scholarly journals BiP/Kar2p serves as a molecular chaperone during carboxypeptidase Y folding in yeast.

1995 ◽  
Vol 130 (1) ◽  
pp. 41-49 ◽  
Author(s):  
J F Simons ◽  
S Ferro-Novick ◽  
M D Rose ◽  
A Helenius
Keyword(s):  
Molecular Chaperone ◽  
Intracellular Transport ◽  
Critical Role ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Yeast Strains ◽  
Mutant Strains

Although transiently associated with numerous newly synthesized proteins, BiP has not been shown to be an essential component directly linked to the folding and oligomerization of newly synthesized proteins in the endoplasmic reticulum. To determine whether it is needed as a molecular chaperone, we analyzed the maturation of an endogenous yeast glycoprotein, carboxypeptidase Y (CPY) in several yeast strains with temperature-sensitive mutations in BiP. These kar2 mutant strains have previously been found to be defective in translocation at the nonpermissive temperature (Vogel, J. P., L. M. Misra, and M. D. Rose, 1990. J. Cell Biol, 110:1885-1895). To circumvent the translocation block, we used DTT at permissive temperature to delay folding and intracellular transport. We then followed the maturation of the ER-retained CPY after shifting to the nonpermissive temperature and dilution of the DTT. Without the functional chaperone, CPY aggregated, failed to be oxidized, and remained in the ER. In contrast to wild-type cells, in which BiP binding was transient with no more than 10-15% of labeled CPY associated at any time, 30-100% of the CPY remained associated with BiP in the mutant strains. In a heterozygous diploid strain, CPY matured and exited the ER normally. Taken together, the results provide clear evidence that BiP plays a critical role as a molecular chaperone in CPY folding.

Mutations in the three largest subunits of yeast RNA polymerase II that affect enzyme assembly

1991 ◽  
Vol 11 (9) ◽  
pp. 4669-4678 ◽  
Author(s):  
P A Kolodziej ◽  
R A Young
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Time Course ◽  
Mutant Enzyme ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Gradient Fractionation ◽  
Mutant Cells

Mutations in the three largest subunits of yeast RNA polymerase II (RPB1, RPB2, and RPB3) were investigated for their effects on RNA polymerase II structure and assembly. Among 23 temperature-sensitive mutations, 6 mutations affected enzyme assembly, as assayed by immunoprecipitation of epitope-tagged subunits. In all six assembly mutants, RNA polymerase II subunits synthesized at the permissive temperature were incorporated into stably assembled, immunoprecipitable enzyme and remained stably associated when cells were shifted to the nonpermissive temperature, whereas subunits synthesized at the nonpermissive temperature were not incorporated into a completely assembled enzyme. The observation that subunit subcomplexes accumulated in assembly-mutant cells at the nonpermissive temperature led us to investigate whether these subcomplexes were assembly intermediates or merely byproducts of mutant enzyme instability. The time course of assembly of RPB1, RPB2, and RPB3 was investigated in wild-type cells and subsequently in mutant cells. Glycerol gradient fractionation of extracts of cells pulse-labeled for various times revealed that a subcomplex of RPB2 and RPB3 appears soon after subunit synthesis and can be chased into fully assembled enzyme. The RPB2-plus-RPB3 subcomplexes accumulated in all RPB1 assembly mutants at the nonpermissive temperature but not in an RPB2 or RPB3 assembly mutant. These data indicate that RPB2 and RPB3 form a complex that subsequently interacts with RPB1 during the assembly of RNA polymerase II.

scholarly journals Mutations in the three largest subunits of yeast RNA polymerase II that affect enzyme assembly.

1991 ◽  
Vol 11 (9) ◽  
pp. 4669-4678 ◽  
Author(s):  
P A Kolodziej ◽  
R A Young
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Time Course ◽  
Mutant Enzyme ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Gradient Fractionation ◽  
Mutant Cells

Mutations in the three largest subunits of yeast RNA polymerase II (RPB1, RPB2, and RPB3) were investigated for their effects on RNA polymerase II structure and assembly. Among 23 temperature-sensitive mutations, 6 mutations affected enzyme assembly, as assayed by immunoprecipitation of epitope-tagged subunits. In all six assembly mutants, RNA polymerase II subunits synthesized at the permissive temperature were incorporated into stably assembled, immunoprecipitable enzyme and remained stably associated when cells were shifted to the nonpermissive temperature, whereas subunits synthesized at the nonpermissive temperature were not incorporated into a completely assembled enzyme. The observation that subunit subcomplexes accumulated in assembly-mutant cells at the nonpermissive temperature led us to investigate whether these subcomplexes were assembly intermediates or merely byproducts of mutant enzyme instability. The time course of assembly of RPB1, RPB2, and RPB3 was investigated in wild-type cells and subsequently in mutant cells. Glycerol gradient fractionation of extracts of cells pulse-labeled for various times revealed that a subcomplex of RPB2 and RPB3 appears soon after subunit synthesis and can be chased into fully assembled enzyme. The RPB2-plus-RPB3 subcomplexes accumulated in all RPB1 assembly mutants at the nonpermissive temperature but not in an RPB2 or RPB3 assembly mutant. These data indicate that RPB2 and RPB3 form a complex that subsequently interacts with RPB1 during the assembly of RNA polymerase II.

scholarly journals Role for adenosine triphosphate in regulating the assembly and transport of vesicular stomatitis virus G protein trimers.

1987 ◽  
Vol 105 (5) ◽  
pp. 1957-1969 ◽  
Author(s):  
R W Doms ◽  
D S Keller ◽  
A Helenius ◽  
W E Balch
Keyword(s):  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Golgi Complex ◽  
Gradient Centrifugation ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Protein Aggregate ◽  
Vesicular Stomatitis

We have characterized the process by which the vesicular stomatitis virus (VSV) G protein acquires its final oligomeric structure using density-gradient centrifugation in mildly acidic sucrose gradients. The mature wild-type VSV G protein is a noncovalently associated trimer. Trimers are assembled from newly synthesized G monomers with a t1/2 of 6-8 min. To localize the site of trimerization and to correlate trimer formation with steps in transport between the endoplasmic reticulum (ER) and Golgi complex, we examined the kinetics of assembly of the temperature-sensitive mutant VSV strain, ts045. At the nonpermissive temperature (39 degrees C), ts045 G protein is not transported from the ER. The phenotypic defect that inhibited export from the ER at the nonpermissive temperature was found to be the accumulation of ts045 G protein in an aggregate. After being shifted to the permissive temperature (32 degrees C), the ts045 G protein aggregate rapidly dissociated (t1/2 less than 1 min) to monomeric G protein which subsequently trimerized with the same kinetics as the wild-type G protein. Only trimers were transported to the Golgi complex. Kinetic studies, as well as the finding that trimerization occurred under conditions which block ER to Golgi transport (at both 15 and 4 degrees C), showed that trimers were formed in the ER. Depletion of cellular ATP inhibited both the dissociation of the aggregated intermediate of ts045 G protein as well as the formation of stable trimers. The results indicate that oligomerization of G protein occurs in several steps, is sensitive to cellular ATP, and is required for transport from the ER.

scholarly journals Construction of In-Frame aroA Deletion Mutants of Mannheimia haemolytica, Pasteurella multocida, and Haemophilus somnus by Using a New Temperature-Sensitive Plasmid

2005 ◽  
Vol 71 (11) ◽  
pp. 7196-7202 ◽  
Author(s):  
Fred M. Tatum ◽  
Robert E. Briggs
Keyword(s):  
Pasteurella Multocida ◽  
Host Cells ◽  
Plasmid Replication ◽  
Deletion Mutants ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Target Species ◽  
Haemophilus Somnus

ABSTRACT A temperature-sensitive (TS) plasmid was generated from the endogenous streptomycin resistance plasmid of Mannheimia hemolytica and used to engineer in-frame aroA deletion mutants of Mannheimia hemolytica, Pasteurella multocida, and Haemophilus somnus. TS replacement plasmids carrying in-frame aroA deletions were constructed for each target species and introduced into host cells by electroporation. After recovery in broth, cells were spread onto plates containing antibiotic and incubated at 30°C, the permissive temperature for autonomous plasmid replication. Transfer of transformants to selective plates cultured at a nonpermissive temperature for plasmid replication selected for single-crossover mutants consisting of replacement plasmids that had integrated into host chromosomes by homologous recombination. Transfer of the single-crossover mutants back to a permissive temperature without antibiotic selection drove plasmid resolution, and, depending on where plasmid excision occurred, either deletion mutants or wild-type cells were generated. The system used here represents a broadly applicable means for generating unmarked mutants of Pasteurellaceae species.

scholarly journals The Myxococcus xanthus Developmental Program Can Be Delayed by Inhibition of DNA Replication

2007 ◽  
Vol 189 (24) ◽  
pp. 8793-8800 ◽  
Author(s):  
Christopher J. Rosario ◽  
Mitchell Singer
Keyword(s):  
Dna Replication ◽  
Developmental Process ◽  
Myxococcus Xanthus ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Temperature Growth ◽  
Developmental Program

ABSTRACT Under conditions of nutrient deprivation, Myxococcus xanthus undergoes a developmental process that results in the formation of a fruiting body containing environmentally resistant myxospores. We have shown that myxospores contain two copies of the genome, suggesting that cells must replicate the genome prior to or during development. To further investigate the role of DNA replication in development, a temperature-sensitive dnaB mutant, DnaBA116V, was isolated from M. xanthus. Unlike what happens in Escherichia coli dnaB mutants, where DNA replication immediately halts upon a shift to a nonpermissive temperature, growth and DNA replication of the M. xanthus mutant ceased after one cell doubling at a nonpermissive temperature, 37°C. We demonstrated that at the nonpermissive temperature the DnaBA116V mutant arrested as a population of 1n cells, implying that these cells could complete one round of the cell cycle but did not initiate new rounds of DNA replication. In developmental assays, the DnaBA116V mutant was unable to develop into fruiting bodies and produced fewer myxospores than the wild type at the nonpermissive temperature. However, the mutant was able to undergo development when it was shifted to a permissive temperature, suggesting that cells had the capacity to undergo DNA replication during development and to allow the formation of myxospores.

scholarly journals Construction and Characterization of a Temperature-Sensitive Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutant

1998 ◽  
Vol 72 (3) ◽  
pp. 2047-2054 ◽  
Author(s):  
Mingjun Huang ◽  
Ralf Zensen ◽  
Michael Cho ◽  
Malcolm A. Martin
Keyword(s):  
Virus Particle ◽  
Particle Production ◽  
Wild Type Virus ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Virus Particle Production

ABSTRACT A temperature-sensitive (ts) human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutant was generated by charged-cluster-to-alanine mutagenesis. The mutant virus, containing three charged residues within the RT finger domain changed to alanine (K64A, K66A, and D67A), replicated normally at 34.5 but not 39.5°C. Quantitating virus particle production by p24 antigen capture or virion-associated RT activity and virus infectivity by the MAGI cell assay, we found that (i) mutant virions produced at the permissive temperature were indistinguishable from wild-type virus in assays performed at the nonpermissive temperature, suggesting that thets mutation did not impair early steps in the virus replication cycle and that the mutant RT enzyme was not ts; and (ii) virus particle production in cells transfected with thets mutant at the nonpermissive temperature was comparable to that of wild-type virus. However, the particle-associated RT activity and infectivity of mutant virions produced at the nonpermissive temperature were greatly reduced when assays were conducted at the permissive temperature. These results are consistent with an irreversible ts event affecting RT that occurs during virus particle production. Radioimmunoprecipitation analyses revealed that both p66 and p51 RT subunits were absent from mutant virions generated at 39.5°C. The presence of normal levels of HIV-1 integrase in mutant particles produced at the nonpermissive temperature was inconsistent with defective Gag-Pol synthesis or Gag-Pol incorporation into progeny virions. Furthermore, wild-type levels of the mutant Pr160 gag-pol were detected in virions produced at the nonpermissive temperature when the HIV-1 protease was inactivated by site-specific mutagenesis. Taken together, these results are most consistent with a ts defect affecting the degradation or aberrant processing of the mutated RT during its processing/maturation within nascent particles.

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.

Growth Properties of Neural Cell Lines Immortalized with the Tsa58 Allele of Sv40 Large T Antigen

1997 ◽  
Vol 6 (3) ◽  
pp. 231-238 ◽  
Author(s):  
M.E. Truckenmiller ◽  
Ora Dillon-Carter ◽  
Carlo Tornatore ◽  
Henrietta Kulaga ◽  
Hidetoshi Takashima ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Line ◽  
Cell Lines ◽  
T Antigen ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Large T Antigen ◽  
Wild Type ◽  
Sv40 Large T Antigen ◽  
Growth Properties

In vitro growth properties of three CNS-derived cell lines were compared under a variety of culture conditions. The M213-20 and J30a cell lines were each derived from embryonic CNS culture with the temperature-sensitive (ts) allele of SV40 large T antigen, tsA58, while the A7 cell line was immortalized using wild-type SV40 large T antigen. Cells immortalized with tsA58 SV40 large T proliferate at the permissive temperature, 33° C, while growth is expected to be suppressed at the nonpermissive temperature, 39.5°C. Both the M213-20 and J30a cell lines were capable of proliferating at 39.5°C continuously for up to 6 mo. All three cell lines showed no appreciable differences in growth rates related to temperature over a 7-day period in either serum-containing or defined serum-free media. The percentage of cells in S-phase of the cell cycle did not decrease or was elevated at 39.5°C for all three cell lines. After 3 wk at 39.5°C, the three cell lines also showed positive immunostaining using two monoclonal antibodies reacting with different epitopes of SV40 large T antigen. Double strand DNA sequence analyses of a 300 base pair (bp) fragment of the large T gene from each cell line, which included the ts locus, revealed mutations in both the J30a and M213-20 cell lines. The J30a cell line ts mutation had reverted to wild type, and two additional loci with bp substitutions with predicted amino acid changes were also found. While the ts mutation of the M213-20 cells was retained, an additional bp substitution with a predicted amino acid change was found. The A7 cell line sequence was identical to the reference wild-type sequence. These findings suggest that (a) nucleic acid sequences in the temperature-sensitive region of the tsA58 allele of SV40 large T are not necessarily stable, and (b) temperature sensitivity of cell lines immortalized with tsA58 is not necessarily retained.

scholarly journals Retrograde Transport of Golgi-localized Proteins to the ER

1998 ◽  
Vol 140 (1) ◽  
pp. 1-15 ◽  
Author(s):  
Nelson B. Cole ◽  
Jan Ellenberg ◽  
Jia Song ◽  
Diane DiEuliis ◽  
Jennifer Lippincott-Schwartz
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Fusion Proteins ◽  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Viral Glycoprotein ◽  
Lumenal Domain

The ER is uniquely enriched in chaperones and folding enzymes that facilitate folding and unfolding reactions and ensure that only correctly folded and assembled proteins leave this compartment. Here we address the extent to which proteins that leave the ER and localize to distal sites in the secretory pathway are able to return to the ER folding environment during their lifetime. Retrieval of proteins back to the ER was studied using an assay based on the capacity of the ER to retain misfolded proteins. The lumenal domain of the temperature-sensitive viral glycoprotein VSVGtsO45 was fused to Golgi or plasma membrane targeting domains. At the nonpermissive temperature, newly synthesized fusion proteins misfolded and were retained in the ER, indicating the VSVGtsO45 ectodomain was sufficient for their retention within the ER. At the permissive temperature, the fusion proteins were correctly delivered to the Golgi complex or plasma membrane, indicating the lumenal epitope of VSVGtsO45 also did not interfere with proper targeting of these molecules. Strikingly, Golgi-localized fusion proteins, but not VSVGtsO45 itself, were found to redistribute back to the ER upon a shift to the nonpermissive temperature, where they misfolded and were retained. This occurred over a time period of 15 min–2 h depending on the chimera, and did not require new protein synthesis. Significantly, recycling did not appear to be induced by misfolding of the chimeras within the Golgi complex. This suggested these proteins normally cycle between the Golgi and ER, and while passing through the ER at 40°C become misfolded and retained. The attachment of the thermosensitive VSVGtsO45 lumenal domain to proteins promises to be a useful tool for studying the molecular mechanisms and specificity of retrograde traffic to the ER.

Correction of the defect in initiation of DNA replication in a temperature-sensitive mutant hamster cell line by in vitro addition of extracts from normal cells

1985 ◽  
Vol 5 (4) ◽  
pp. 902-905
Author(s):  
M Narkhammar ◽  
R Hand
Keyword(s):  
Cell Line ◽  
Nuclear Extract ◽  
Cell Extract ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
Whole Cell ◽  
Mutant Cells

ts BN-2 is a temperature-sensitive hamster cell line that is defective in DNA synthesis at the restrictive temperature. The mutant expresses its defect during in vitro replication in whole-cell lysates. Addition of a high-salt-concentration extract from wild-type BHK-21, revertant RBN-2, or CHO cells to mutant cells lysed with 0.01% Brij 58 increased the activity in the mutant three- to fourfold, so that it reached 85% of the control value, and restored replicative synthesis. The presence of extract had an insignificant effect on wild-type and revertant replication and on mutant replication at the permissive temperature. Extract prepared from mutant cells was less effective than the wild-type cell extract was. Also, the stimulatory activity was more heat labile in the mutant than in the wild-type extract. Nuclear extract was as active as whole-cell extract.

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