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1.
We previously described the isolation and preliminary characterization of a Chinese hamster ovary cell mutant, termed G.7.1, that carried a temperature-sensitive, conditional-lethal lesion affecting the acidification of vesicles in crude cellular extracts (Marnell, M. H., Mathis, L. S., Stookey, M., Shia, S.-P., Stone, D. K., and Draper, R. K. (1984) J. Cell Biol. 99, 1907-1916). In the present report, we have separated lysosomal vesicles from more buoyant nonlysosomal vesicles by centrifuging cell extracts with Percoll and correlated the acidification defect with nonlysosomal vesicles, including endosomes, but not with secondary lysosomes. Moreover, the acidification of nonlysosomal vesicles prepared from mutant cells grown at the permissive temperature was more sensitive to thermal inactivation than similar vesicles from parental cells, implying that a heat-sensitive component is a normal resident of nonlysosomal vesicles in the mutant. This heat-sensitive component is apparently not associated with lysosomes, or if it is, it does not inhibit lysosomal acidification at the nonpermissive temperature. We also found that the transferrin-mediated uptake of iron is inhibited by 50% in the mutant cells at the nonpermissive temperature and that the inhibition cannot be accounted for by reduced binding or internalization of transferrin.  相似文献   

2.
tsJT60 cells are G0-specific temperature-sensitive mutants of the cell cycle from Fischer rats i.e., they grow exponentially at both 34 degrees and 39.5 degrees C, but when stimulated with fetal bovine serum (FBS) from the resting state (G0) they enter S phase at 34 degrees C but not at 39.5 degrees C. Epidermal growth factor (EGF) also induced DNA synthesis, although weakly, in G0-arrested tsJT60 cells at 34 degrees C but failed at 39.5 degrees C. When G0-arrested tsJT60 cells were stimulated at 39.5 degrees C with FBS plus EGF, they entered S phase and divided. Somatomedin C, insulin, or transferrin had a weak effect in inducing DNA synthesis in G0-arrested cells when applied at 34 degrees C or with FBS at 39.5 degrees C. Fibroblast growth factor, platelet-derived growth factor, or 12-O-tetradecanoylphorbol 13-acetate had no such stimulatory effect at 39.5 degrees C. Binding of 125I-somatomedin C was not temperature-sensitive. Several other ts mutant cells that were blocked at 39.5 degrees C from entering S phase from the resting state following FBS addition were stimulated by FBS plus EGF at 34 degrees C but not at 39.5 degrees C.  相似文献   

3.
We have recently described a mutant of Chinese hamster ovary cells, termed G.7.1, that contains a temperature-sensitive, conditionally lethal mutation resulting in defective vacuolar acidification (Marnell, M. H., Mathis, L. S., Stookey, M., Shia, S.-P., Stone, D.K., and Draper, R. K. (1984) J. Cell Biol. 99, 1907-1916). To further characterize the lesion, clathrin-coated vesicles were partially purified from wild type and G.7.1 cells, and the thermolabilities of vanadate and oligomycin-insensitive, N-ethylmaleimide-sensitive, H+-ATPase activity, 32Pi-ATPase exchange activity, and proton pumping were compared. All three parameters of H+ pump activity were markedly diminished by preincubation at 44 degrees C for vesicles harvested from the G.7.1 cells, but not for those from wild type cells. Phosphatidylserine did not protect against heat inactivation in vesicle fractions prepared from G.7.1 cells. The results suggest that the mutation responsible for defective acidification in G.7.1 cells is expressed at the level of the proton pump of organelles present in our clathrin-coated vesicle-enriched preparation.  相似文献   

4.
Vacuolar acidification has been proposed to play a key role in a number of cellular processes, including protein sorting, zymogen activation, and maintenance of intracellular pH. We investigated the significance of vacuolar acidification by cloning and mutagenizing the gene for the yeast vacuolar proton-translocating ATPase 60-kilodalton subunit (VAT2). Cells carrying a vat2 null allele were viable; however, these cells were severely defective for growth in medium buffered at neutral pH. Vacuoles isolated from cells bearing the vat2 null allele were completely devoid of vacuolar ATPase activity. The pH of the vacuolar lumen of cells bearing the vat2 mutation was 7.1, compared with the wild-type pH of 6.1, as determined by a flow cytometric pH assay. These results indicate that the vacuolar proton-translocating ATPase complex is essential for vacuolar acidification and that the low-pH state of the vacuole is crucial for normal growth. The vacuolar acidification-defective vat2 mutant exhibited normal zymogen activation but displayed a minor defect in vacuolar protein sorting.  相似文献   

5.
Two heat-sensitive (reversibly arrested in G1 phase at 39.5 degrees C, multiplying at 33 degrees C) and two cold-sensitive (reversibly arrested in G1 phase at 33 degrees C, multiplying at 39.5 degrees C) cell-cycle mutants of the P-815-X2 murine mastocytoma line were tested for ribonucleotide reductase activity, using cells made permeable to nucleotides. After transfer of the heat-sensitive mutant cells to 39.5 degrees C, ribonucleotide reductase activity, similar to thymidine kinase (Schneider, E., Müller, B. and Schindler, R. (1983) Biochim. Biophys. Acta 741, 77-85), but unlike DNA polymerase alpha (Schneider, E., Müller, B. and Schindler, R. (1985) Biochim. Biophys. Acta 825, 375-383), decreased rapidly and in parallel with numbers of cells in S phase, whereas in the cold-sensitive mutant cells brought to 33 degrees C, ribonucleotide reductase activity decreased approx. 8 h later than numbers of DNA-synthesizing cells. When arrested heat- or cold-sensitive mutant cells were returned to the permissive temperature, ribonucleotide reductase activities, similar to DNA polymerase alpha and to thymidine kinase in heat-sensitive mutants, increased essentially in parallel with reentry of cells into S phase, whereas the increase in thymidine kinase activity in the cold-sensitive mutants was previously shown to occur approx. one cell-cycle time later. This indicates that ribonucleotide reductase and thymidine kinase are coordinately expressed in the heat-sensitive, but independently regulated in the cold-sensitive mutants.  相似文献   

6.
tsJT60, a temperature-sensitive (ts) mutant cell line of Fischer rat, is viable at both permissive (34 degrees C) and non-permissive (39.5 degrees C) temperatures. The cells grow normally in exponential growth phase at both temperatures, but when stimulated with fetal bovine serum (FBS) from G0 phase they re-enter S phase at 34 degrees C but not at 39.5 degrees. When tsJT60 cells were transformed with adenovirus (Ad) 5 wild type, they grew well at both temperatures, expressed E1A and E1B genes, and formed colonies in soft agar. When tsJT60 cells were transformed with Ad5 dl313, that lacks E1B gene, the transformed cells grew well at 34 degrees C but failed to form colony in soft agar. They died very soon at 39.5 degrees C. 3Y1 cells (a parental line of tsJT60) transformed with dl313 grew well at both temperatures, although neither expressed E1B gene nor formed colonies in soft agar. The phenotype of being lethal at 39.5 degrees C of dl313-transformed tsJT60 cells was complemented by cell fusion with 3Y1BUr cells (5-BrdU-resistant 3Y1), but not with tsJT60TGr cells (6-thioguanine resistant tsJT60). These results indicate that the lethal phenotype is related to the ts mutation of tsJT60 cells and also to the deletion of E1B gene of Ad5.  相似文献   

7.
Two heat-sensitive (arrested in G1 at 39.5°C) and two cold-sensitive (arrested in G1 at 33°C) clonal cell-cycle mutants that had been isolated from the same clone (K 21), of the murine mastocytoma P-815 cell line, were tested for thymidine kinase (EC 2.7.1.21) activity. After shift of mutant cells to the nonpermissive temperature, thymidine kinase activity decreased, and minimal levels (i.e., less than 3% of those observed for ‘wild-type’ K 21 cells at the respective temperature) were attained within 16 h in heat-sensitive and after 3–4 days in cold-sensitive mutants, which is in good agreement with kinetics of accumulation of heat-sensitive and cold-sensitive cells in G1 phase. After return of arrested mutant cells to the permissive temperature, thymidine kinase of heat-sensitive cells increased rapidly and in parallel with entry of cells into the S phase. In cultures of cold-sensitive cells, however, initiation of DNA synthesis preceded the increase of thymidine kinase activity by approx. one cell-cycle time. Thymidine kinase activities in revertants of the heat-sensitive and cold-sensitive mutants were similar to those of ‘wild-type’ cells. In ‘wild-type’ K 21 cells incubated at 39.5°C, thymidine kinase activity was approx. 30% of that at 33°C. This difference is attributable, at least in part, to a higher rate of inactivation of the enzyme at 39.5°C, as determined in cultures incubated with cycloheximide. The rapid increase of thymidine kinase activity that occurred after shift of K 21 cells and of arrested heat-sensitive mutant cells from 39.5°C to 33°C was inhibited by actinomycin D and cycloheximide.  相似文献   

8.
We have used the abnormal form of conjugation known as "genomic exclusion" to isolate a collection of heat-sensitive mutants of Tetrahymena pyriformis, syngen 1. Growth at room temperature in bacterized medium and no growth at 40 degrees C in the same medium was the criterion used for the isolation. The mutant strains were tested for growth in pure (axenic) culture in proteose peptone medium; of the 31 strains which grew normally at room temperature and not at 40 degrees C in that medium, 21 also failed to grow at 37 degrees C. Preliminary results of complementation tests suggest that most, if not all, the mutations are recessive and that a variety of genes was affected. A detailed genetic analysis was performed on one mutant (H9). The results are all consistent with the idea that the heat-sensitive phenotype of this mutant is determined by a single recessive mutation, designated ts-2. Heterozygotes ts-2/+ yield heat-sensitive segregants during vegetative growth; we interpret this finding as another example of allelic exclusion, a phenomenon universally encountered among heterozygotes in syngen 1 of T. pyriformis. Our results are discussed in the context of some questions of current interest in Tetrahymena genetics.  相似文献   

9.
K Miyashita  T Kakunaga 《Cell》1975,5(2):131-138
A clone of spontaneously transformed Chinese hamster lung cells was exposed to N-methyl-N'-nitro-N-nitroso-guanidine (MNNG), and six heat-sensitive and three cold-sensitive mutants were isolated after selection for inability to form colonies in soft agar at 39.5 degrees C and 34.5 degrees C, respectively. The heat-sensitive mutants had growth characteristics of transformed cells at 34.5 degrees C, but exhibited a normal phenotype at 39.5 degrees C. By contrast, cold-sensitive mutants displayed the characteristics of the normal cells at 34.5 degrees C and converted to a transformed phenotype at 39.5 degrees C. Transformed parent cells exhibited no obvious temperature-dependent properties. Temperature shift experiments showed that the colony-forming ability of both types of mutants was fully reversible. All of the mutants were able to grow well at both permissive and nonpermissive temperatures when grown on the surface of plastic dishes. Such mutants will be useful in analysis of factors involved in the expression of the transformed state or the maintenance of the nontransformed state.  相似文献   

10.
tsJT60 cells, a temperature-sensitive G0 mutant of a Fischer rat cell line, grew normally in an exponential growth phase at both permissive (34 degrees C) and nonpermissive (39.5 degrees C) temperatures, but when stimulated with fetal bovine serum in the growth-arrested state (G0 phase) they entered S phase at 34 degrees C but not at 39.5 degrees C. Infection of G0-arrested tsJT60 cells with SV40, adenovirus (Ad) 5 wild type and its E1B mutant dl313, and Ad12 wild type and its E1B mutants in205B, in205C, dl205, and in206B induced DNA synthesis at both temperatures. The DNA synthesized after virus infection was shown to be cellular by Hirt separation of DNA from SV40-infected cells and by CsCl equilibrium density gradient centrifugation of DNA from Ad5-infected cells.  相似文献   

11.
Of 625 aphidicolin-resistant clones selected at 33.5°C from mutagenized mouse FM3A cells, 13 clones could not grow at 39.5°C. Five of these clones, chosen at random, resumed growth at 39.5°C when thymidine was added to the culture medium. In hybrids, conditional thymidine auxotrophy was a recessive trait, but aphidicolin-resistance was either a codominant or recessive one depending on the mutant clone used.Thymidylate synthetase activity in crude extracts of these mutants was completely inactivated by preincubation for 30 min at 42°C, whereas that of the parent cells was not affected by the same treatment. Thus, the temperature-sensitive growth of the mutants described here seems to be due to this heat-sensitive thymidylate synthetase.  相似文献   

12.
A fibroblast mutant cell line lacking the Na+/H+ antiporter was used to study the influence of low cytoplasmic pH on membrane transport in the endocytic and exocytic pathways. After being loaded with protons, the mutant cells were acidified at pH 6.2 to 6.8 for 20 min while the parent cells regulated their pH within 1 min. Cytoplasmic acidification did not affect the level of intracellular ATP or the number of clathrin-coated pits at the cell surface. However, cytosolic acidification below pH 6.8 blocked the uptake of two fluid phase markers, Lucifer Yellow and horseradish peroxidase, as well as the internalization and the recycling of transferrin. When the cytoplasmic pH was reversed to physiological values, both fluid phase endocytosis and receptor-mediated endocytosis resumed with identical kinetics. Low cytoplasmic pH also inhibited the rate of intracellular transport from the Golgi complex to the plasma membrane. This was shown in cells infected by the temperature-sensitive mutant ts 045 of the vesicular stomatitis virus (VSV) using as a marker of transport the mutated viral membrane glycoprotein (VSV-G protein). The VSV-G protein was accumulated in the trans-Golgi network (TGN) by an incubation at 19.5 degrees C and was transported to the cell surface upon shifting the temperature to 31 degrees C. This transport was arrested in acidified cells maintained at low cytosolic pH and resumed during the recovery phase of the cytosolic pH. Electron microscopy performed on epon and cryo-sections of mutant cells acidified below pH 6.8 showed that the VSV-G protein was present in the TGN. These results indicate that acidification of the cytosol to a pH less than 6.8 inhibits reversibly membrane transport in both endocytic and exocytic pathways. In all likelihood, the clathrin and nonclathrin coated vesicles that are involved in endo- and exocytosis cannot pinch off from the cell surface or from the TGN below this critical value of internal pH.  相似文献   

13.
Transmembrane movement of phosphatidylserine (PS) and various PS analogs at the plasma membrane is thought to occur by an ATP-dependent, protein-mediated process. To isolate mutant CHO cells defective in this activity, we first obtained conditions which inhibited the endocytic, but not the non-endocytic pathway of lipid internalization since PS may enter cells by a combination of these two pathways. We found that acidic treatment of cells, which blocks clathrin-dependent endocytosis, enhanced the energy-dependent uptake of 1-palmitoyl-2-(6-[(7-nitrobenz- 2-oxa-1,3-diazol-4-yl)amino]caproyl -sn- glycero-3-phosphoserine (C6- NBD-PS) in CHO cells from donor vesicles (liposomes) by about twofold. Control experiments demonstrated that the enhanced uptake of C6-NBD-PS at acidic pH was not due to: (a) an increase in the capacity of the plasma membrane to incorporate C6-NBD-PS from the donor vesicles; (b) a decrease in the rate of loss of C6-NBD-PS from the cells; or (c) fusion or engulfment of the donor vesicles. When cytosolic acidification (to pH 6.3) was imposed without acidification of the extracellular medium, C6-NBD-PS uptake by intact cells was increased by about 50% compared to control values determined in the absence of acidification. These results suggested that a protein and energy dependent system(s) for transbilayer movement of the fluorescent PS was stimulated by cytosolic acidification. A screening method for mutant cells defective in the non- endocytic uptake of fluorescent PS analogs with replica cell colonies at acidic pH was then devised. After selection of mutagenized CHO-K1 cells by in situ screening, we obtained a mutant cell line in which uptake of fluorescent PS analogs was reduced to about 25% of the wild type level at either pH 6.0 or 7.4. Control experiments demonstrated that the reduced uptake of fluorescent PS analogs in the mutant cells was unrelated to multidrug resistance, and that endocytosis of another plasma membrane lipid marker occurred normally in the mutant cells. These results suggested that a non-endocytic pathway responsible for uptake of fluorescent PS analogs was specifically affected in the mutant cells.  相似文献   

14.
The vacuole of the yeast Saccharomyces cerevisiae contains a proton-translocating ATPase that acidifies the vacuolar lumen and generates a pH gradient across the vacuole membrane. We have investigated the role of compartment acidification of the vacuolar system in the sorting of vacuolar proteins. Strains with chromosomal disruptions of the genes encoding the A, B, or c subunit of the vacuolar ATPase are unable to acidify their vacuoles. These vat mutant strains accumulate and secrete precursor forms of the soluble vacuolar hydrolases carboxypeptidase Y and proteinase A. The kinetics of secretion suggests that missorting occurs in the Golgi complex or in post-Golgi vesicles. The presence of mature forms of the vacuolar proteins within the cell indicates that vat mutations do not cause defects in zymogen processing. Precursor forms of the membrane-associated vacuolar hydrolase alkaline phosphatase are also accumulated in vat mutant cells but to a lesser extent, suggesting that sorting of vacuolar membrane proteins is less sensitive to changes in the lumenal pH. A similar type of missorting defect can be induced in wild-type cells at pH 7.5. These results indicate that acidification of the vacuolar system is important for efficient sorting of proteins to the vacuole.  相似文献   

15.
A heat-sensitive (hs, arrested at 39.5 degrees C, termed 21-Ta) and a cold-sensitive (cs, arrested at 33 degrees C, termed 21-Fb) clonal cell cycle variant were isolated from the same clone of the P-815 murine mastocytoma line. At the respective nonpermissive temperatures, both the hs and the cs variant were reversibly arrested in G1 phase, and numbers of cells forming colonies upon reincubation at the permissive temperature remained nearly constant for at least 6 days. Cells arrested in G1 by incubation at the respective nonpermissive temperatures were fused to cells of another P-815 clone (31-S) that had been arrested by serum deprivation. Upon reincubation in medium containing 10% serum for 48 h at 39.5 degrees C, 21-Ta x 31-S heterokaryons, similar to 31-S x 31-S homokaryons, entered the S phase, whereas at 33 degrees C, 21-Fb x 31-S heterokaryons, similar to 21-Fb x 21-Fb homokaryons, remained arrested in G1, indicating a recessive expression of the hs and a dominant expression of the cs phenotype.  相似文献   

16.
The putative role of sorting early endosomes (EEs) in synaptic-like microvesicle (SLMV) formation in the neuroendocrine PC12 cell line was investigated by quantitative immunoelectron microscopy. By BSA-gold internalization kinetics, four distinct endosomal subcompartments were distinguished: primary endocytic vesicles, EEs, late endosomes, and lysosomes. As in other cells, EEs consisted of vacuolar and tubulovesicular subdomains. The SLMV marker proteins synaptophysin and vesicle-associated membrane protein 2 (VAMP-2) localized to both the EE vacuoles and associated tubulovesicles. Quantitative analysis showed that the transferrin receptor and SLMV proteins colocalized to a significantly higher degree in primary endocytic vesicles then in EE-associated tubulovesicles. By incubating PC12 cells expressing T antigen-tagged VAMP (VAMP-TAg) with antibodies against the luminal TAg, the recycling pathway of SLMV proteins was directly visualized. At 15 degrees C, internalized VAMP-TAg accumulated in the vacuolar domain of EEs. Upon rewarming to 37 degrees C, the labeling shifted to the tubular part of EEs and to newly formed SLMVs. Our data delineate a pathway in which SLMV proteins together with transferrin receptor are delivered to EEs, where they are sorted into SLMVs and recycling vesicles, respectively.  相似文献   

17.
tsJT60, a temperature-sensitive (ts) G0-mutant cell line from a Fischer rat, grows normally in the exponential growth phase at 34 degrees C and 39.5 degrees C, but when stimulated with fetal bovine serum (FBS), from the G0 phase they reenter the S phase at 34 degrees C but not at 39.5 degrees C. The ts-block was bypassed when G0-arrested tsJT60 cells were stimulated at 39.5 degrees C with FBS plus epidermal growth factor (EGF). The presence of EGF for the first 6 h after serum stimulation caused tsJT60 cells to enter the S phase in the presence of FBS at 39.5 degrees C. When EGF was added 6 h after serum stimulation, entrance into the S phase was delayed by about 6 h. The sequential presence of two growth factors, EGF without FBS for 6 h then FBS without EGF, or the reversed sequence, failed to initiate DNA synthesis at 39.5 degrees C. The binding of EGF was not temperature sensitive. The amounts of RNA and protein present doubled after stimulation with both FBS and EGF at 39.5 degrees C. These and other findings suggest that EGF bypasses only some specific event in the entire prereplicative process that operates operating in serum-stimulated cells at 39.5 degrees C.  相似文献   

18.
We have investigated the expression of growth-regulated genes in tsJT60 cells, a temperature-sensitive (ts) mutant of Fischer rat cells, which, on the basis of its kinetic behavior, can be classified as a G0 mutant. It grows normally at 34 degrees C and also at 39.5 degrees C if shifted to the higher temperature during exponential growth. However, if the cell population is first made quiescent by serum deprivation, subsequent stimulation by serum induces the cells to enter S phase at 34 degrees C but not at 39.5 degrees C. A panel of growth-regulated genes was used that included three protooncogenes (c-fos, c-myc, and p53), several genes that are induced in G0 cells stimulated by growth factors (beta-actin, 2A9, 2F1, vimentin, JE-3, KC-1, and ornithine decarboxylase), and an S-phase gene (histone H3). The expression of these growth-regulated genes was studied in both tsJT60 cells and its parental cell line, rat 3Y1 cells. All the genes tested, except histone H3, are similarly induced when quiescent tsJT60 cells are stimulated by serum at either permissive or restrictive temperatures. These results raise intriguing questions on the nature of quiescence and the relationship between G0 and G1 in cells in culture.  相似文献   

19.
Cardiolipin (CL) is an anionic phospholipid with a dimeric structure predominantly localized in the mitochondrial inner membrane, where it is closely associated with mitochondrial function, biogenesis, and genome stability (Daum, 1985; Janitor and Subik, 1993; Jiang et al., 2000; Schlame et al., 2000; Zhong et al., 2004). Previous studies have shown that yeast mutant cells lacking CL due to a disruption in CRD1, the structural gene encoding CL synthase, exhibit defective colony formation at elevated temperature even on glucose medium (Jiang et al., 1999; Zhong et al., 2004), suggesting a role for CL in cellular processes apart from mitochondrial bioenergetics. In the current study, we present evidence that the crd1Delta mutant exhibits severe vacuolar defects, including swollen vacuole morphology and loss of vacuolar acidification, at 37 degrees C. Moreover, vacuoles from crd1Delta show decreased vacuolar H(+)-ATPase activity and proton pumping, which may contribute to loss of vacuolar acidification. Deletion mutants in RTG2 and NHX1, which mediate vacuolar pH and ion homeostasis, rescue the defective colony formation phenotype of crd1Delta, strongly suggesting that the temperature sensitivity of crd1Delta is a consequence of the vacuolar defects. Our results demonstrate the existence of a novel mitochondria-vacuole signaling pathway mediated by CL synthesis.  相似文献   

20.
Isolation of a G0-specific ts mutant from a Fischer rat cell line, 3Y1   总被引:2,自引:0,他引:2  
A ts mutant clone, tsJT60, was isolated from Fisher rat cell line, 3Y1. During the exponential growth at both 34 and 39.5 degrees C, tsJT60 did not appear as ts mutant cells. However, once entered resting state (G0) under serum deprivation at the confluent state, they could re-enter S phase at 34 degrees C but could not at 39.5 degrees C following the stimulation of cells either by the addition of fetal bovine serum or by trypsinization and replating. These and other results suggested that tsJT60 is a G0-specific ts mutant, i.e., the cells have ts defect(s) in the function which is required for the stimulation from the resting state to S phase but not for the progression of the cell cycle in an exponential growth phase.  相似文献   

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