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M G Roth

Publications and source records attributed to M G Roth.

At least 55 records · Page 3Linked to original sources
PubMed1993

Basis for selective incorporation of glycoproteins into the influenza virus envelope.

The ability of mutant or chimeric A/Japan hemagglutinins (HAs) to compete for space in the envelope of A/WSN influenza viruses was investigated with monkey kidney fibroblasts that were infected with recombinant simian virus 40 vectors expressing the Japan proteins and superinfected with A/WSN influenza virus. Wild-type Japan HA assembled into virions as well as WSN HA did. Japan HA lacking its cytoplasmic sequences, HAtail-, was incorporated into influenza virions at half the efficiency of wild-type Japan HA. Chimeric HAs containing the 11 cytoplasmic amino acids of the herpes simplex virus type 1gC glycoprotein or the 29 cytoplasmic amino acids of the vesicular stomatitis virus G protein were incorporated into virions at less than 1% the efficiency of HAtail-. Thus, the cytoplasmic domain of HA was not required for the selection process; however, foreign cytoplasmic sequences, even short ones, were excluded. A chimeric HA having the gC transmembrane domain and the HA cytoplasmic domain (HgCH) was incorporated at 4% the efficiency of HAtail-. When expressed from simian virus 40 recombinants in this system, vesicular stomatitis virus G protein with or without (Gtail-) its cytoplasmic domain was essentially excluded from influenza virions. Taken together, these data indicate that the HA transmembrane domain is required for incorporation of HA into influenza virions. The slightly more efficient incorporation of HgCH than G or Gtail- could indicate that the region important for assembling HA into virions extends into part of the cytoplasmic domain.

Amino Acid Sequence↗
PubMed1992

The cytoplasmic domain of the polymeric immunoglobulin receptor contains two internalization signals that are distinct from its basolateral sorting signal.

The C-terminal cytoplasmic domain of the polymeric immunoglobulin receptor (pIgR) contains two tyrosine residues, Tyr668 and Tyr734. Previous work identifying Tyr734 as a critical residue in the endocytosis of the pIgR in Madin-Darby canine kidney (MDCK) cells also suggested that a second functional internalization signal was present (Breitfeld, P. P., Casanova, J. E., McKinnon, W. C., and Mostov, K. E. (1990) J. Biol. Chem. 265, 13750-13757). To test this hypothesis, Tyr668 and Tyr734 were mutated singly or together by oligonucleotide-directed mutagenesis of pIgR cDNA, and the mutants were expressed in MDCK cells. The amount of ligand internalized within 5 min from the basolateral membrane by the pIgR in which cytoplasmic tyrosines were mutated separately to Cys668 or Ser734 or together to Cys668, Ser734 was 58, 39, and 20%, respectively, of the internalized by the wild-type pIgR. The cytoplasmic and transmembrane domains of the pIgR, when joined to the external domain of the influenza virus hemagglutinin, retained the capacity to mediate rapid internalization. As with the full-length pIgR, mutation of either tyrosine in the chimera resulted in impairment of endocytosis, with mutation of Tyr734 having a significantly greater effect than mutation on Tyr668 on the initial rate of endocytosis (3 and 44% of control values, respectively). However, unlike the full-length pIgR, mutation of both tyrosines together in the chimera did not reduce internalization further. The two tyrosines in the cytoplasmic sequence of the pIgR, although widely separated in the linear amino acid sequence, both contribute to internalization of the protein, suggesting that both can function as internalization signals. In addition, the correlation between endocytosis and basolateral targeting of the pIgR in MDCK cells was investigated. Neither tyrosine of the cytoplasmic domain was necessary for basolateral targeting of the pIgR.

Amino Acid Sequence↗
PubMed1992

Action of brefeldin A blocked by activation of a pertussis-toxin-sensitive G protein.

In many mammalian cells brefeldin A interferes with mechanisms that keep the Golgi appartus separate from the endoplasmic reticulum. The earliest effect of brefeldin A is release of the coat protein beta-COP from the Golgi. This release is blocked by pretreatment with GTP-gamma S or AlF4- (ref. 12). The AlF4- ion activates heterotrimeric G proteins but not proteins of the ras superfamily, suggesting that a heterotrimeric G protein might control membrane transfer from the endoplasmic reticulum to the Golgi. We report here that mastoparan, a peptide that activates heterotrimeric G proteins, promotes binding of beta-COP to Golgi membranes in vitro and antagonizes the effect of brefeldin A on beta-COP in perforated cells and on isolated Golgi membranes. This inhibition is greatly diminished if cells are pretreated with pertussis toxin before perforation. Thus, a heterotrimeric G protein of the Gi/Go subfamily regulates association of coat components with Golgi membranes.

Brefeldin A↗
PubMed1992

A chimeric avian retrovirus containing the influenza virus hemagglutinin gene has an expanded host range.

We have investigated what protein sequences are necessary for glycoprotein incorporation into Rous sarcoma virus (RSV) virions by utilizing the hemagglutinin (HA) protein of influenza virus. Two chimeric HA genes were constructed. In the first the coding sequence for the signal peptide of the RSV env gene product was fused in frame to the entire HA structural gene, and in the second the hydrophobic anchor and cytoplasmic domain sequences of the HA gene were also replaced with those from the RSV env gene. Both chimeric genes, expressed from a simian virus 40 expression vector in CV-1 cells, yielded functional HA proteins that were transported to the cell surface and were able to bind to erythrocytes. When the genes were expressed in combination with the RSV gag-pol gene region in QT6 cells by using a vaccinia virus-T7 expression/complementation system, virions that efficiently incorporated either chimeric protein were assembled. This result indicated that the presence of the RSV env membrane anchor and cytoplasmic sequences did not facilitate HA glycoprotein incorporation into virions. The presence of the RSV env signal sequence allowed the chimeric HA genes to be substituted into the RSV-derived BH-RCAN.HiSV viral genome in place of the RSV env gene. Both chimeric genomes yielded infectious virus that could infect human and avian cells with equal efficiency. These experiments demonstrate that a foreign glycoprotein, efficiently incorporated into virions lacking a native glycoprotein, can confer a broadened host range on the virus. Moreover, because the HA of influenza virus requires the acidic pH of the endosome in order to be activated, these results imply that foreign proteins can modify the normal route of entry of this avian retrovirus.

Amino Acid Sequence↗
PubMed1991

PtK1 cells contain a nondiffusible, dominant factor that makes the Golgi apparatus resistant to brefeldin A.

Brefeldin A (BFA) was shown in earlier studies of numerous cell types to inhibit secretion, induce enzymes of the Golgi stacks to redistribute into the ER, and to cause the Golgi cisternae to disappear. Here, we demonstrate that the PtK1 line of rat kangaroo kidney cells is resistant to BFA. The drug did not disrupt the morphology of the Golgi complex in PtK1 cells, as judged by immunofluorescence using antibodies to 58- (58K) and 110-kD (beta-COP) Golgi proteins, and by fluorescence microscopy of live cells labeled with C6-NBD-ceramide. In addition, BFA did not inhibit protein secretion, not alter the kinetics or extent of glycosylation of the vesicular stomatitis virus (VSV) glycoprotein (G-protein) in VSV-infected PtK1 cells. To explore the mechanism of resistance to BFA, PtK1 cells were fused with BFA-sensitive CV-1 cells that had been infected with a recombinant SV-40 strain containing the gene for VSV G-protein and, at various times following fusion, the cultures were exposed to BFA. Shortly after cell fusion, heterokaryons contained one Golgi complex associated with each nucleus. Golgi membranes derived from CV-1 cells were sensitive to BFA, whereas those of PtK1 origin were BFA resistant. A few hours after fusion, most heterokaryons contained a single, large Golgi apparatus that was resistant to BFA and contained CV-1 galactosyltransferase. In unfused cells that had been perforated using nitrocellulose filters, retention of beta-COP on the Golgi was optimal in the presence of cytosol, ATP, and GTP. In perforated cell models of the BFA-sensitive MA104 line, BFA caused beta-COP to be released from the Golgi complex in the presence of nucleotides, and either MA104 or PtK1 cytosol. In contrast, when perforated PtK1 cells were incubated with BFA, nucleotides, and cytosol from either cell type, beta-COP remained bound to the Golgi complex. We conclude that PtK1 cells contain a nondiffusible factor, which is located on or very close to the Golgi complex, and confers a dominant resistance to BFA. It is possible that this factor is homologous to the target of BFA in cells that are sensitive to the drug.

Animals↗
PubMed1991

A single amino acid change in the cytoplasmic domain alters the polarized delivery of influenza virus hemagglutinin.

In the polarized kidney cell line MDCK, the influenza virus hemagglutinin (HA) has been well characterized as a model for apically sorted membrane glycoproteins. Previous work from our laboratory has shown that a single amino acid change in the cytoplasmic sequence of HA converts it from a protein that is excluded from coated pits to one that is efficiently internalized. Using trypsin or antibodies to mark protein on the surface, we have shown in MDCK cells that HA containing this mutation is no longer transported to the apical surface but instead is delivered directly to the basolateral plasma membrane. We propose that a cytoplasmic feature similar to an endocytosis signal can cause exclusive basolateral delivery.

Animals↗
PubMed1991

Evidence from lateral mobility studies for dynamic interactions of a mutant influenza hemagglutinin with coated pits.

Replacement of cysteine at position 543 by tyrosine in the influenza virus hemagglutinin (HA) protein enables the endocytosis of the mutant protein (Tyr 543) through coated pits (Lazarovits, J., and M. G. Roth. 1988. Cell. 53:743-752). To investigate the interactions between Tyr 543 and the clathrin coats in the plasma membrane of live cells, we performed fluorescence photobleaching recovery measurements comparing the lateral mobilities of Tyr 543 (which enters coated pits) and wild-type HA (HA wt, which is excluded from coated pits), following their expression in CV-1 cells by SV-40 vectors. While both proteins exhibited the same high mobile fractions, the lateral diffusion rate of Tyr 543 was significantly slower than that of HA wt. Incubation of the cells in a sucrose-containing hypertonic medium, a treatment that disperses the membrane-associated coated pits, resulted in similar lateral mobilities for Tyr 543 and HA wt. These findings indicate that the lateral motion of Tyr 543 (but not of HA wt) is inhibited by transient interactions with coated pits (which are essentially immobile on the time scale of the lateral mobility measurements). Acidification of the cytoplasm by prepulsing the cells with NH4Cl (a treatment that arrests the pinching-off of coated vesicles from the plasma membrane and alters the clathrin lattice morphology) led to immobilization of a significant part of the Tyr 543 molecules, presumably due to their entrapment in coated pits for the entire duration of the lateral mobility measurement. Furthermore, in both untreated and cytosol-acidified cells, the restrictions on Tyr 543 mobility were less pronounced in the cold, suggesting that the mobility-restricting interactions are temperature dependent and become weaker at low temperatures. From these studies we conclude the following. (a) Lateral mobility measurements are capable of detecting interactions of transmembrane proteins with coated pits in intact cells. (b) The interactions of Tyr 543 with coated pits are dynamic, involving multiple entries of Tyr 543 molecules into and out of coated pits. (c) Alterations in the clathrin lattice structure can modulate the above interactions.

Biological Transport↗
PubMed1990

The effects of foreign transmembrane domains on the biosynthesis of the influenza virus hemagglutinin.

Eleven chimeric proteins were created in which the transmembrane, the cytoplasmic, or both topological domains of the influenza virus hemagglutinin (HA) were replaced with those from five other glycoproteins. All of the chimeric HAs reached the cell surface but appeared to differ in the degree to which they were stably folded. Comparisons of the rates of folding, passage into the Golgi, and arrival at the plasma membrane of wild-type HA and the chimeric proteins suggest that formation of a stable HA trimer is not an absolute requirement for export from the endoplasmic reticulum. In addition, there appear to be at least two steps at which the rate of transport can be altered during exocytosis, one occurring before and the other after the trimming of oligosaccharides by Golgi mannosidases. Certain of the chimeras differed from HA in their ability to pass through each of these steps. Replacement of the HA transmembrane domain with the analogous sequences from other proteins affected folding and transport of the chimeric HAs in ways that suggest that the HA transmembrane sequences form a specific structure in the membrane that differs from that formed by analogous sequences from the other proteins.

Amino Acid Sequence↗
PubMed1990

Characteristics of the tyrosine recognition signal for internalization of transmembrane surface glycoproteins.

A tyrosine residue in the cytoplasmic domain of a class of cell surface receptors is necessary, but not sufficient, for internalization through coated pits. To identify the amino acid context enabling a tyrosine to serve as a signal for endocytosis, we mutated the short cytoplasmic domain of a mutant influenza virus hemagglutinin that is competent for internalization, HA-Y543, and determined the effect of each change on internalization. From these results and a comparison of sequences of other proteins recognized by coated pits, a "tyrosine internalization signal" was proposed. Site-directed mutagenesis was employed to insert complete, or incomplete "tyrosine internalization signals" into the cytoplasmic domain of a protein normally not endocytosed, human glycophorin A. Only the complete signal caused internalization of mutant glycophorins by coated pits. The signal is formed by a short amino acid sequence, with polar or basic residues preferred at certain positions on either side of the tyrosine. Amino acids, which in proteins of known structure are frequently found in turns, are clustered near the tyrosine on the side of the signal nearest the transmembrane domain.

Amino Acid Sequence↗
PubMed1989

Differential extractability of influenza virus hemagglutinin during intracellular transport in polarized epithelial cells and nonpolar fibroblasts.

Biochemical changes in the influenza virus hemagglutinin during intracellular transport to the apical plasma membrane of epithelial cells were investigated in Madin-Darby canine kidney (MDCK) cells and in LLC-PK1 cells stably transfected with a hemagglutinin gene. After pulse-labeling a substantial fraction of hemagglutinin was observed to become insoluble in isotonic solutions of Triton X-100. Insolubility of hemagglutinin was detected late in the transport pathway after addition of complex sugars in the Golgi complex but before insertion of the protein in the plasma membrane. Insolubility was not dependent on oligosaccharide modification since deoxymannojirimycin (dMM), which inhibits mannose trimming, failed to prevent its onset. Insolubility was not due to assembly of virus particles at the plasma membrane because insoluble hemagglutinin was also observed in transfected cells. Hemagglutinin insolubility was also seen in MDCK cells cultured in suspension and in chick embryo fibroblasts, indicating that insolubility and plasma membrane polarity are not simply correlated. In addition to insolubility, an apparent transport-dependent reduction of the disulfide bond linking HA1 and HA2 in hemagglutinin was detected. Because of the timing of both insolubility and the loss of the disulfide bond, these modifications may be important in the delivery of the hemagglutinin to the cell surface.

1-Deoxynojirimycin↗
PubMed1989

Molecular biological approaches to protein sorting.

The use of recombinant DNA technology to introduce specifically altered genes into epithelial cells has passed through its introductory phase. Enough data have accumulated to indicate the major problems and opportunities for this approach. The greatest opportunity is for locating the features of glycoproteins that are recognized during sorting through the expression of specifically altered proteins in epithelial cells. The results of experiments of this kind briefly summarized here emphasize the need for detailed analyses of the effects of specific mutations on the structure of the mutant proteins. In addition, progress will require quantitative results that have been difficult to obtain to date. To accomplish this, the level of expression of mutant proteins in polarized cells must be higher than previously has been the case. This in turn requires that an effort be made to identify vectors specifically adapted for use in epithelial cells.

Animals↗
PubMed1989

Evaluation and characterization of the hyt/hyt hypothyroid mouse. II. Abnormalities of TSH and the thyroid gland.

The hyt/hyt mouse (BALB/cBY-hyt, C.hytRF) provides a useful model for exploring the effect of inherited severe primary hypothyroidism. Studies were undertaken to try to define the basis of the primary hypothyroidism in mice homozygous for the autosomal recessive gene, hyt. These mice had congenital hypothyroidism of fetal onset after 15 days post conception. Through their lifetime, the hyt/hyt mice had reduced serum thyroxine (T4), triiodothyronine (T3), reduced thyroid gland intralumenal colloid on electron microscopy and a 100-fold elevation of TSH-like activity compared to hyt/+ littermates. Thyroglobulin made in hyt/hyt animals was similar in size to normal thyroglobulin which was inconsistent with a major structural thyroglobulin gene defect. The thyroglobulin was iodinated. Marked, erratic dilation of rough endoplasmic reticulum (RER) was noted in hyt/hyt mouse follicular cells. Despite these ultrastructural findings, pulse chase and immunoprecipitation studies with isolated hyt/hyt and normal thyroid glands indicated that normal thyroglobulin processing occurred in the RER and Golgi of the hyt/hyt mice. The hyt/hyt thyroid glands were hypoplastic compared to hyt/+ littermates. Histologically, the hyt/hyt thyroid glands demonstrated an increase in smaller follicular cells, and greater variability in follicular size compared to hyt/+ littermates. Histological and ultrastructural abnormalities in the gland were similar to those seen in certain cases of human congenital hypothyroidism with TSH receptor insensitivity of the thyroid gland. These findings along with the significant TSH elevation, the reduction in colloid and in serum T3 and T4, the efficacy of the hypothalamo-pituitary-thyroid feedback system, and previous observations of reduced iodine uptake and intrathyroidal T4, suggested that primary hypothyroidism in the hyt/hyt mouse might be due to a defect in TSH responsivity of the thyroid gland.

Animals↗
PubMed1987

The large external domain is sufficient for the correct sorting of secreted or chimeric influenza virus hemagglutinins in polarized monkey kidney cells.

MA104.11 rhesus kidney cells express several characteristics of polarized epithelial cells, including the formation of "domes" on impermeable substrates, the establishment of a transmonolayer electrical resistance when grown on collagen gels, the polarized maturation of influenza and vesicular stomatitis viruses, and the expression of the glycoproteins of those viruses at a single surface domain. The polarized expression of the influenza virus hemagglutinin (HA) is maintained in MA104.11 cells infected with SV40-derived vectors carrying a cDNA gene for either the wild-type influenza virus HA, a truncated HA gene encoding a secreted form of HA (HAsec), or a chimeric gene encoding a hybrid protein with the external domain of the HA and the transmembrane and cytoplasmic domains of the vesicular stomatitis virus G protein (HAG). Thus, the recognition event separating glycoproteins, such as HA, destined for the apical surface from proteins, such as G, destined for the basolateral membranes involves features of the external domains of the proteins. The transmembrane and cytoplasmic domains of HA have no role in this process.

Animals↗
PubMed1986

Heterologous transmembrane and cytoplasmic domains direct functional chimeric influenza virus hemagglutinins into the endocytic pathway.

Chimeric genes were created by fusing DNA sequences encoding the ectodomain of the influenza virus hemagglutinin (HA) to DNA coding for the transmembrane and cytoplasmic domains of either the G glycoprotein of vesicular stomatitis virus or the gC glycoprotein of Herpes simplex virus 1. CV-1 cells infected with SV40 vectors carrying the recombinant genes expressed large amounts of the chimeric proteins, HAG or HAgC on their surfaces. Although the ectodomains of HAG and HAgC differed in their immunological properties from that of HA, the chimeras displayed the biological functions characteristic of the wild-type protein. Both HAG and HAgC bound erythrocytes as efficiently as HA did and, after brief exposure to an acidic environment, induced the fusion of erythrocyte and CV-1 cell membranes. However, the behavior of HAG and HAgC at the cell surface differed from that of HA in several important respects. HAG and HAgC were observed to collect in coated pits whereas wild-type HA was excluded from those structures. In the presence of chloroquine, which inhibits the exit of receptors from endosomes, HAG and HAgC accumulated in intracellular vesicles. By contrast, chloroquine had no effect on the location of wild-type HA. HAG and HAgC labeled at the cell surface exhibited a temperature-dependent acquisition of resistance to extracellular protease at a rate similar to the rates of internalization observed for many cell surface receptors. HA acquired resistance to protease at a rate at least 20-fold slower. We conclude that HAG and HAgC are efficiently routed into the endocytic pathway and HA is not. However, like HA, HAG was degraded slowly, raising the possibility that HAG recycles to the plasma membrane.

Animals↗
PubMed1985

Mutations in the cytoplasmic domain of the influenza virus hemagglutinin affect different stages of intracellular transport.

Mutations have been introduced into the cloned DNA sequences coding for influenza virus hemagglutinin (HA), and the resulting mutant genes have been expressed in simian cells by the use of SV40-HA recombinant viral vectors. In this study we analyzed the effect of specific alterations in the cytoplasmic domain of the HA molecule on its rate of biosynthesis and transport, cellular localization, and biological activity. Several of the mutants displayed abnormalities in the pathway of transport from the endoplasmic reticulum to the cell surface. One mutant HA remained within the endoplasmic reticulum; others were delayed in reaching the Golgi apparatus after core glycosylation had been completed in the endoplasmic reticulum, but then progressed at a normal rate from the Golgi apparatus to the cell surface; another was delayed in transport from the Golgi apparatus to the plasma membrane. However, two mutants were indistinguishable from wild-type HA in their rate of movement from the endoplasmic reticulum through the Golgi apparatus to the cell surface. We conclude that changes in the cytoplasmic domain can powerfully influence the rate of intracellular transport and the efficiency with which HA reaches the cell surface. Nevertheless, absolute conservation of this region of the molecule is not required for maturation and efficient expression of a biologically active HA on the surface of infected cells.

Animals↗
PubMed1983

Influenza virus hemagglutinin expression is polarized in cells infected with recombinant SV40 viruses carrying cloned hemagglutinin DNA.

Primary cell cultures of African Green monkey kidney (AGMK) contain polarized epithelial cells in which influenza virus matures predominantly at the apical surfaces above tight junctions. Influenza virus glycoproteins were found to be localized at the same membrane domain from which the virus budded. When polarized primary AGMK cells were infected with recombinant SV40 viruses containing DNA coding for either an influenza virus H1 or H2 subtype hemagglutinin (HA), the HA proteins were preferentially expressed at the apical surface in a manner identical to that observed in influenza virus-infected cells. Thus, cellular mechanisms for sorting membrane glycoproteins recognize some structural feature of the HA glycoprotein itself, and other viral proteins are not necessary for this process.

Animals↗
PubMed1983

Basolateral maturation of retroviruses in polarized epithelial cells.

We have investigated the maturation sites of avian and mammalian C-type retroviruses in polarized epithelial cells. Examination of thin sections of Madin Darby canine kidney cells infected with RD114 or avian reticuloendotheliosis virus revealed that these viruses mature from the basolateral membrane domains. Similar results were obtained with a continuous line of mouse mammary epithelial cells infected with Friend, Moloney, Rauscher, or Kirsten murine leukemia viruses, or Friend virus-related or Moloney virus-related mink cell focus-forming viruses. Immunofluorescence observations indicate that viral glycoproteins are inserted only at the basolateral membranes in these cells. Because of the availability of DNA and protein sequence data, and of molecularly cloned viruses, these virus systems offer advantages for molecular studies on directional transport of plasma membrane glycoproteins.

Animals↗