scholarly journals Inhibition of Endosome Function in CHO Cells Bearing a Temperature-sensitive Defect in the Coatomer (COPI) Component ε-COP

1997 ◽  
Vol 139 (7) ◽  
pp. 1747-1759 ◽  
Author(s):  
Elizabeth Daro ◽  
David Sheff ◽  
Marie Gomez ◽  
Thomas Kreis ◽  
Ira Mellman
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Semliki Forest Virus ◽  
Temperature Shift ◽  
Endocytic Pathway ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Intact Cells ◽  
Early Endosomes ◽  
Mutant Phenotypes

Recent evidence has suggested that subunits of the coatomer protein (COPI) complexes are functionally associated with endosomes in mammalian cells. We now provide genetic evidence that COPI plays a role in endocytosis in intact cells. The ldlF mutant CHO cell line bears a temperature-sensitive defect in the COPI subunit ε-COP. In addition to exhibiting conditional defects in the secretory pathway, we find that the cells are also defective at mediating endosome-associated functions. As found for cells microinjected with anti-COPI antibodies, ldlF cells at the restrictive temperature could not be infected by vesicular stomatitis (VSV) or Semliki Forest virus (SFV) that require delivery to acidic endosomes to penetrate into the cytosol. Although there was no temperature-sensitive defect in the internalization of receptor-bound transferrin (Tfn), Tfn recycling and accumulation of HRP were markedly inhibited at the restrictive temperature. Sorting of receptor-bound markers such as EGF to lysosomes was also reduced, although delivery of fluid-phase markers was only partially inhibited. In addition, lysosomes redistributed from their typical perinuclear location to the tips of the ldlF cells. Mutant phenotypes began to emerge within 2 h of temperature shift, the time required for the loss of detectable ε-COP, suggesting that the endocytic defects were not secondary to a block in the secretory pathway. Importantly, the mutant phenotypes were also corrected by transfection of wild-type ε-COP cDNA demonstrating that they directly or indirectly reflected the ε-COP defect. Taken together, the results suggest that ε-COP acts early in the endocytic pathway, most likely inhibiting the normal sorting and recycling functions of early endosomes.

scholarly journals Effects of a temperature-sensitiveMinutemutation on gene expression inDrosophila melanogaster

1984 ◽  
Vol 43 (3) ◽  
pp. 257-275 ◽  
Author(s):  
Donald A. R. Sinclair ◽  
Thomas A. Grigliatti ◽  
Thomas C. Kaufman
Keyword(s):  
Gene Expression ◽  
Temperature Shift ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Gene Products ◽  
The Past ◽  
Reciprocal Temperature ◽  
Sex Comb ◽  
Mutant Phenotypes

SUMMARYMinute(M) lesions exhibit a striking propensity for interacting with many different mutations. In the past, few attempts have been made to explain these diverse phenomena. This study describes a variety of temperature-sensitive (ts) interactions exhibited by the ts third chromosomeMinutemutationM(3)LS4Q-III(Q-III). Most of these interactions (i.e. those involvingvg, cp, Dl, DfdorLy) reflectQ-III-induced enhancement of the respective mutant phenotypes at the restrictive temperature. However,Q-IIIalso suppresses the extra-sex-comb phenotypes ofPcandMscat 29 °C and evokes lethal and bristle traits when combined withJ34eat the restrictive temperature. All of these interactions are characteristic of non-tsMinutelesions and thus they appear to be correlated with general physiological perturbations associated with theMsyndrome. In addition, our findings show that mutations that affect ribosome production and/or function, namelysu(f)ts67gandbbts−1, exhibit interactions comparable to those elicited byQ-III. Hence, in accordance with previous findings, we argue that most of theQ-IIIinteractions can be attributed to reduced translational capacity at the restrictive temperature. Finally, reciprocal temperature shift studies were used to delineate TSPs for interactions betweenQ-IIIandvg(mid to late second instar),cp(about mid-third instar),Dfd(early third instar) andDl(late second to mid third instar). We believe that these TSPs represent developmental intervals during which the respective gene products are utilized.

scholarly journals Incubation at the nonpermissive temperature induces deficiencies in UV resistance and mutagenesis in mouse mutant cells expressing a temperature-sensitive ubiquitin-activating enzyme (E1).

1997 ◽  
Vol 17 (3) ◽  
pp. 1484-1489 ◽  
Author(s):  
H Ikehata ◽  
S Kaneda ◽  
F Yamao ◽  
T Seno ◽  
T Ono ◽  
...  
Keyword(s):  
Dna Repair ◽  
Mammalian Cells ◽  
Uv Light ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Nonpermissive Temperature ◽  
Conjugation System ◽  
Ubiquitin Conjugating Enzyme ◽  
Mutant Phenotypes ◽  
Mutant Cells

In temperature-sensitive (ts) mutants of mouse FM3A cells, the levels of mutagenesis and survival of cells treated with DNA-damaging agents have been difficult to assess because they are killed after their mutant phenotypes are expressed at the nonpermissive temperature. To avoid this difficulty, we incubated the ts mutant cells at the restrictive temperature, 39 degrees C, for only a limited period after inducing DNA damage. We used ts mutants defective in genes for ubiquitin-activating enzyme (E1), DNA polymerase alpha, and p34(cdc2) kinase. Whereas the latter two showed no effect, E1 mutants were sensitized remarkably to UV light if incubated at 39 degrees C for limited periods after UV exposure. Eighty-five percent of the sensitization occurred within the first 12 h of incubation at 39 degrees C, and more than 36 h at 39 degrees C did not produce any further sensitization. Moreover, while the 39 degrees C incubation gave E1 mutants a moderate spontaneous mutator phenotype, the same treatment significantly diminished the level of UV-induced 6-thioguanine resistance mutagenesis and extended the time necessary for expression of the mutation phenotype. These characteristics of E1 mutants are reminiscent of the defective DNA repair phenotypes of Saccharomyces cerevisiae rad6 mutants, which have defects in a ubiquitin-conjugating enzyme (E2), to which E1 is known to transfer ubiquitin. These results demonstrate the involvement of E1 in eukaryotic DNA repair and mutagenesis and provide the first direct evidence that the ubiquitin-conjugation system contributes to DNA repair in mammalian cells.

Conditional absence of mitosis-specific antigens in a temperature-sensitive embryonic-arrest mutant of Caenorhabditis elegans

1987 ◽  
Vol 87 (2) ◽  
pp. 305-314 ◽  
Author(s):  
R.M. Hecht ◽  
M. Berg-Zabelshansky ◽  
P.N. Rao ◽  
F.M. Davis
Keyword(s):  
Caenorhabditis Elegans ◽  
Mammalian Cells ◽  
Temperature Shift ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
C Elegans ◽  
Phosphatase Treatment ◽  
M Phase ◽  
A Cell ◽  
Embryonic Arrest

A monoclonal antibody, specific to phosphoproteins in mitotic HeLa cells was found to crossreact with a similar set of proteins in embryos of the nematode, Caenorhabditis elegans. In C. elegans, as in mammalian cells, the highly conserved antigenic epitope is associated with a family of high molecular weight polypeptides. The antigenic reactivity of these multiple proteins also depends on their phosphorylation, since antibody binding is reduced after alkaline phosphatase treatment. The antigens are detected at the centrosomes, and in the nuclear region and surrounding cytoplasm of mitotic cells. The significance of these antigens is emphasized by their absence at restrictive temperature in embryos of the temperature-sensitive embryonic-arrest mutant, emb-29V. Furthermore, temperature shift-down experiments suggest that the emb-29 mutation defines a cell division cycle function that affects an essential activity required for progression into M phase.

Growth-dependent regulation of system A in SV40-transformed fetal rat hepatocytes

1988 ◽  
Vol 255 (3) ◽  
pp. C261-C270 ◽  
Author(s):  
M. E. Handlogten ◽  
M. S. Kilberg
Keyword(s):  
Cell Density ◽  
De Novo ◽  
Membrane Vesicles ◽  
Rat Hepatocytes ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Plasma Membrane Vesicles ◽  
Intact Cells ◽  
System A

Fetal RLA209-15 hepatocytes, transformed with a temperature-sensitive SV40 mutant, behave like fully differentiated cells at the growth-restrictive temperature of 40 degrees C. Conversely, incubation at the growth-permissive temperature of 33 degrees C results in a transformed phenotype characterized by rapid cell division and decreased production of liver-specific proteins. The results presented here demonstrate that the cells at 33 degrees C exhibited high rates of system A transport, but transfer to 40 degrees C reduced the activity greater than 50% within 24 h. This decline in transport was independent of cell density, although the basal rate of uptake was inversely proportional to cell density in rapidly dividing cells. Transfer of cells from 40 to 33 degrees C resulted in an enhancement of system A activity that was blocked by tunicamycin. Plasma membrane vesicles from cells maintained at either 33 or 40 degrees C retained uptake rates proportional to those in the intact cells; this difference in transport activity could also be demonstrated after detergent solubilization and reconstitution. Collectively, these data indicate that de novo synthesis of the system A carrier is regulated in conjunction with temperature-dependent cell growth in RLA209-15 hepatocytes.

scholarly journals Enzymatic Defects of the nsP2 Proteins of Semliki Forest Virus Temperature-Sensitive Mutants

2007 ◽  
Vol 81 (6) ◽  
pp. 2849-2860 ◽  
Author(s):  
Giuseppe Balistreri ◽  
Javier Caldentey ◽  
Leevi Kääriäinen ◽  
Tero Ahola
Keyword(s):  
Protease Activity ◽  
Semliki Forest Virus ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Helicase Domain ◽  
Permissive Temperature ◽  
Protease Domain ◽  
Rna Triphosphatase ◽  
Interdomain Interactions

ABSTRACT We have analyzed the biochemical consequences of mutations that affect viral RNA synthesis in Semliki Forest virus temperature-sensitive (ts) mutants. Of the six mutations mapping in the multifunctional replicase protein nsP2, three were located in the N-terminal helicase region and three were in the C-terminal protease domain. Wild-type and mutant nsP2s were expressed, purified, and assayed for nucleotide triphosphatase (NTPase), RNA triphosphatase (RTPase), and protease activities in vitro at 24°C and 35°C. The protease domain mutants (ts4, ts6, and ts11) had reduced protease activity at 35°C but displayed normal NTPase and RTPase. The helicase domain mutation ts1 did not have enzymatic consequences, whereas ts13a and ts9 reduced both NTPase and protease activities but in different and mutant-specific ways. The effects of these helicase domain mutants on protease function suggest interdomain interactions within nsP2. NTPase activity was not directly required for protease activity. The similarities of the NTPase and RTPase results, as well as competition experiments, suggest that these two reactions utilize the same active site. The mutations were also studied in recombinant viruses first cultivated at the permissive temperature and then shifted up to the restrictive temperature. Processing of the nonstructural polyprotein was generally retarded in cells infected with viruses carrying the ts4, ts6, ts11, and ts13a mutations, and a specific defect appeared in ts9. All mutations except ts13a were associated with a large reduction in the production of the subgenomic 26S mRNA, indicating that both protease and helicase domains influence the recognition of the subgenomic promoter during virus replication.

scholarly journals Molecular Defects Caused by Temperature-Sensitive Mutations in Semliki Forest Virus nsP1

2008 ◽  
Vol 82 (18) ◽  
pp. 9236-9244 ◽  
Author(s):  
Valeria Lulla ◽  
Dorothea L. Sawicki ◽  
Stanley G. Sawicki ◽  
Aleksei Lulla ◽  
Andres Merits ◽  
...  
Keyword(s):  
Sindbis Virus ◽  
Rna Synthesis ◽  
Semliki Forest Virus ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Minus Strand ◽  
Nonstructural Proteins ◽  
Mutant Proteins ◽  
Recombinant Viruses ◽  
Mrna Capping

ABSTRACT Alphavirus replicase protein nsP1 has multiple functions during viral RNA synthesis. It catalyzes methyltransferase and guanylyltransferase activities needed in viral mRNA capping, attaches the viral replication complex to cytoplasmic membranes, and is required for minus-strand RNA synthesis. Two temperature-sensitive (ts) mutations in Semliki Forest virus (SFV) were previously identified within nsP1: ts10 (E529D) and ts14 (D119N). Recombinant viruses containing these individual mutations reproduced the features of the original ts strains. We now find that the capping-associated enzymatic activities of recombinant nsP1, containing ts10 or ts14 lesions, were not ts. The mutant proteins and polyproteins also were membrane bound, mutant nsP1 interacted normally with the other nonstructural proteins, and there was no major defect in nonstructural polyprotein processing in the mutants, although ts14 surprisingly displayed slightly retarded processing. The two mutant viruses were specifically defective in minus-strand RNA synthesis at the restrictive temperature. Integrating data from SFV and Sindbis virus, we discuss the domain structure of nsP1 and the relative positioning of and interactions between the replicase proteins. nsP1 is suggested to contain a specific subdomain involved in minus-strand synthesis and interaction with the polymerase nsP4 and the protease nsP2.

scholarly journals Sec34p, a Protein Required for Vesicle Tethering to the Yeast Golgi Apparatus, Is in a Complex with Sec35p

1999 ◽  
Vol 147 (4) ◽  
pp. 729-742 ◽  
Author(s):  
Susan M. VanRheenen ◽  
Xiaochun Cao ◽  
Stephanie K. Sapperstein ◽  
Elbert C. Chiang ◽  
Vladimir V. Lupashin ◽  
...  
Keyword(s):  
Golgi Complex ◽  
Secretory Pathway ◽  
Rab Gtpase ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Dominant Form ◽  
Vesicle Tethering ◽  
Protein Receptor ◽  
Complex Target

A screen for mutants of Saccharomyces cerevisiae secretory pathway components previously yielded sec34, a mutant that accumulates numerous vesicles and fails to transport proteins from the ER to the Golgi complex at the restrictive temperature (Wuestehube, L.J., R. Duden, A. Eun, S. Hamamoto, P. Korn, R. Ram, and R. Schekman. 1996. Genetics. 142:393–406). We find that SEC34 encodes a novel protein of 93-kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec34-2 is suppressed by the rab GTPase Ypt1p that functions early in the secretory pathway, or by the dominant form of the ER to Golgi complex target-SNARE (soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor)–associated protein Sly1p, Sly1-20p. Weaker suppression is evident upon overexpression of genes encoding the vesicle tethering factor Uso1p or the vesicle-SNAREs Sec22p, Bet1p, or Ykt6p. This genetic suppression profile is similar to that of sec35-1, a mutant allele of a gene encoding an ER to Golgi vesicle tethering factor and, like Sec35p, Sec34p is required in vitro for vesicle tethering. sec34-2 and sec35-1 display a synthetic lethal interaction, a genetic result explained by the finding that Sec34p and Sec35p can interact by two-hybrid analysis. Fractionation of yeast cytosol indicates that Sec34p and Sec35p exist in an ∼750-kD protein complex. Finally, we describe RUD3, a novel gene identified through a genetic screen for multicopy suppressors of a mutation in USO1, which suppresses the sec34-2 mutation as well.

scholarly journals Schizosaccharomyces pombe ypt5: a homologue of the rab5 endosome fusion regulator.

1993 ◽  
Vol 4 (6) ◽  
pp. 583-592 ◽  
Author(s):  
J Armstrong ◽  
M W Craighead ◽  
R Watson ◽  
S Ponnambalam ◽  
S Bowden
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Mammalian Cells ◽  
Normal Growth ◽  
Endocytic Pathway ◽  
Rab Proteins ◽  
Gene Encoding ◽  
Rich Media ◽  
Early Endosomes ◽  
Molecular Aspects ◽  
Mammalian Protein

The ypt/rab proteins are a family of small GTP-binding proteins thought to be required for different stages of membrane traffic. From the fission yeast Schizosaccharomyces pombe we have isolated and characterized ypt5, a gene encoding a homologue of rab5, a mammalian protein apparently involved in regulating fusion of early endosomes. Recombinant ypt5 protein bound GTP. The ypt5 gene was found to be essential for viability on minimal media, but ypt5-disrupted cells grew slowly on some rich media and accumulated a population of small vesicles not observed in wild-type cells. Canine rab5 cDNA could replace the ypt5 gene in S. pombe and restore normal growth and viability. Ypt5 protein expressed in mammalian cells colocalized with the transferrin receptor to early endosomes. Thus, molecular aspects of the early endocytic pathway may be conserved between mammalian cells and S. pombe and hence may be amenable to genetic analysis.

scholarly journals The Ypt1 GTPase is essential for the first two steps of the yeast secretory pathway.

1995 ◽  
Vol 131 (3) ◽  
pp. 583-590 ◽  
Author(s):  
G Jedd ◽  
C Richardson ◽  
R Litt ◽  
N Segev
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Small Gtpases ◽  
Rab Proteins ◽  
Restrictive Temperature ◽  
Rab Gtpases ◽  
Curr Opin Cell Biol ◽  
Rab Family ◽  
One Step ◽  
Mutant Cells

Small GTPases of the rab family are involved in the regulation of vesicular transport. The restricted distribution of each of these proteins in mammalian cells has led to the suggestion that different rab proteins act at different steps of transport (Pryer, N. K., L. J. Wuestehube, and R. Sheckman. 1992. Annu Rev. Biochem. 61:471-516; Zerial, M., and H. Stenmark. 1993. Curr. Opin. Cell Biol. 5:613-620). However, in this report we show that the Ypt1-GTPase, a member of the rab family, is essential for more than one step of the yeast secretory pathway. We determined the secretory defect conferred by a novel ypt1 mutation by comparing the processing of several transported glycoproteins in wild-type and mutant cells. The ypt1-A136D mutant has a change in an amino acid that is conserved among rab GTPases. This mutation leads to a rapid and tight secretory block upon a shift to the restrictive temperature, and allows for the identification of the specific steps in the secretory pathway that directly require Ypt1 protein (Ypt1p). The ypt1-A136D mutant exhibits tight blocks in two secretory steps, ER to cis-Golgi and cis- to medial-Golgi, but later steps are unaffected. Thus, it is unlikely that Ypt1p functions as the sole determinant of fusion specificity. Our results are more consistent with a role for Ypt1/rab proteins in determining the directionality or fidelity of protein sorting.

scholarly journals The role of Myo2, a yeast class V myosin, in vesicular transport.

1995 ◽  
Vol 128 (6) ◽  
pp. 1055-1068 ◽  
Author(s):  
B Govindan ◽  
R Bowser ◽  
P Novick
Keyword(s):  
Mother Cell ◽  
Secretory Pathway ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Transit Times ◽  
Cargo Proteins ◽  
Section Electron ◽  
Synthetically Lethal ◽  
Actin Cables ◽  
Exocytic Pathway

Previous studies have shown that temperature-sensitive, myo2-66 yeast arrest as large, unbudded cells that accumulate vesicles within their cytoplasm (Johnston, G. C., J. A. Prendergast, and R. A. Singer. 1991. J. Cell Biol. 113:539-551). In this study we show that myo2-66 is synthetically lethal in combination with a subset of the late-acting sec mutations. Thin section electron microscopy shows that the post-Golgi blocked secretory mutants, sec1-1 and sec6-4, rapidly accumulate vesicles in the bud, upon brief incubations at the restrictive temperature. In contrast, myo2-66 cells accumulate vesicles predominantly in the mother cell. Double mutant analysis also places Myo2 function in a post-Golgi stage of the secretory pathway. Despite the accumulation of vesicles in myo2-66 cells, pulse-chase studies show that the transit times of several secreted proteins, including invertase and alpha factor, as well as the vacuolar proteins, carboxy-peptidase Y and alkaline phosphatase, are normal. Therefore the vesicles which accumulate in this mutant may function on an exocytic pathway that transports a set of cargo proteins that is distinct from those analyzed. Our observations are consistent with a role for Myo2 in transporting a class of secretory vesicles from the mother cell along actin cables into the bud.

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