreased for the mutant with a glutamic acid substitution. Posttranslational, ribosome independent, translocation was observed for all the mutants tested, with the same dependence on N-terminal charge but with much lower efficiency than cotranslational translocation. These studies show that the presence of basic amino acids in the N-terminal domain of a eukaryotic signal sequence is not required for its activity. 相似文献
12.
The Bcl-2 oncogenic protein was synthesized in vitro and shown to post-translationally integrate asymmetrically into microsomal membranes with no requirement for an amino-terminal signal sequence. Instead, a carboxyl-terminal hydrophobic domain of Bcl-2 served as an insertion sequence essential for membrane assembly since a Bcl-2 mutant lacking this domain completely lost its ability to associate with microsomal membranes. The data demonstrate that Bcl-2 is tightly associated with the lipid bilayer with the nature of an integral membrane protein. The membrane orientation of Bcl-2 was determined using a protease protection assay, which showed that it is predominantly localized to the cytoplasmic face of membranes. A similar type of membrane processing has been shown for cytochrome b5 and also suggested for the viral oncogenic protein polyoma middle-T antigen. 相似文献
13.
The twin-arginine translocation (Tat) system can translocate folded proteins across biological membranes. Among the known Tat-system components in Escherichia coli, TatC is the only protein with multiple trans-membrane domains. TatC is important for translocon interactions with Tat substrates. The knowledge of its membrane topology is therefore crucial for the understanding of substrate binding and translocon function. Recently, based on active PhoA reporter fusions to the second predicted cytoplasmic loop of TatC, a topology with four trans-membrane domains has been suggested, calling in silico predictions of six trans-membrane domains into question. Here we report studies with translational fusions of TatC to the topological marker enzymes PhoA and LacZ which provide strong evidence for a six-trans-membrane domain topology. The stop transfer capacity of the fourth trans-membrane domain was found to be strongly influenced by the succeeding cytoplasmic domain. The presence of linker sequences at PhoA-fusion sites of the cytoplasmic domain induced PhoA leakage. In the case of one tested fusion (S185-PhoA), the stop-transfer efficiency was already low due to the negative charge in the center of the fourth trans-membrane domain (E170). The results point to the importance of cytoplasmic loops for the stabilization of stop-transfer sequences and revoke evidence for only four trans-membrane domains of TatC. 相似文献
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Identification of basolateral membrane targeting signal of human sodium-dependent dicarboxylate transporter 3 总被引:5,自引:0,他引:5
Sodium-dependent dicarboxylate transporters (NaDC) include low-affinity NaDC1 and high-affinity NaDC3. Despite high similarities structurally and functionally, both are localized to opposite surfaces of renal tubular cells. The molecular mechanisms and localization signals leading to this polarized distribution remain unknown. In this study, distribution of NaDC3 in human kidney tissue was firstly observed by immunohistochemistry and immunofluorescence. Then, EGFP-fused wild-type, NH2- and COOH-terminal deletion and point mutants of NaDC3, and chimera between NaDC3 and NaDC1, were generated and transfected into polarized renal cells lines, LLC-PK1 and MDCK. Their subcellular localizations were analyzed by laser confocal microscopy. Immunolocalization results revealed that NaDC3 was expressed at basolateral membrane of human renal proximal tubular epithelia. Confocal examinations showed that wild-type NaDC3 was targeted to the basolateral membrane of MDCK and LLC-PK1. Deletion mutations indicated that the basolateral targeting signal of NaDC3 located within a short sequence AKKVWSARR of its amino-terminal cytoplasmic domain. Addition of this sequence could redirect apical NaDC1 to the basolateral membrane of LLC-PK1. Point mutagenesis revealed that mutation of either of two hydrophobic amino acids V and W in this short sequence largely redirected NaDC3 to both apical and basolateral surfaces of LLC-PK, indicating that the two hydrophobic amino acids are critical for the basolateral targeting of NaDC3. Our studies provide direct evidence of the localization of NaDC3 at the basolateral membrane of human renal proximal tubule cells and identify a di-hydrophobic amino acid motif VW as basolateral localization signal in the N-terminal cytoplasmic domain of NaDC3. 相似文献
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E(rns) is a structural glycoprotein of pestiviruses found to be attached to the virion and to membranes within infected cells via its COOH terminus, although it lacks a hydrophobic anchor sequence. The COOH-terminal sequence was hypothesized to fold into an amphipathic alpha-helix. Alanine insertion scanning revealed that the ability of the E(rns) COOH terminus to bind membranes is considerably reduced by the insertion of a single amino acid at a wide variety of positions. Mutations decreasing the hydrophobicity of the apolar face of the putative helix led to reduction of membrane association. Proteinase K protection assays showed that E(rns) translated in vitro in the presence of microsomal membranes was protected, whereas a mutant with an artificial transmembrane region and a short cytosolic tag was shortened by the protease treatment. A tag fused to the COOH terminus of wild type E(rns) was not accessible for antibodies within digitonin-permeabilized cells, but the variant with the tag located downstream of the artificial transmembrane region was detected under the same conditions. These results are in accordance with the model that the COOH-terminal membrane anchor of E(rns) represents an amphipathic helix embedded in plane into the membrane. The integrity of the membrane anchor was found to be important for recovery of infectious virus. 相似文献
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Localization of the immunity protein-reactive domain in unmodified and chemically modified COOH-terminal peptides of colicin E1. 总被引:14,自引:4,他引:10
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The region of the colicin E1 polypeptide that interacts with immunity protein has been localized to a 168-residue COOH-terminal peptide. This is the length of a proteolytically generated peptide fragment of colicin E1 against which imm+ function can be demonstrated in osmotically shocked cells. The role of particular amino acids of the COOH-terminal peptide in the expression of the immune phenotype was studied. Chemical modification showed that the two histidine residues (His 427 and His 440) and the single cysteine residue (Cys 505) present in the COOH-terminal peptide were not necessary for the colicin-immunity protein interaction. The immunity protein was localized in the cytoplasmic membrane fraction, consistent with previous work of others on the colicin Ia immunity protein and the prediction from the immunity protein amino acid sequence that it is a hydrophobic protein. The distribution of hydrophobic residues along the immunity polypeptide was calculated. 相似文献
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Isolation and sequencing of a cDNA clone encoding 96 kDa sialoglycoprotein in rat liver lysosomal membranes 总被引:4,自引:0,他引:4
Y Noguchi M Himeno H Sasaki Y Tanaka A Kono Y Sakaki K Kato 《Biochemical and biophysical research communications》1989,164(3):1113-1120
We isolated and sequenced LGP 96, a cDNA clone corresponding to the entire coding sequence of the rat liver lysosomal membrane sialoglycoprotein with an apparent Mr of 96 K, LGP 96. The deduced amino acid sequence indicates that LGP 96 consists of 411 amino acid residues (Mr 45,163) and the 26 NH2-terminal residues presumably constitute a cleavable signal peptide. The major portion of LGP 96 resides on the luminal side of the lysosome and bears a large number of N-linked heavily sialylated complex type carbohydrate chains, giving the mature molecule of 96 kDa. The protein has 17 potential N-glycosylation sites and 32.1 and 65.3% sequence similarities in amino acid to LGP 107 and human lamp-2, respectively. The glycosylation sites are clustered into two domains separated by a hinge-like structure enriched with proline and threonine. LGP 96 possesses one putative transmembrane domain consisting of 24 hydrophobic amino acids near the COOH-terminus and contains a short cytoplasmic segment constituting 12 amino acid residues at the COOH-terminal end. Comparison of LGP 96 and recently cloned lysosomal membrane glycoprotein sequences reveals strong similarity in the putative transmembrane domain and cytoplasmic tail. It is very likely that these portions are important for the targeting of molecules to lysosomes. A comparison of LGP 96 and LGP 107 showed numerous structural similarities. 相似文献