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1.
The B-fragment of diphtheria toxin binds to cell surface receptors and facilitates entry of the enzymatically active A-fragment into the cytosol. The roles of the amino- and carboxyl-terminal regions of the B-fragment in interactions with the cell membrane were studied by measuring specific binding, insertion into membranes at low pH, and formation of cation-selective channels, as well as by toxicity measurements after association with active A-fragment. Deletion of the amino-terminal 12 amino acids of the B-fragment did not affect its ability to bind to receptors and to form ion channels at low pH, whereas both abilities were strongly impaired when one more amino acid (Trp206) was removed. Replacement of the amino-terminal 31 residues with an amphipathic sequence from human apolipoprotein A1 restored receptor binding but not ion channel formation. The binding to cells was virtually abolished when 9 residues were deleted from the carboxyl terminus. Deletion of only 4 residues or extension by 12 residues did not prevent specific binding, but reduced insertion, channel formation, and toxicity. Those deletions that reduced receptor binding ability increased the trypsin sensitivity of the B-fragment. The results indicate that the amino- and carboxyl-terminal regions of diphtheria toxin B-fragment are important for receptor binding, possibly because they contribute to keep the B-fragment in a binding-competent conformation. Small alterations in the carboxyl-terminal end reduced insertion, channel formation, and toxicity more than the ability of the B-fragment to bind to cells.  相似文献   

2.
Deletion and insertion mutants of the multidrug transporter   总被引:5,自引:0,他引:5  
The multidrug transporter is a 170,000-dalton membrane glycoprotein which confers multidrug resistance through its activity as an ATP-dependent efflux pump for hydrophobic, cytotoxic drugs. To determine the essential structural components of this complex membrane transporter we have altered an MDR1 cDNA in an expression vector by deletion and insertion mutations. The structure of the transporter deduced from its amino acid sequence suggests that it consists of two homologous, perhaps functionally autonomous, halves each with six transmembrane segments and a cytoplasmic ATP-binding domain. However, several carboxyl-terminal deletions, one involving 53 amino acids, the second removing 253 amino acids, and an internal deletion within the carboxyl-terminal half of the molecule, totally eliminate the ability of the mutant transporter to confer drug resistance. An internal deletion of the amino-terminal half, which removed residues 140-229, is also nonfunctional. Small carboxylterminal deletions of up to 23 amino acids leave a functional transporter, although the removal of 23 COOH-terminal amino acids reduces its ability to confer colchicine resistance. Insertions of 4 amino acids in a transmembrane domain, and in one of the two ATP-binding regions, have no effect on activity. These studies define some of the limits of allowable deletions and insertions in the MDR1 gene, and demonstrate the requirement for two intact halves of the molecule for a functional multidrug transporter.  相似文献   

3.
Human asialoglycoprotein receptor H1 is a single-spanning membrane protein with an amino-terminal domain of 40 residues exposed to the cytoplasm and the carboxyl-terminal domain translocated to the exoplasmic side of the membrane. It has been shown earlier that the transmembrane segment functions as an internal uncleaved signal sequence for insertion into the endoplasmic reticulum. In a deletion protein lacking almost the entire cytoplasmic domain, the signal sequence is cleaved at the carboxyl-terminal end of the transmembrane segment. All available criteria suggest that the protein is processed by signal peptidase. The cytoplasmic domain of the receptor does not directly inhibit signal cleavage since it does not detectably hinder cleavage of the normally amino-terminal signal sequence of influenza hemagglutinin in fusion proteins. We suggest that by its size or structure it affects the position of the receptor in the membrane and thus the accessibility of the potential cleavage site to signal peptidase.  相似文献   

4.
A rat testicular luteinizing hormone (LH) receptor cDNA containing a 266-base pair deletion resulting in the omission of the 1st transmembrane region and truncation of the open reading frame was isolated using a rat ovarian LH receptor cDNA probe. Comparison of this clone with a restriction fragment from the LH receptor genomic DNA revealed potential alternative splice sites following the consensus sequence TTXCAG that is present at an intron acceptor splice site and also within the next exon, accounting for the specific deletion mutation observed in this cDNA. Expression of the testicular cDNA in COS1 cells resulted in synthesis and secretion of a soluble binding protein with high affinity and specificity for LH and human chorionic gonadotropin. These studies have demonstrated that the LH receptor gene contains intron(s) within the region coding for the extracellular domain of the molecule, which determine the nature and generation of LH receptor isoforms. Expression of the soluble form of the LH receptor has indicated that the amino-terminal extracellular region plays a major role in gonadotropin binding. These features of the LH receptor are distinct from those of most other G protein-coupled receptors, which are intronless and contain their binding sites within the transmembrane region rather than the extracellular domain.  相似文献   

5.
Structure and expression of the rat inositol 1,4,5-trisphosphate receptor   总被引:23,自引:0,他引:23  
The complete primary structure of the inositol 1,4,5-trisphosphate receptor from rat brain was elucidated using a series of overlapping cDNA clones. Two different sets of clones that either contain or lack a 45-nucleotide sequence in the amino-terminal third of the protein were isolated, suggesting a differential splicing event that results in the biosynthesis of either a 2734- or 2749-amino acid receptor protein. Hydrophobicity analysis demonstrates the presence of a cluster of hydrophobic sequences in the carboxyl-terminal third of the protein that probably comprise eight transmembrane regions and that may form the calcium channel intrinsic to the receptor. The receptor was universally expressed at low levels in all tissues and cultured cells tested. Transfection of a full-length expression construct of the inositol 1,4,5-trisphosphate receptor into COS cells resulted in the biosynthesis of a 260-kDa protein that bound inositol 1,4,5-trisphosphate and formed high molecular weight complexes similar to the native receptor as analyzed by sucrose gradient centrifugations. On the other hand, the protein product synthesized by a mutant receptor construct in which the amino-terminal 418 amino acids were deleted failed to bind inositol 1,4,5-trisphosphate. The mutant receptor still formed high molecular weight complexes, suggesting that it folded normally and that the amino-terminal sequences of the receptor are part of the ligand binding domain.  相似文献   

6.
Amino acid substitutions were made in the heat-labile enterotoxin signal sequence of Escherichia coli by recombinant DNA techniques, and their influence on the secretion of recombinant human epidermal growth factor by E. coli was examined. The heat-labile enterotoxin signal sequence is an amino-terminal extension of the octadecapeptide chain and is comprised of three distinct regions: a positively charged amino-terminal region, a central hydrophobic region, and a carboxyl-terminal region with the cleavage site recognized by the signal peptidase. Some alterations in the signal sequence caused a 1.5-3.5-fold increase in the secretion of recombinant human epidermal growth factor. These were the introduction of: (i) polar and small residues into the carboxyl-terminal region (replacement of Pro-1 Leu-3 with Asn-Ala or Ser-Ala), which may give a favorable structure for the recognition and cleavage by the signal peptidase; and (ii) a polar residue into the central hydrophobic region (replacement of Ile-9 with Ser), which may cause an increase of the affinity to the cytoplasmic membrane. In the latter case, a large amount of the unprocessed "precursor" was accumulated. The combination of these modifications, however, did not work additively. An increase in the amino-terminal positive charge (insertion of Lys) had no effect on secretion. These results prove that the level of protein secretion is greatly dependent on the polarity of the carboxyl-terminal region and the hydrophobicity and/or the amphiphilicity of the central region. Moreover, the overall balance of the physicochemical properties of respective regions is important.  相似文献   

7.
The vesicle-associated membrane proteins (Vamp(s)) function as soluble N-ethylmaleimide-sensitive factor attachment receptor proteins in the intracellular trafficking of vesicles. The membrane attachment of Vamps requires a carboxyl-terminal hydrophobic sequence termed an insertion sequence. Unlike other insertion sequence-containing proteins, targeting of the highly homologous Vamp1 and Vamp2 to the endoplasmic reticulum requires ATP and a membrane-bound receptor. To determine if this mechanism of targeting to the endoplasmic reticulum extends to other Vamps, we compared the membrane binding of Vamp1 and Vamp2 with the distantly related Vamp8. Similar to the other Vamps, Vamp8 requires both ATP and a membrane component to target to the endoplasmic reticulum. Furthermore, binding curves for the three Vamps overlap, suggesting a common receptor-mediated process. We identified a minimal endoplasmic reticulum targeting domain that is both necessary and sufficient to confer receptor-mediated, ATP-dependent, binding of a heterologous protein to microsomes. Surprisingly, this conserved sequence includes four positively charged amino acids spaced along an amphipathic sequence, which unlike the carboxyl-terminal targeting sequence in mitochondrial Vamp isoforms, is amino-terminal to the insertion sequence. Because Vamps do not bind to phospholipid vesicles, it is likely that these residues mediate an interaction with a protein, rather than bind to acidic phospholipids. Therefore, we suggest that a bipartite motif is required for the specific targeting and integration of Vamps into the endoplasmic reticulum with receptor-mediated recognition of specifically configured positive residues leading to the insertion of the hydrophobic tail into the membrane.  相似文献   

8.
We have isolated and sequenced cDNA clones encoding the entire sequence of the bovine cation-independent mannose 6-phosphate receptor. The deduced 2499-amino acid precursor has a calculated molecular mass of 275 kDa. Analysis of the sequence indicates that the protein has a 44-residue amino-terminal signal sequence, a 2269-residue extracytoplasmic region, a single 23-residue transmembrane region, and a 163-residue carboxyl-terminal cytoplasmic region. The extra-cytoplasmic region consists of 15 contiguous repeating domains, one of which contains a 43-residue insertion that is similar to the type II repeat of fibronectin. The 15 domains have an average size of 147 amino acids and a distinctive pattern of 8 cysteine residues. Alignment of the 15 domains and the extracytoplasmic domain of the cation-dependent mannose 6-phosphate receptor shows that all have sequence similarities and suggests that all are homologous.  相似文献   

9.
We report here the isolation and DNA sequence of a cDNA clone encoding a 252-amino acid non-muscle or cytoskeletal tropomyosin (cTm) isoform from Drosophila. The Drosophila cTm shows considerable homology with vertebrate cTm throughout the middle portion of the molecule. The amino-terminal end of the molecule, however, shows less homology and contains five more amino acids than the equine platelet and human tropomyosins. There is also a proline at position 6 in the Drosophila protein. The carboxyl-terminal 27 amino acids also show little homology with vertebrate non-muscle tropomyosins. This is a region of the molecule that shows considerably diversity among other Drosophila tropomyosins and vertebrate tropomyosins. A comparison of the DNA sequence of the cTm cDNA and a previously reported muscle tropomyosin II cDNA sequence shows regions of identical DNA sequence alternating with regions of nonidentical sequence, suggesting that both mRNAs are produced by alternate splicing of the same gene.  相似文献   

10.
The phorbol ester receptor protein kinase C (PKC) gene family encodes essential mediators of eukaryotic cellular signals. Molecular dissection of their mechanisms of action has been limited in part by the lack of random mutagenesis approaches and by the complexity of signaling pathways in mammalian cells which involve multiple PKC isoforms. Here we present a rapid screen which permits the quantification of mammalian PKC activity phenotypically in the yeast Saccharomyces cerevisiae. Bovine PKC alpha cDNA is functionally expressed in S. cerevisiae. This results in a phorbol ester response: a fourfold increase in the cell doubling time and a substantial decrease in yeast colony size on agar plates. We have expressed pools of bovine PKC alpha cDNAs mutagenized by Bal 31 deletion of internal, amino-terminal, or carboxyl-terminal sequences and have identified three classes of mutants on the basis of their distinct yeast phenotypes. Representatives of each class were analyzed. An internal deletion of amino acids (aa) 172 to 225 displayed ligand-dependent but reduced catalytic activity, an amino-terminal truncation of aa 1 to 153 displayed elevated and ligand-independent activity, and a carboxyl-terminal 26-aa truncation (aa 647 to 672) lacked activity under any conditions. Additional mutations confirmed the distinct functional characteristics of these classes. Our data show that deletion of the V1 and C1 regions results in elevated basal catalytic activity which is still Ca2+ responsive. Internal deletions in the V2 and C2 regions do not abolish phorbol ester or Ca2+ regulation of PKC activity, suggesting that most of the C2 domain is not essential for phorbol ester stimulation and most of the regulatory domain is dispensable for Ca2+ regulation of PKC activity. These distinct activities od the PKC mutants correlate with a specific and proportional yeast phenotype and are quantified on agar plates by yeast colony size. This provides a phenotypic screen which is suitable to identity rare, randomly altered but active mammalian PKC mutants. It quantifies their catalytic and biological activities in response to PKC activators or inhibitors for a systematic mapping of PKC structure and function or PKC-drug interaction.  相似文献   

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