Isolation and characterization of a viable adenovirus mutant defective in nuclear transport of the DNA-binding protein.

The isolation and characterization of an adenovirus mutant, Ad5dl802r1, containing two independent deletions in the 72-kilodalton (kDa) DNA-binding protein (DBP) gene is described. The two deletions remove amino acids 23 through 105 of DBP, resulting in the production of a 50-kDa product. Expression of this truncated DBP was delayed 12 to 24 h compared with that of the 72-kDa protein produced by wild-type adenovirus type 5. The DBP was located primarily in the cytoplasm of infected cells, whereas the wild-type product was predominantly nuclear. Therefore, DBP appears to contain a nuclear localization signal within the deleted region. Ad5dl802r1 DNA synthesis, viral late gene expression, and virus production were all delayed 12 to 24 h and were approximately 10-fold lower than with wild-type adenovirus type 5. These phenotypic properties can be accounted for by the delay in synthesis and the inefficient accumulation of the 50-kDa DBP within the nucleus of infected cells. The truncated DBP also lacks the majority of amino acids which are phosphorylated in the normal protein. The loss of these phosphorylation sites does not appear to seriously impair the ability of the protein to carry out its functions.     

A toxin-resistant mouse L-cell mutant defective in protein transport along the secretory pathway.

Using methods designed for isolation of mutants defective in receptor-mediated endocytosis, a novel L-cell mutant was obtained that exhibits resistance to three different protein toxins as well as alterations in secretion. This mutant, LEFIC, is resistant to modeccin, Pseudomonas exotoxin, and ricin. These toxins, which enter the cytoplasm via receptor-mediated endocytosis, are thought to penetrate into cells at the level of late endosomes or the trans Golgi network. Early endosomal acidification appears to be normal in the mutant based on its accumulation of iron from transferrin and its sensitivity to diphtheria toxin A chain-transferrin conjugate. Within the secretory pathway two delays in transport of vesicular stomatitis virus (VSV) G protein were observed in LEFIC: a 20-30 min delay in acquisition of Endo H resistance and a 1-2 hr delay in appearance of newly synthesized G protein on the cell surface. Movement of endogenous proteins along the secretory pathway was also affected in LEFIC. Fibronectin secretion was delayed by 15 min, and membrane proteins were delayed in arrival at the cell surface. The phenotype of LEFIC is consistent with a defect in a component or compartment shared by both the late endocytic and constitutive secretory pathways.     

Isolation and characterization of a yeast mutant defective in cholinephosphotransferase

A yeast mutant defective in cholinephosphotransferase (cpt) was isolated as a revertant from a choline-sensitive mutant, which exhibited lowered phosphatidylinositol synthesis. A block at the cholinephosphotransferase step in the mutant was indicated by the enzyme defect and the accumulation of CDP-choline in the cells with a decrease in phosphatidylcholine synthesis. The defect was due to a single recessive mutation in a nuclear gene. The residual activity in the mutant showed an increased apparent Km for CDP-choline and an altered sensitivity to Tween 20. Thus the structural gene may be affected in the mutant. The occurrence of an intact ethanolaminephosphotransferase in the mutant indicates the distinctness of the genes encoding cholinephosphotransferase and ethanolaminephosphotransferase in yeast. The present selection method was also effective for isolating mutants defective in the other steps of the CDP-choline pathway and choline transport.     

Isolation and initial characterization of a temperature-sensitive mutant of mouse FM3A cells defective in cytokinesis

A temperature-sensitive mutant, designated tsFT101, was isolated from a mouse mammary carcinoma cell line, FM3A, and given an initial characterization. In this cell line, cytokinesis was blocked at a non-permissive temperature (39 degrees C), but DNA synthesis and nuclear division proceeded normally for at least 24 h at 39 degrees C as detected respectively by autoradiography and cytofluorometric analysis. As a result, multinucleate cells accumulated at 39 degrees C (more than 95% in 36 h). When the culture was returned to a permissive temperature (33 degrees C) after 24 h of arrest at 39 degrees C, cytokinesis was resumed and there was a rapid decrease in the number of multinucleate cells. At 39 degrees C, tsFT101 cells had less F-actin than cells at 33 degrees C, indicative of the existence of an abnormality in actin polymerization in this mutant.     

Construction and characterization of an SV40 mutant defective in nuclear transport of T antigen

An SV40-adenovirus 7 hybrid virus, PARA(cT), has been described that is defective for the nuclear transport of SV40 large tumor antigen. An SV40(cT) mutant was constructed using SV40 early and late region DNA fragments derived from PARA(cT) and wild-type SV40 respectively. The SV40(cT)-3 construct is defective for viral replication, but can be propagated in COS-1 cells. T antigen induced by SV40(cT)-3 is localized in the cytoplasm of infected cells. The cT mutation also inhibits the transport of wild-type T antigen; COS-1 cells lose their constitutive expression of nuclear T antigen after infection with SV40(cT)-3. Sequence analysis revealed that the cT mutation results in the replacement of a positively charged lysine in wild-type T antigen with a neutral asparagine at amino acid number 128, demonstrating that the alteration of a single amino acid is sufficient to abolish nuclear transport. Implications of the cT mutation on possible mechanisms for the transport of proteins to the nucleus are discussed.     

Bromoconduritol treatment delays intracellular transport of secretory glycoproteins in human hepatoma cell cultures

Previous studies in our laboratory have shown that specific glycan structures are required for the normal secretion of some glycoproteins. Bromoconduritol is known to inhibit the removal of the innermost glucose moiety from the Glc3Man9(GlcNAc)2 precursor of N-linked glycoproteins. We have used this inhibitor to investigate the possible role of glycan structure in the intracellular transport of secretory glycoproteins of Hep G2 cultures. Cells were pretreated with 1mM bromoconduritol for 1h, pulsed with [35S]-methionine for 10min and chased for varying intervals. Specific glycoproteins and albumin were immunoprecipitated from the cell lysate and medium. We found that bromoconduritol-treatment inhibited the secretion of alpha 1-protease inhibitor, ceruloplasmin, alpha 2-macroglobulin, transferrin, and alpha-fetoprotein. Apparently, the glucosylated high-mannose intermediate is not secreted, since glycoproteins in the medium are of complex form. We conclude that the removal of the innermost glucose residue from secretory glycoprotein represents an important regulatory step in the intracellular transport pathway.     

Changes in cell surface and intracellular glycoproteins of trophoblastic giant cells during mouse placentation

Summary Changes in lectin bindings of mouse trophoblastic giant cells (TGCs) were examined by light and electron microscopy. Neither Griffonia simplicifolia agglutinin (GS)-II nor succinyl-wheat germ agglutinin (s-WGA) bound to the 1st and 2nd TGCs on day 6.5 post coitum (p.c.), but did so from days 8.5 to 12.5 p.c. Positive reactions with s-WGA were localized in the perinuclear region and cell surface of both 1st and 2nd TGCs; while GS-II bound only to the perinuclear region, where it appeared as network-like deposits. This region was identified as well-developed Golgi lamellae by electron microscopy. Moreover, SDS-PAGE and lectin-blot analysis of the 1st TGCs indicated that the intensity of s-WGA and GS-II bindings increased in the glycoproteins of approximately 43, 40, 37, and 26 kDa and in those of 43 and 38 kDa, respectively, during the 8.5th to 10.5th day p.c. The reaction with GS-I was detected on cell surface of both the 1st and 2nd TGCs on day 6.5 p.c. The reaction in the 1st TGCs was intensely positive throughout their development, whereas the reactivity decreased in the 2nd TGCs on day 10.5 p.c. and completely disappeared on day 12.5 p.c. The GS-I reaction in TGCs was more intense at the maternal side than at the embryonic side. These results suggest that certain Gal and/or GlcNAc glycoproteins on the cell surface and in Golgi lamellae of TGCs dynamically change from the 8.5th to 10.5th day p.c. in association with mouse placentation.     

Changes in cell surface and intracellular glycoproteins of trophoblastic giant cells during mouse placentation

Changes in lectin bindings of mouse trophoblastic giant cells (TGCs) were examined by light and electron microscopy. Neither Griffonia simplicifolia agglutinin (GS)-II nor succinyl-wheat germ agglutinin (s-WGA) bound to the 1st and 2nd TGCs on day 6.5 post coitum (p.c.), but did so from days 8.5 to 12.5 p.c. Positive reactions with s-WGA were localized in the perinuclear region and cell surface of both 1st and 2nd TGCs; while GS-II bound only to the perinuclear region, where it appeared as network-like deposits. This region was identified as well-developed Golgi lamellae by electron microscopy. Moreover, SDS-PAGE and lectin-blot analysis of the 1st TGCs indicated that the intensity of s-WGA and GS-II bindings increased in the glycoproteins of approximately 43, 40, 37, and 26 kDa and in those of 43 and 38 kDa, respectively, during the 8.5th to 10.5th day p.c. The reaction with GS-I was detected on cell surface of both the 1st and 2nd TGCs on day 6.5 p.c. The reaction in the 1st TGCs was intensely positive throughout their development, whereas the reactivity decreased in the 2nd TGCs on day 10.5 p.c. and completely disappeared on day 12.5 p.c. The GS-I reaction in TGCs was more intense at the maternal side than at the embryonic side. These results suggest that certain Gal and/or GlcNAc glycoproteins on the cell surface and in Golgi lamellae of TGCs dynamically change from the 8.5th to 10.5th day p.c. in association with mouse placentation.     

Membrane association and defective transport of spleen focus-forming virus glycoproteins

The gp52 glycoprotein of the spleen focus-forming virus found in the Friend and Rauscher complexes of murine leukemia viruses (MuLV) has been previously identified as a recombinant molecule involving substitutions and deletions of the MuLV env gene. Unlike the MuLV structural glycoproteins, gp52 is defective in its transport to the cell surface. We have studied aspects of the intracellular transport and membrane association of gp52 to investigate the possible mechanisms underlying the defective transport process. It was found that a panel of monoclonal antibodies to different epitopes of p 15E, as well as an antiserum to a synthetic peptide corresponding to the carboxy terminus of MuLV envelope precursors, failed to react with gp52. Despite the possible absence of membrane-anchoring regions of MuLV envelope proteins known to reside on p 15E, gp52 was not found to be secreted into the culture fluids. Detergent extraction studies indicated that gp52 is associated with the membranes and not the contents of microsomal vesicles in speen focus-forming virus-infected cells. gp65, the processed form of gp52, could be labeled with [3H]palmitic acid, suggesting a membrane association. To determine whether a spontaneous denaturation occurs leading to aggregation and defective transport of gp52, we studied the surface expression of gp52 in cells grown at different temperatures, as well as the solubility of gp52 in low concentrations of Triton X-100. No evidence of aggregation or of a temperature-dependent difference in transport was obtained. gp52 appears to be a monotopic integral membrane protein, unlike MuLV envelope proteins which are bitopic integral membrane proteins; proteolytic digestion of intact microsomal vesicles did not reveal a detectable cytoplasmic tail under conditions where this could be demonstrated on MuLV envelope precursors. We suggest that a loss of putative signals involved in mediating intracellular transport is a likely cause for the defective transport of the spleen focus-forming virus glycoproteins.     

Temperature-sensitive transport of glycoproteins to the surface of a variant mouse lymphoma cell line.

The expression of mouse mammary tumor virus (MMTV) glycoproteins on the surface of stably infected mouse lymphoma cell line W7MG1 is dramatically increased by glucocorticoid hormones. A variant cell line, W7M.TS1, was selected from W7MG1 for its lack of expression of MMTV glycoproteins on the cell surface in response to treatment with glucocorticoid. Hormonal stimulation of MMTV RNA levels and hormone-induced cytolysis occurred normally in the variant cells. Furthermore, the rates of production of the precursor and mature forms of MMTV glycoproteins in the presence of glucocorticoid were similar in variant and wild-type cells. However, the accumulation of MMTV glycoproteins on the cell surface after hormone treatment was delayed by about 8 h in the variant relative to wild-type cells. The steady-state level of a constitutively expressed cellular protein, T200, on the variant cell surface was comparable to that on wild-type cells. However, in pulse-chase experiments, the appearance of newly synthesized T200 on the cell surface was delayed in the variant compared with wild-type cells. Another glucocorticoid hormone response, removal of H-2 class I antigens from the cell surface, was also delayed in the variant relative to wild-type cells, suggesting that turnover or internalization of cell surface glycoproteins may also be affected in the variant. The defects in the variant cell line were observed at 37 degrees C, but not at 31 degrees C; the variant cells grew normally at both temperatures. This variant phenotype defines a new genetic entity that is important for transport of glycoproteins between internal microsomal compartments and the cell surface.     

Posttranslational modification and intracellular transport of mumps virus glycoproteins

Analysis of the pronase-derived glycopeptides of isolated mumps virus glycoproteins revealed the presence of both complex and high-mannose-type oligosaccharides on the HN and F1 glycoproteins, whereas only high-mannose-type glycopeptides were detected on F2. Endoglycosidase F, a newly described glycosidase that cleaves N-linked high mannose as well as complex oligosaccharides, appeared to completely cleave the oligosaccharides linked to HN and F2, whereas F1 was resistant to the enzyme. Two distinct cleavage products of F2 were observed, suggesting the presence of two oligosaccharide side chains. Tunicamycin was found to reduce the infectious virus yield and inhibit mumps virus particle formation. The two glycoproteins, HN and F, were not found in the presence of the glycosylation inhibitor. However, two new polypeptides were detected, with molecular weights of 63,000 (HNT) and 53,000 (FT), respectively, which may represent nonglycosylated forms of the glycoproteins. Synthesis of the nonglycosylated virus-coded proteins (L, NP, P, M, pI, and pII) was not affected by tunicamycin. The formation of HN oligomers and the proteolytic cleavage of the F protein were found to occur with the same kinetics. Analysis of the time course of appearance of mumps virus glycoproteins on the cell surface suggested that dimerization of HN and cleavage of F occur immediately after their exposure on the plasma membrane.     

Identification and characterization of glycoproteins secreted by the skin of the day 16 fetal mouse

The secretion of tissue-specific proteins during mouse skin development, was investigated by incubating day 16 fetal skin explants in the presence of [35S]methionine and analyzing the medium electrophoretically. The medium was found to contain five proteins, which could be classified into two groups according to molecular weight. The kinetics of release of these proteins indicated that they were specifically secreted and not released by cytolysis. Mapping of the proteins by partial proteolytic digestion revealed that although the digestion patterns between the two molecular weight groups were different, within each group similar patterns were seen, suggesting that they were structurally related. Incubation in the presence of tunicamycin resulted in the decrease in molecular weight of the secreted proteins, indicating that the proteins were glycosylated. The results suggest that the two groups of structurally related glycoproteins were secreted by the peridermal layer of the fetal skin.     

Isolation and characterization of Chinese hamster ovary cell mutants defective in glucose transport

Cultured Chinese hamster ovary (CHO) cells possess an insulin-sensitive facilitated diffusion system for glucose transport. Mutant clones of CHO cells defective in glucose transport were obtained by repeating the selection procedure, which involved mutagenesis with ethyl methanesulfonate, radiation suicide with tritiated 2-deoxy-D-glucose, the polyester replica technique and in situ autoradiographic assaying for glucose accumulation. On the first selection, we obtained mutants exhibiting about half the glucose uptake activity of parental CHO-K1 cells and half the amount of a glucose transporter, the amount of which was determined by immunoblotting with an antibody to the human erythrocyte glucose transporter. The second selection, starting from one of the mutants obtained in the first-step selection, yielded a strain, GTS-31, in which both glucose uptake activity and the quantity of the glucose transporter were 10-20% of the levels in CHO-K1 cells, whereas the responsiveness of glucose transport to insulin, and the activities of leucine uptake and several glycolytic enzymes remained unchanged. GTS-31 cells grew slower than CHO-K1 cells at both 33 and 40 degrees C, and in a medium containing a low concentration of glucose (0.1 mM), the mutant cells lost the ability to form colonies. All the three spontaneous GTS-31 cell revertants, which were isolated by growing the mutant cells in medium containing 0.1 mM glucose, exhibited about half the glucose uptake activity and about half the amount of glucose transporter, as compared to in CHO-K1 cells, these characteristics being similar to those of the first-step mutant. These results indicate that the decrease in glucose uptake activity in strain GTS-31 is due to a mutation which induces a reduction in the amount of the glucose transporter, providing genetic evidence that the glucose transporter functions as a major route for glucose entry into CHO-K1 cells.     

Isolation and characterization of a mammalian cell mutant defective in 3-hydroxy-3-methylglutaryl coenzyme A synthase

A somatic cell mutant of the Chinese hamster ovary (CHO)-K1 cell auxotrophic for mevalonic acid has been isolated by means of the bromodeoxyuridine-visible light technique. This mutant can incorporate labeled mevalonate but not labeled acetate into cholesterol and, thus, is apparently defective in mevalonate biosynthesis. The mutant is recessive with respect to the parental cell phenotype. Assessment of the in vitro activities of the enzymes responsible for mevalonate biosynthesis under varying growth conditions indicates that the mutant, Mev-1, is defective in 3-hydroxy-3-methylglutaryl coenzyme A synthase.     

Identification and preliminary characterization of a mutant defective in the bacteriophage T4-induced unfolding of the Escherichia coli nucleoid.

The nucleoids of Escherichia coli S/6/5 cells are rapidly unfolded at about 3 min after infection with wild-type T4 bacteriophage or with nuclear disruption deficient, host DNA degradation-deficient multiple mutants of phage T4. Unfolding does not occur after infection with T4 phage ghosts. Experiments using chloramphenicol to inhibit protein synthesis indicate that the T4-induced unfolding of the E. coli chromosomes is dependent on the presence of one or more protein synthesized between 2 and 3 min after infection. A mutant of phage T4 has been isolated which fails to induce this early unfolding of the host nucleoids. This mutant has been termed "unfoldase deficient" (unf-) despite the fact that the function of the gene product defective in this strain is not yet known. Mapping experiments indicate that the unf- mutation is located near gene 63 between genes 31 and 63. The folded genomes of E. coli S/6/5 cells remain essentially intact (2,000-3,000S) at 5 min after infection with unfoldase-, nuclear disruption-, and host DNA degradation-deficient T4 phage. Nuclear disruption occurs normally after infection with unfoldase- and host DNA degradation-deficient but nuclear disruption-proficient (ndd+), T4 phage. The host chromosomes remain partially folded (1,200-1,800S) at 5 min after infection with the unfoldase single mutant unf39 x 5 or an unfoldase- and host DNA degradation-deficient, but nuclear disruption-proficient, T4 strain. The presence of the unfoldase mutation causes a slight delay in host DNA degradation in the presence of nuclear disruption but has no effect on the rate of host DNA degradation in the absence of nuclear disruption. Its presence in nuclear disruption- and host DNA degradation-deficient multiple mutants does not alter the shutoff to host DNA or protein synthesis.     

Monensin and FCCP inhibit the intracellular transport of alphavirus membrane glycoproteins

Temperature-sensitive mutants of semliki forest virus (SFV) and sindbis virus (SIN) were used to study the intracellular transport of virus membrane glycoproteins in infected chicken embryo fibroblasts. When antisera against purified glycoproteins and (125)I- labeled protein A from staphylococcus aureus were used only small amounts of virus glycoproteins were detected at the surface of SFV ts-1 and SIN Ts-10 infected cells incubated at the restrictive temperature (39 degrees C). When the mutant-infected cells were shifted to the permissive temperature (28 degrees C), in the presence of cycloheximide, increasing amounts of virus glycoproteins appeared at the cell surface from 20 to 80 min after the shift. Both monensin (10muM) and carbonylcyanide-p- trifluoromethoxyphenylhydrazone (FCCP; 10-20 muM) inhibited the appearance of virus membrane glycoproteins at the cell surface. Vinblastine sulfate (10 μg/ml) inhibited the transport by approximately 50 percent, whereas cytochalasin B (1 μg/ml) had only a marginal effect. Intracellular distribution of virus glycoproteins in the mutant-infected cells was visualized in double-fluorescence studies using lectins as markers for endoplasmic reticulum and Golgi apparatus. At 39 degrees C, the virus membrane glycoproteins were located at the endoplasmic reticulum, whereas after shift to 28 degrees C, a bright juxtanuclear reticular fluorescence was seen in the location of the Golgi apparatus. In the presence of monensin, the virus glycoproteins could migrate to the Golgi apparatus, although transport to the cell surface did not take place. When the shift was carried out in the presence of FCCP, negligible fluorescence was seen in the Golgi apparatus and the glycoproteins apparently remained in the rough endoplasmic reticulum. A rapid inhibition in the accumulation of virus glycoproteins at the cell surface was obtained when FCCP was added during the active transport period, whereas with monensin there was a delay of approximately 10 min. These results suggest a similar intracellular pathway in the maturation of both plasma membrane and secretory glycoproteins.     

Preliminary characterization of the temperature-sensitive defect in DNA replication in a mutant mouse L cell

Temperature-sensitive ts A1S9 mouse L cells synthesize DNA apparently normally for 6–8 hr upon incubation at 38.5°C. Thereafter, these cells are able to perform limited polydeoxyribonucleotide chain synthesis at the high temperature, but are unable to convert newly replicated small single-strand segments of DNA (of the order of molecular weight 10⁶ daltons) to large molecular weight chromosomal DNA. Data obtained are compatible with a model which suggests that ts A1S9 cells are able to carry out most individual reactions of DNA synthesis at the high temperature, but are temperature-sensitive in a protein which participates in the joining of small DNA segments to make chromosomal DNA strands. When cells are reincubated at a permissive temperature, after the temperature-sensitive lesion has been established, they recover the latter capability several hours before they are able once again to synthesize DNA at normal rates.