scholarly journals Characterization of yeast Vps33p, a protein required for vacuolar protein sorting and vacuole biogenesis.

1990 ◽  
Vol 10 (9) ◽  
pp. 4638-4649 ◽  
Author(s):  
L M Banta ◽  
T A Vida ◽  
P K Herman ◽  
S D Emr
Keyword(s):  
Sequence Similarity ◽  
Protein Sorting ◽  
Atp Binding ◽  
Temperature Sensitive ◽  
Cell Fractionation ◽  
Permissive Temperature ◽  
Sensitive Mutant ◽  
Temperature Sensitive Mutant ◽  
Vacuolar Protein ◽  
Vacuole Biogenesis

vps33 mutants missort and secrete multiple vacuolar hydrolases and exhibit extreme defects in vacuolar morphology. Toward a molecular understanding of the role of the VPS33 gene in vacuole biogenesis, we have cloned this gene from a yeast genomic library by complementation of a temperature-sensitive vps33 mutation. Gene disruption demonstrated that VPS33 was not essential but was required for growth at high temperatures. At the permissive temperature, vps33 null mutants exhibited defects in vacuolar protein localization and vacuole morphology similar to those seen in most of the original mutant alleles. Sequence analysis revealed a putative open reading frame sufficient to encode a protein of 691 amino acids. Hydropathy analysis indicated that the deduced product of the VPS33 gene is generally hydrophilic, contains no obvious signal sequence or transmembrane domains, and is therefore unlikely to enter the secretory pathway. Polyclonal antisera raised against TrpE-Vps33 fusion proteins recognized a protein in yeast cells of the expected molecular weight, approximately 75,000. In cell fractionation studies, Vps33p behaved as a cytosolic protein. The predicted VPS33 gene product possessed sequence similarity with a number of ATPases and ATP-binding proteins specifically in their ATP-binding domains. One vps33 temperature-sensitive mutant contained a missense mutation near this region of sequence similarity; the mutation resulted in a Leu-646----Pro substitution in Vps33p. This temperature-sensitive mutant strain contained normal vacuoles at the permissive temperature but lacked vacuoles specifically in the bud at the nonpermissive temperature. Our data suggest that Vps33p acts in the cytoplasm to facilitate Golgi-to-vacuole protein delivery. We propose that as a consequence of the vps33 protein-sorting defects, abnormalities in vacuolar morphology and vacuole assembly result.

scholarly journals Characterization of yeast Vps33p, a protein required for vacuolar protein sorting and vacuole biogenesis

1990 ◽  
Vol 10 (9) ◽  
pp. 4638-4649
Author(s):  
L M Banta ◽  
T A Vida ◽  
P K Herman ◽  
S D Emr
Keyword(s):  
Sequence Similarity ◽  
Protein Sorting ◽  
Atp Binding ◽  
Temperature Sensitive ◽  
Cell Fractionation ◽  
Permissive Temperature ◽  
Sensitive Mutant ◽  
Temperature Sensitive Mutant ◽  
Vacuolar Protein ◽  
Vacuole Biogenesis

vps33 mutants missort and secrete multiple vacuolar hydrolases and exhibit extreme defects in vacuolar morphology. Toward a molecular understanding of the role of the VPS33 gene in vacuole biogenesis, we have cloned this gene from a yeast genomic library by complementation of a temperature-sensitive vps33 mutation. Gene disruption demonstrated that VPS33 was not essential but was required for growth at high temperatures. At the permissive temperature, vps33 null mutants exhibited defects in vacuolar protein localization and vacuole morphology similar to those seen in most of the original mutant alleles. Sequence analysis revealed a putative open reading frame sufficient to encode a protein of 691 amino acids. Hydropathy analysis indicated that the deduced product of the VPS33 gene is generally hydrophilic, contains no obvious signal sequence or transmembrane domains, and is therefore unlikely to enter the secretory pathway. Polyclonal antisera raised against TrpE-Vps33 fusion proteins recognized a protein in yeast cells of the expected molecular weight, approximately 75,000. In cell fractionation studies, Vps33p behaved as a cytosolic protein. The predicted VPS33 gene product possessed sequence similarity with a number of ATPases and ATP-binding proteins specifically in their ATP-binding domains. One vps33 temperature-sensitive mutant contained a missense mutation near this region of sequence similarity; the mutation resulted in a Leu-646----Pro substitution in Vps33p. This temperature-sensitive mutant strain contained normal vacuoles at the permissive temperature but lacked vacuoles specifically in the bud at the nonpermissive temperature. Our data suggest that Vps33p acts in the cytoplasm to facilitate Golgi-to-vacuole protein delivery. We propose that as a consequence of the vps33 protein-sorting defects, abnormalities in vacuolar morphology and vacuole assembly result.

scholarly journals Tracking the Cartoon mouse phenotype: Hemopexin domain–dependent regulation of MT1-MMP pericellular collagenolytic activity

2018 ◽  
Vol 293 (21) ◽  
pp. 8113-8127 ◽  
Author(s):  
Moustafa Sakr ◽  
Xiao-Yan Li ◽  
Farideh Sabeh ◽  
Tamar Y. Feinberg ◽  
John J. G. Tesmer ◽  
...  
Keyword(s):  
Cell Surface ◽  
Critical Role ◽  
Collagenolytic Activity ◽  
Temperature Sensitive ◽  
Type I ◽  
Permissive Temperature ◽  
Sensitive Mutant ◽  
Temperature Sensitive Mutant ◽  
Golgi Network ◽  
Hemopexin Domain

Following ENU mutagenesis, a phenodeviant line was generated, termed the “Cartoon mouse,” that exhibits profound defects in growth and development. Cartoon mice harbor a single S466P point mutation in the MT1-MMP hemopexin domain, a 200-amino acid segment that is thought to play a critical role in regulating MT1-MMP collagenolytic activity. Herein, we demonstrate that the MT1-MMPS466P mutation replicates the phenotypic status of Mt1-mmp–null animals as well as the functional characteristics of MT1-MMP−/− cells. However, rather than a loss-of-function mutation acquired as a consequence of defects in MT1-MMP proteolytic activity, the S466P substitution generates a misfolded, temperature-sensitive mutant that is abnormally retained in the endoplasmic reticulum (ER). By contrast, the WT hemopexin domain does not play a required role in regulating MT1-MMP trafficking, as a hemopexin domain-deletion mutant is successfully mobilized to the cell surface and displays nearly normal collagenolytic activity. Alternatively, when MT1-MMPS466P–expressing cells are cultured at a permissive temperature of 25 °C that depresses misfolding, the mutant successfully traffics from the ER to the trans-Golgi network (ER → trans-Golgi network), where it undergoes processing to its mature form, mobilizes to the cell surface, and expresses type I collagenolytic activity. Together, these analyses define the Cartoon mouse as an unexpected gain-of-abnormal function mutation, wherein the temperature-sensitive mutant phenocopies MT1-MMP−/− mice as a consequence of eliciting a specific ER → trans-Golgi network trafficking defect.

Aggregation properties of chondrocytes infected with a temperature-sensitive mutant of Rous sarcoma virus

1980 ◽  
Vol 43 (1) ◽  
pp. 407-417
Author(s):  
A. Tanaka ◽  
A. Kaji
Keyword(s):  
Normal Level ◽  
Rous Sarcoma Virus ◽  
Cell Aggregation ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Sensitive Mutant ◽  
Temperature Sensitive Mutant ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Level Of Aggregation

Aggregation capacity of chicken embryo chondrocytes decreases when transformed by Rous sarcoma viruses. Cell-to-cell aggregation capacity of chondrocytes infected with a T class temperature-sensitive mutant (tsNY68) (with the temperature-sensitive lesion at the src gene) of Rous sarcoma virus is dependent upon the temperature at which these cells are grown. When grown at the permissive temperature (36 degrees C), where the transforming gene is expressed, aggregation capacity was lower than normal while infected cells grown at the non-permissive temperature (41.5 degrees C) had similar capacity to aggregate to that of normal chondrocytes. However, after a prolonged period of culture (10 days), chondrocytes transformed by wild type SR-RSV regained the normal level of aggregation capacity. Cells transformed by tsNY68 and incubated at the permissive temperature for 10 days also regained the normal level of aggregation capacity. It appears therefore that RSV-transformed chondrocytes first become less adhesive but during long-term cultivation they regain their property to aggregate. The decrease of aggregation capacity due to T class mutants of RSV at 36 degrees C is dependent on constant maintenance of protein synthesis because addition of cycloheximide restored the aggregation capacity even at the permissive temperature.

scholarly journals Infection of chick limb bud presumptive chondroblasts by a temperature-sensitive mutant of Rous sarcoma virus and the reversible inhibition of their terminal differentiation in culture.

1983 ◽  
Vol 3 (8) ◽  
pp. 1518-1526 ◽  
Author(s):  
D Boettiger ◽  
R Soltesz ◽  
H Holtzer ◽  
M Pacifici
Keyword(s):  
Rous Sarcoma Virus ◽  
Limb Bud ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Sensitive Mutant ◽  
Temperature Sensitive Mutant ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Infected Cells

Stage 21 to 22 chicken embryo limb bud cells were infected with a temperature-sensitive mutant of Rous sarcoma virus and were grown in culture. Although control, uninfected cells yielded definitive chondroblasts (by day 4) which initiated the synthesis of the cartilage-characteristic proteoglycan, the transformed cells grown at the permissive temperature failed to do so. These effects were fully reversible after a shift to the nonpermissive temperature. In addition, infected cells at the nonpermissive temperature expressed traits of terminal chondrogenic maturation 2 to 3 days earlier than parallel, uninfected cells. Thus, Rous sarcoma virus-induced transformation reversibly blocks terminal limb bud cell chondrogenesis in culture, at the nonpermissive temperature, viral infection may also induce intracellular or extracellular conditions which favor or accelerate the process of chondrogenic cell maturation.

Infection of chick limb bud presumptive chondroblasts by a temperature-sensitive mutant of Rous sarcoma virus and the reversible inhibition of their terminal differentiation in culture

1983 ◽  
Vol 3 (8) ◽  
pp. 1518-1526
Author(s):  
D Boettiger ◽  
R Soltesz ◽  
H Holtzer ◽  
M Pacifici
Keyword(s):  
Rous Sarcoma Virus ◽  
Limb Bud ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Sensitive Mutant ◽  
Temperature Sensitive Mutant ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Infected Cells

Stage 21 to 22 chicken embryo limb bud cells were infected with a temperature-sensitive mutant of Rous sarcoma virus and were grown in culture. Although control, uninfected cells yielded definitive chondroblasts (by day 4) which initiated the synthesis of the cartilage-characteristic proteoglycan, the transformed cells grown at the permissive temperature failed to do so. These effects were fully reversible after a shift to the nonpermissive temperature. In addition, infected cells at the nonpermissive temperature expressed traits of terminal chondrogenic maturation 2 to 3 days earlier than parallel, uninfected cells. Thus, Rous sarcoma virus-induced transformation reversibly blocks terminal limb bud cell chondrogenesis in culture, at the nonpermissive temperature, viral infection may also induce intracellular or extracellular conditions which favor or accelerate the process of chondrogenic cell maturation.

scholarly journals Mutations That Affect Vacuole Biogenesis Inhibit Proliferation of the Endoplasmic Reticulum inSaccharomyces cerevisiae

Genetics ◽  
2002 ◽  
Vol 160 (4) ◽  
pp. 1335-1352 ◽  
Author(s):  
Ann J Koning ◽  
Lynnelle L Larson ◽  
Emily J Cadera ◽  
Mark L Parrish ◽  
Robin L Wright
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Sorting ◽  
Temperature Sensitive ◽  
Biosynthetic Enzyme ◽  
Temperature Sensitive Mutants ◽  
Coa Reductase ◽  
Vacuolar Protein ◽  
Vacuole Biogenesis ◽  
Global Defect ◽  
Assembly Defect

AbstractIn yeast, increased levels of the sterol biosynthetic enzyme, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase isozyme, Hmg1p, induce assembly of nuclear-associated ER membranes called karmellae. To identify additional genes involved in karmellae assembly, we screened temperature-sensitive mutants for karmellae assembly defects. Two independently isolated, temperature-sensitive strains that were also defective for karmellae biogenesis carried mutations in VPS16, a gene involved in vacuolar protein sorting. Karmellae biogenesis was defective in all 13 other vacuole biogenesis mutants tested, although the severity of the karmellae assembly defect varied depending on the particular mutation. The hypersensitivity of 14 vacuole biogenesis mutants to tunicamycin was well correlated with pronounced defects in karmellae assembly, suggesting that the karmellae assembly defect reflected alteration of ER structure or function. Consistent with this hypothesis, seven of eight mutations causing defects in secretion also affected karmellae assembly. However, the vacuole biogenesis mutants were able to proliferate their ER in response to Hmg2p, indicating that the mutants did not have a global defect in the process of ER biogenesis.

scholarly journals Characterization of the herpes simplex virus (HSV)-1 tegument protein VP1-2 during infection with the HSV temperature-sensitive mutant tsB7

2009 ◽  
Vol 90 (10) ◽  
pp. 2353-2363 ◽  
Author(s):  
F. Abaitua ◽  
R. N. Souto ◽  
H. Browne ◽  
T. Daikoku ◽  
P. O'Hare
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Current Evidence ◽  
Tegument Protein ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Sensitive Mutant ◽  
Temperature Sensitive Mutant ◽  
Simplex Virus ◽  
Hsv 1

VP1-2, encoded by the UL36 gene of herpes simplex virus (HSV), is a large structural protein, conserved across the family Herpesviridae, that is assembled into the tegument and is essential for virus replication. Current evidence indicates that VP1-2 is a central component in the tegumentation and envelopment processes and that it also possesses important roles in capsid transport and entry. However, any detailed mechanistic understanding of VP1-2 function(s) remains limited. This study characterized the replication of HSV-1 tsB7, a temperature-sensitive mutant restricted at the non-permissive temperature due to a defect in VP1-2 function. A tsB7 virus expressing green fluorescent protein-fused VP16 protein was used to track the accumulation and location of a major tegument protein. After infection at the permissive temperature and shift to the non-permissive temperature, the production of infectious virus ceased. VP1-2 accumulated in altered cytosolic clusters, together with VP16 and other virion proteins. Furthermore, correlating with the results of immunofluorescence, electron microscopy demonstrated abnormal cytosolic capsid clustering and a block in envelopment. As VP1-2 encompasses a ubiquitin-specific protease domain, the occurrence of ubiquitin-conjugated proteins during tsB7 infection was also examined at the non-permissive temperature. A striking overaccumulation was observed of ubiquitin-specific conjugates in cytoplasmic clusters, overlapping and adjacent to the VP1-2 clusters. These results are discussed in relation to the possible functions of VP1-2 in the assembly pathway and the nature of the defect in tsB7.

scholarly journals Rapid and reversible reduction of junctional permeability in cells infected with a temperature-sensitive mutant of avian sarcoma virus.

1981 ◽  
Vol 91 (2) ◽  
pp. 573-578 ◽  
Author(s):  
M M Atkinson ◽  
A S Menko ◽  
R G Johnson ◽  
J R Sheppard ◽  
J D Sheridan
Keyword(s):  
Rat Kidney ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Avian Sarcoma Virus ◽  
Dye Transfer ◽  
Sensitive Mutant ◽  
Temperature Sensitive Mutant ◽  
Sarcoma Virus ◽  
Infected Cells ◽  
Avian Sarcoma

The transformed or normal phenotype of cultured normal rat kidney cells infected with a temperature-sensitive mutant of avian sarcoma virus is conditional on the temperature at which the cells are grown. Using dye injection techniques, we show that junction-mediated dye transfer is also temperature-sensitive. The extent and rate of transfer between infected cells grown at the transformation-permissive temperature (35 degrees C) is significantly reduced when compared to infected cells grown at the nonpermissive temperature (40.5 degrees C) or uninfected cells grown at either temperature. Infected cells subjected to reciprocal temperature shifts express rapid and reversible alterations of dye transfer capacities, with responses evident by 15 min and completed by 60 min for temperature shifts in either direction. These results suggest that altered junctional capacities may be fundamental to the expression of the ASV-induced, transformed phenotype.

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