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At least 19 recordsLinked to original sources
PubMed1998

Isolation and characterization of pediocin AcH chimeric protein mutants with altered bactericidal activity.

A collection of pediocin AcH amino acid substitution mutants was generated by PCR random mutagenesis of DNA encoding the bacteriocin. Mutants were isolated by cloning mutagenized DNA into an Escherichia coli malE plasmid that directs the secretion of maltose binding protein-pediocin AcH chimeric proteins and by screening transformant colonies for bactericidal activity against Lactobacillus plantarum NCDO955 (K. W. Miller, R. Schamber, Y. Chen, and B. Ray, 1998. Appl. Environ. Microbiol. 64:14-20, 1998). In all, 17 substitution mutants were isolated at 14 of the 44 amino acids of pediocin AcH. Seven mutants (N5K, C9R, C14S, C14Y, G37E, G37R, and C44W) were completely inactive against the pediocin AcH-sensitive strains L. plantarum NCDO955, Listeria innocua Lin11, Enterococcus faecalis M1, Pediococcus acidilactici LB42, and Leuconostoc mesenteroides Ly. A C24S substitution mutant constructed by other means also was inactive against these bacteria. Nine other mutants (K1N, W18R, I26T, M31T, A34D, N41K, H42L, K43N, and K43E) retained from <1% to approximately 60% of wild-type activity when assayed against L. innocua Lin11. One mutant, K11E, displayed approximately 2. 8-fold-higher activity against this indicator. About one half of the mutations mapped to amino acids that are conserved in the pediocin-like family of bacteriocins. All four cysteines were found to be required for activity, although only C9 and C14 are conserved among pediocin-like bacteriocins. Several basic amino acids as well as nonpolar amino acids located within the hydrophobic C-terminal region also were found to be important. The mutations are discussed in the context of structural models that have been proposed for the bacteriocin.

ATP-Binding Cassette Transporters↗
PubMed1992

Pseudomonas exotoxin A-epidermal growth factor (EGF) mutant chimeric protein as an indicator for identifying amino acid residues important in EGF-receptor interaction.

Epidermal growth factor (EGF) was fused to the carboxyl end of a modified pseudomonas exotoxin A that has its toxin binding domain deleted. This chimeric toxin designated as PE(delta Ia)-EGF kills A431 cells through the EGF receptor-mediated pathway. In this study, we used a random mutagenesis approach to make point mutations on EGF, followed by replacing the wild type EGF in PE(delta Ia)-EGF with these EGF mutants. We have constructed 14 different PE(delta Ia)-EGFmutants, and examined their EGF receptor binding activity as well as their cytotoxicity to A431 cells. Our results showed that individual mutations of Val19 to Glu and Val34 to Asp in the EGF domain of PE(delta Ia)-EGFmutants resulted in an increase in the binding affinity to EGF receptor and cytotoxicity to A431 cells. On the other hand, individual mutations of His16 to Asp and Gly18 to Ala in the EGF domain of PE(delta Ia)-EGFmutants lead to a decrease in the binding affinity to EGF receptor and cytotoxicity to A431 cells. In addition, mutations of any of the cysteine residues of EGF in PE(delta Ia)-EGFmutants resulted in the loss of their binding activity to EGF receptor and a corresponding loss of their cytotoxicity. This study indicates that the cytotoxicity of PE(delta Ia)-EGFmutant to EGF receptor-bearing cells may be used as an indicator to screen mutations of EGF important in EGF-receptor interactions.

ADP Ribose Transferases↗
PubMed1995

Functional dissection of P-glycoprotein nucleotide-binding domains in chimeric and mutant proteins. Modulation of drug resistance profiles.

We wished to determine if the two nucleotide-binding domains (NBD) of P-glycoprotein are functionally equivalent and interchangeable, and if not, which segments and amino acids are important for proper function of each NBD within the context of the C- or N-terminal P-glycoprotien halves. For this, we constructed and tested the biological activity in yeast and mammalian cells of a series of chimeric mdr3 cDNAs in which discrete domains of the N-terminal NBD (NBD1) were replaced by the homologous segments of the C-terminal NBD (NBD2). Although most NBD1 segments could be replaced without loss of P-glycoprotein function, exchange of small segments near the Walker B motif caused a dramatic reduction in Adriamycin, actinomycin D, and colchicine resistance in LR73 cells, as well as in FK506 resistance and STE6 complementation in yeast. Site-directed mutagenesis identified amino acid positions 522-525 (ERGA-->DKGT) and 578 (Thr-->Cys) as essential for proper function of NBD1 in the context of the N-terminal half P-glycoprotein. In addition, the observed phenotype of the mutants (altered drug resistance profile) suggests that these residues may participate directly or indirectly in substrate interactions and are possibly implicated in signal transduction from NBDs to transmembrane domains, the primary sites of drug binding in P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Expression of high-affinity interleukin 4 receptors on murine sarcoma cells and receptor-mediated cytotoxicity of tumor cells to chimeric protein between interleukin 4 and Pseudomonas exotoxin.

The presence of interleukin 4 receptor (IL-4R) on methylcholanthrene (MCA-106, MCA-102, and MC-38)- and viral DNA (G-2TS and 14-2TS)-induced murine sarcoma cells was demonstrated. MCA-106 tumor cells express about 500 to 1348 (median, 800) interleukin 4 (IL-4) binding sites/cell with a dissociation constant (Kd) of 115 +/- 26 pM (mean +/- SD, n = 4). By Northern blot analysis, tumor cells exhibited a single mRNA species of 3.9 kilobases. Other murine sarcoma (MCA-102), colon adenocarcinoma (MC-38), G-2TS, and 14-2TS tumor cells express low numbers of IL-4R. By immunoperoxidase staining, 81 to 92% of the cells from fresh MCA-106 tumors were positive for IL-4 receptors, while only 7 to 10% of tumor-infiltrating cells were Thy 1.2 and less than 1% Mac-1 positive. Using a chimeric protein composed of IL-4 and Pseudomonas exotoxin (IL-4-PE40), we observed that IL-4-PE40 was cytotoxic (determined by inhibition of protein synthesis by [3H]leucine uptake) to MCA-106 tumor cells in a dose-dependent manner. A nonchimeric protein (PE40) that cannot bind to the IL-4R did not inhibit protein synthesis in tumor cells. A chimeric mutant protein (IL4-PE40 asp553) that can bind to IL-4 receptors but does not have the capability to inhibit protein synthesis was not cytotoxic to tumor cells. These studies strongly suggest that IL-4R on murine MCA-106 sarcoma cells is internalized when occupied by IL-4 PE40. Furthermore, a neutralizing antibody (11B11) to IL-4 completely abolished the protein synthesis-inhibitory activity of IL-4-PE40. G-2TS tumor cells which expressed low numbers of IL-4 receptors were not vulnerable to cytotoxicity by IL-4-PE40. Taken together, these data suggest that IL-4 receptor may be a target for IL-4-toxin therapy.

Animals↗
PubMed1996

Lactacystin, an inhibitor of the proteasome, blocks the degradation of a mutant precursor of glycosylphosphatidylinositol-linked protein in a pre-Golgi compartment.

When transiently expressed in the COS-1 cell, a mutant chimeric protein with an uncleavable glycosylphosphatidylinositol (GPI) -anchor signal failed to be modified by GPI and undergoes rapid degradation in a pre-Golgi compartment. Among several protease inhibitors, 3,4-dichloroisocoumarin and N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal, potent inhibitors of the proteasome, strongly inhibited the degradation of the mutant protein. Furthermore, lactacystin, a highly specific inhibitor of the proteasome, was found to block the degradation. These results suggest that the pre-Golgi degradation pathway is functionally linked to the proteolytic system dependent on the proteasome, which hitherto was believed to play a role mostly in the cytoplasm and nucleus.

Acetylcysteine↗
PubMed1994

Human renal cell carcinoma cells are sensitive to the cytotoxic effect of a chimeric protein composed of human interleukin-4 and Pseudomonas exotoxin.

We have previously demonstrated that functional high-affinity interleukin-4 receptors (IL-4R) are expressed on human renal cell carcinoma (RCC) cells (N. I. Obiri et al., J. Clin. Invest. 91, 88, 1993). In the present study, we examined the cytotoxic effect (determined by inhibition of protein synthesis) of a chimeric protein composed of human IL-4 and Pseudomonas exotoxin (PE) on human RCC tumor samples obtained from patients undergoing nephrectomy. The chimeric gene encoding hIL4-PE4E was constructed by fusing a cDNA clone for human IL-4 to the 5' end of a mutated cDNA encoding a full-length PE molecule. This gene was expressed in Escherichia coli, and large quantities of this recombinant protein were isolated to more than 95% purity. This chimeric protein, hIL4-PE4E, was highly cytotoxic to all six RCC cell lines examined. The concentration of hIL4-PE4E at which 50% inhibition of protein synthesis was obtained ranged from < 1 ng/ml (12 pM) to 10 ng/ml (120 pM) in five of the six isolates of RCC and 40-70 ng/ml in one other. A mutant chimeric protein which can bind to IL-4R but lacks the ADP ribosylation activity of PE was not cytotoxic to the RCC cells. The cytotoxic effect of hIL4-PE4E was IL-4R mediated because a fourfold molar excess of IL-4 abrogated the cytotoxic effect of hIL4-PE4E. A neutralizing monoclonal antibody to IL-4 also abrogated the cytotoxic effect of hIL4-PE4E. hIL4-PE4E showed very little cytotoxic activity to a normal human umbilical vein endothelial cell line (ID50 = 1000 ng/ml) and a human fibroblast cell line (ID50 approximately 400 ng/ml). Nonactivated human peripheral blood lymphocytes (PBL) were also insensitive to hIL4-PE4E (ID50, approximately 500 ng/ml), whereas phytohemagglutinin-activated PBL were highly susceptible to the cytotoxic effect of hIL4-PE4E (ID50, approximately 4 ng/ml). These data indicate that hIL4-PE4E may be a useful agent for the treatment of human RCC without affecting normal and resting immune cells.

ADP Ribose Transferases↗
PubMed2001

Functional expression and cellular localization of a green fluorescent protein-tagged proline transporter in Aspergillus nidulans.

The PrnB protein is a highly specific proline transporter that belongs to an amino acid transporter family conserved in both prokaryotes and eukaryotes. In this work, we detected and analyzed the cellular localization of PrnB in vivo by means of green fluorescent protein (GFP) fusions. Several prnB-gfp gene fusions, driven by prnB native promoter sequences, were constructed and targeted to the genomic locus of a prnB null mutant. Chimeric proteins containing GFP fused to the C terminus of PrnB through a linker of two, four, or eight amino acids, with low potential to form secondary structure elements, were shown to be functional in vivo. A two-linker fusion results in partial complementation at both 25 and 37 degrees C. A four-linker fusion affords almost full complementation at 25 degrees C but partial complementation at 37 degrees C, whereas the eight-linker fusion results in partial complementation at both temperatures but in no GFP fluorescence. These results show that the number of linker amino acids is critical for the correct expression and/or translocation of PrnB-GFP fused proteins to the plasma membrane and for the fluorescence of the GFP. The expression of the four-linker PrnB-GFP transporter was detected and analyzed in vivo by both conventional fluorescence and confocal laser microscopy. This chimeric protein is localized in the plasma membrane, secondarily in large vacuoles found in the swollen conidial end of the germlings, and in other small intracellular structures observed as fluorescent granules. A strong correlation between known patterns of PrnB expression and intensity of PrnB-GFP fluorescence was observed. This work also demonstrates that the GFP fusion technology is a unique tool with which to study the expression and cellular localization of low-abundance transmembrane transporters expressed from their native promoters.

Amino Acid Transport Systems, Neutral↗
PubMed1994

A chimeric protein comprised of IL-4 and Pseudomonas exotoxin is cytotoxic for activated human lymphocytes.

IL4-Pseudomonas exotoxin (IL4-PE4E) is a chimeric molecule in which human IL-4 is genetically fused to the mutated binding domain of Pseudomonas exotoxin. This molecule binds specifically to human IL-4 receptor-bearing cells. IL4-PE4E was extremely cytotoxic to highly purified anti-CD3-activated CD8+ T lymphocytes. The cytotoxic activity of this molecule was dependent on the activation state of CD8+ T cells: 3- and 4-day activated T cells were very susceptible to the cytotoxic activity of IL4-PE4E compared with 0- to 2-day activated cells. PHA-activated lymphocytes and PBL activated in mixed lymphocyte reaction were also highly sensitive to IL4-PE4E. CD16+ and/or CD56+ highly purified NK cells or highly purified, IL-2-activated NK cells were also very sensitive to the cytotoxic effect of IL4-PE4E. IL-2-activated LAK cells had little susceptibility after 1 day but were very sensitive to IL4-PE4E after 3 days. The cytotoxic effects of IL4-PE4E were mediated through a ligand receptor interaction because excess rIL-4 abrogated these effects as did a neutralizing Ab to human IL-4. A chimeric mutant protein that can bind to IL-4 receptors but lacks the ability to inhibit protein synthesis was not cytotoxic to activated lymphocytes. The IL4-PE4E-mediated cytotoxicity of activated T cells correlated with the level of expression of IL-4 receptors on these cells. CD8+ T cells activated for 3 days expressed the highest density of IL-4 receptors compared with 1- or 2-day activated cells. Among two chimeric toxins tested only IL4-PE4E was cytotoxic to 2-day anti-CD3-activated CD8+ T lymphocytes, whereas IL6-PE4E was not active at all. These studies suggest that human IL4 toxin could be a potent agent for the elimination of activated lymphocytes in allograft rejection, some autoimmune diseases, or treatment of lymphomas and leukemias.

ADP Ribose Transferases↗
PubMed1993

The subcellular distribution of early endosomes is affected by the annexin II2p11(2) complex.

The tyrosine kinase substrate annexin II is a member of a multigene family of Ca2+ and lipid-binding proteins which have been implicated in a number of membrane-related events. We have analyzed the subcellular distribution of annexin II in relation to other cellular components in normal and specifically manipulated MDCK cells. In a polarized monolayer of MDCK cells annexin II and its cellular ligand p11 are restricted almost exclusively to the cortical regions of the cells which also contain peripheral early endosomes. Treatment of the polarized cells with low Ca2+ medium leads to a disintegration of the cortical cytoskeleton and a translocation of both, the annexin II2p11(2) complex and early endosomes, to the cytoplasm. A similar translocation which is however specific for the annexin II2p11(2) complex and early endosomes and does not affect other elements of the cell cortex is observed in cells expressing a trans-dominant annexin II-p11 mutant. This chimeric mutant protein causes the aggregation of endogenous annexin II and p11 and the simultaneous detachment of early endosomes from the cell periphery resulting in the binding of the early endosomes but no other components of the endocytotic or biosynthetic pathways to the annexin II/p11 aggregates. The specificity of this effect argues for the association of the annexin II2p11(2) complex with early endosomes and suggests that this association contributes to establish the peripheral localization of early endosomal structures.

Actins↗
PubMed1991

Epitope mapping of the Syrian hamster prion protein utilizing chimeric and mutant genes in a vaccinia virus expression system.

The cellular prion protein (PrPc) is a host-encoded sialoglycoprotein bound to the external surface of the cell membrane by a glycosyl phosphatidylinositol anchor. A posttranslationally modified PrP isoform (PrPSc) is a component of the infectious particle causing scrapie and the other prion diseases. mAb have been raised against the protease-resistant core of Syrian hamster (SHa) PrPSc designated PrP 27-30. To map the epitopes within PrP reacting to these antibodies, we have expressed wild-type, chimeric mouse (Mo)/SHa and mutant MoPrP genes using recombinant vaccinia virus systems. The fidelity of the expression of recombinant PrPC was examined using vaccinia viruses expressing SHa-PrPC. It is full length, possesses Asn-linked carbohydrates and is attached to the external surface of the cell membrane by a glycosyl phosphatidylinositol anchor that is sensitive to cleavage by phosphatidylinositol-specific phospholipase C. We have tested 18 mAb for their ability to bind to chimeric prion proteins on immunoblots. Three distinct epitopes were identified that mapped to amino acid differences between SHa and MoPrP sequences. The first epitope, recognized by three of the antibodies tested, was defined by methionines at amino acids 108 and 111 in the mouse protein. The second epitope was dependent upon the presence of asparagines at positions 154 and 174 in MoPrP and was recognized by four of the antibodies tested. The third epitope mapped to a single amino acid substitution at residue 138 in MoPrP. mAb raised against SHaPrP 27-30 specific for this epitope are able to bind MoPrPC which has a single amino acid change (Ile to Met) at position 138. Eleven of the 18 antibodies tested mapped to this immunodominant epitope. It is located within a postulated amphipathic helix, a structure associated with immunodominant Ag. Inasmuch as PrPC, in its native form on the cell surface, is detected by the mAb 13A5 (a prototypic antibody of the immunodominant third epitope class), it is likely that this epitope is accessible in the native conformation of this protein.

Animals↗
PubMed1998

Domain-structure of cytoplasmic border region is main determinant for palmitoylation of influenza virus hemagglutinin (H7).

We have shown previously that the length of cytoplasmic tails influences the selection of lipid substrates for palmitoylation of influenza viral hemagglutinin esterase fusion (HEF) and hemagglutinin (HA) glycoproteins [Veit et al. (1996) Biochem. J. 318, 163-172; Reverey et al. (1996) J. Biol. Chem. 271, 23607-23610]. Using a series of new chimeric mutant proteins derived from acylated influenza virus HA (subtype H7) and from nonacylated Sendai virus fusion protein (F, strain Z), we report here that palmitoylation levels depend on the type of transmembrane or cytoplasmic domain, or both, present in the expression products and that cysteine residues placed close to the cytoplasmic membrane border are not sufficient for acylation. By inserting stretches of the HA transmembrane domain into a nonacylated mutant of Sendai F (FCys), we induce palmitoylation after expression in CV.1 cells, and the level of fatty acid transfer increases with the length of the HA-derived insert. A five-amino-acid shift of the HA transmembrane domain severely augments fatty acid transfer. Our data suggest that the influenza virus HA contains complex conformational signals for palmitoylation that are mainly located within the transmembrane domain but also involve the C-tail region, whereas the extracellular (luminal) domain has only marginal influence on palmitoylation.

Acylation↗
PubMed2006

Directed evolution of a bacterial efflux pump: adaptation of the E. coli TolC exit duct to the Pseudomonas MexAB translocase.

Bacterial multidrug efflux pumps operate by periplasmic recruitment and opening of TolC family outer membrane exit ducts by cognate inner membrane translocases. Directed evolution of active hybrid pumps was achieved by challenging a library of mutated, shuffled TolC variants to adapt to the non-cognate Pseudomonas MexAB translocase, and confer resistance to the efflux substrate novobiocin. Amino acid substitutions in MexAB-adapted TolC variants that endowed high resistance were recreated independently, and revealed that MexAB-adaptation was conferred only by substitutions located in the lower alpha-helical barrel of TolC, specifically the periplasmic equatorial domain and entrance coiled coils. These changes converge to the native MexAB partner OprM, and indicate an interface key to the function and diversity of efflux pumps.

Anti-Bacterial Agents↗
PubMed2005

Subunits of the yeast mitochondrial ADP/ATP carrier: cooperation within the dimer.

The mitochondrial ADP/ATP carrier, or Ancp, is a member of the mitochondrial carrier family (MCF). It exchanges ADP and ATP between matrix and intermembrane space. It is postulated from numerous experiments that the inactive Ancp bound to one of its inhibitors (CATR or BA) is a dimer, and it is inferred that the active unit is a dimer, too. However, the structure of beef Ancp bound to CATR obtained at high resolution is that of a monomer. To ascertain the dimeric organization of Ancp, we have constructed covalent tandem dimers of which one "subunit" (protomer) is the wild type and the other is inactive for ADP/ATP exchange. We have chosen either the op1 mutant or another member of the MCF, the phosphate carrier (Picp). Activities of the chimeras were first evaluated in vivo. The Ancp/op1 constructs exchange the adenine nucleotides. The Anc/Pic chimeras are considered as bifunctional forms since they exchange ADP and ATP and transport P(i) within the same cells. We have then controlled the fact that the chimeras are stable in vivo and in vitro. Proteinase K digestion showed that both protomers of Ancp/op1 have similar organization in the membrane. Analyses of kinetic properties indicated that protomers of Ancp/op1 chimeras crosstalk during the nucleotide exchange unlike those of Anc/Pic. However, full inhibition of phosphate uptake by CATR, a very specific inhibitor of Ancp, strongly suggests that the native functional unit of Ancp, and thus of Picp, is a dimer.

Atractyloside↗
PubMed2005

A genome-wide visual screen reveals a role for sphingolipids and ergosterol in cell surface delivery in yeast.

Recently synthesized proteins are sorted at the trans-Golgi network into specialized routes for exocytosis. Surprisingly little is known about the underlying molecular machinery. Here, we present a visual screen to search for proteins involved in cargo sorting and vesicle formation. We expressed a GFP-tagged plasma membrane protein in the yeast deletion library and identified mutants with altered marker localization. This screen revealed a requirement of several enzymes regulating the synthesis of sphingolipids and ergosterol in the correct and efficient delivery of the marker protein to the cell surface. Additionally, we identified mutants regulating the actin cytoskeleton (Rvs161p and Vrp1p), known membrane traffic regulators (Kes1p and Chs5p), and several unknown genes. This visual screening method can now be used for different cargo proteins to search in a genome-wide fashion for machinery involved in post-Golgi sorting.

Biological Transport↗
PubMed2004

Nuclear export of the oncoprotein v-ErbA is mediated by acquisition of a viral nuclear export sequence.

v-ErbA, an oncogenic derivative of the thyroid hormone receptor alpha (TRalpha) carried by the avian erythroblastosis virus, contains several alterations including fusion of a portion of avian erythroblastosis virus Gag to its N terminus, N- and C-terminal deletions, and 13 amino acid substitutions. Nuclear export of v-ErbA occurs through a CRM1-mediated pathway. In contrast, nuclear export of TRalpha and another isoform, TRbeta, is CRM1-independent. To determine which amino acid changes in v-ErbA confer CRM1-dependent nuclear export, we expressed a panel of green and yellow fluorescent protein-tagged mutant and chimeric proteins in mammalian cells. The sensitivity of subcellular trafficking of these mutants to leptomycin B (LMB), a specific inhibitor of CRM1, was assessed by fluorescence microscopy. Our data showed that a nuclear export sequence resides within a 70-amino acid domain in the C-terminal portion of the p10 region of Gag, and in vitro binding assays demonstrated that Gag interacts directly with CRM1. However, a panel of ligand-binding domain mutants of v-ErbA lacking the Gag sequence exhibited greater nuclear localization in the presence of LMB, suggesting that the various amino acid substitutions/deletions may cause a conformation shift, unmasking an additional CRM1-dependent nuclear export sequence. In contrast, the altered DNA-binding domain of the oncoprotein did not contribute to CRM1-dependent nuclear export. Heterokaryon experiments revealed that v-ErbA did not undergo nucleocytoplasmic shuttling when the CRM1 export pathway was blocked by LMB treatment, suggesting that the ability to follow the export pathway used by TRalpha has been lost by the oncoprotein during its evolution. Our findings thus point to the intriguing possibility that acquisition of altered nuclear export capabilities contributes to the oncogenic properties of v-ErbA.

Active Transport, Cell Nucleus↗
PubMed1995

Construction of murine coronavirus mutants containing interspecies chimeric nucleocapsid proteins.

Targeted RNA recombination was used to construct mouse hepatitis virus (MHV) mutants containing chimeric nucleocapsid (N) protein genes in which segments of the bovine coronavirus N gene were substituted in place of their corresponding MHV sequences. This defined portions of the two N proteins that, despite evolutionary divergence, have remained functionally equivalent. These regions included most of the centrally located RNA-binding domain and two putative spacers that link the three domains of the N protein. By contrast, the amino terminus of N, the acidic carboxy-terminal domain, and a serine- and arginine-rich segment of the central domain could not be transferred from bovine coronavirus to MHV, presumably because these parts of the molecule participate in protein-protein interactions that are specific for each virus (or, possibly, each host). Our results demonstrate that targeted recombination can be used to make extensive substitutions in the coronavirus genome and can generate recombinants that could not otherwise be made between two viruses separated by a species barrier. The implications of these findings for N protein structure and function as well as for coronavirus RNA recombination are discussed.

Amino Acid Sequence↗
PubMed1994

Membrane anchoring domain of herpes simplex virus glycoprotein gB is sufficient for nuclear envelope localization.

We have used the glycoprotein gB of herpes simplex virus type 1 (gB-1), which buds from the inner nuclear membrane, as a model protein to study localization of membrane proteins in the nuclear envelope. To determine whether specific domains of gB-1 glycoprotein are involved in localization in the nuclear envelope, we have used deletion mutants of gB-1 protein as well as chimeric proteins constructed by replacing the domains of the cell surface glycoprotein G of vesicular stomatitis virus with the corresponding domains of gB. Mutant and chimeric proteins expressed in COS cells were localized by immunoelectron microscopy. A chimeric protein (gB-G) containing the ectodomain of gB and the transmembrane and cytoplasmic domains of G did not localize in the nuclear envelope. When the ectodomain of G was fused to the transmembrane and cytoplasmic domains of gB, however, the resulting chimeric protein (G-gB) was localized in the nuclear envelope. Substitution of the transmembrane domain of G with the 69 hydrophobic amino acids containing the membrane anchoring domain of gB allowed the hybrid protein (G-tmgB) to be localized in the nuclear envelope, suggesting that residues 721 to 795 of gB can promote retention of proteins in the nuclear envelope. Deletion mutations in the hydrophobic region further showed that a transmembrane segment of 21 hydrophobic amino acids, residues 774 to 795 of gB, was sufficient for localization in the nuclear envelope. Since wild-type gB and the mutant and chimeric proteins that were localized in the nuclear envelope were also retained in the endoplasmic reticulum, the membrane spanning segment of gB could also influence retention in the endoplasmic reticulum.

Animals↗
PubMed1986

Synthesis of growth hormone-prolactin chimeric proteins and processing mutants by the exchange and deletion of genomic exons.

To test the feasibility of synthesizing recombinant peptide hormones by exon deletion and exchange, we have constructed and expressed hybrid human growth hormone (hGH)-rat prolactin (rPrl) genes in which the third and fourth exons of the hGH gene are deleted and separately replaced by the corresponding exons of the rPrl gene. These exon deletion and exon exchange genes were inserted into an SV40 viral vector, packaged, and expressed following acute viral infection of monkey kidney cells. Expression of the hGH gene lacking the third exon (hGHd3) was not detectable at the level of protein production. However, replacement of the deleted third exon in the hGHd3 gene with exon 3 of the rPrl gene (hGHP3) resulted in the efficient synthesis of a secreted chimeric protein. When the fourth exon of the hGH gene was deleted (hGHd4), the encoded protein was found only in the cytosol despite signal sequence processing. Replacement of the deleted fourth exon in this hGHd4 gene with exon 4 of rPrl resulted in the synthesis and secretion of both a chimeric protein (hGHP4) as well as a larger protein corresponding in size to prehGHP4. These results suggest that exon exchange among distantly related genes in the GH family may be used to produce high levels of chimeric GH-related proteins, and regions internal to the hGH protein may be critical in establishing normal protein processing and secretion.

Chimera↗