Chemical chaperone-mediated protein folding: stabilization of P22 tailspike folding intermediates by glycerol

2007 ◽  
Vol 388 (8) ◽  
pp. 797-804 ◽  
Author(s):  
Rajesh Mishra ◽  
Rajiv Bhat ◽  
Robert Seckler
Keyword(s):  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Chemical Chaperone ◽  
Preferential Hydration ◽  
Denatured State ◽  
Folding Intermediates ◽  
Fluorescence Measurements ◽  
Phage P22 ◽  
The Stability ◽  
P22 Tailspike

Abstract Polyol co-solvents such as glycerol increase the thermal stability of proteins. This has been explained by preferential hydration favoring the more compact native over the denatured state. Although polyols are also expected to favor aggregation by the same mechanism, they have been found to increase the folding yields of some large, aggregation-prone proteins. We have used the homotrimeric phage P22 tailspike protein to investigate the origin of this effect. The folding of this protein is temperature-sensitive and limited by the stability of monomeric folding intermediates. At non-permissive temperature (≥35°C), tailspike refolding yields were increased significantly in the presence of 1–4 m glycerol. At low temperature, tailspike refolding is prevented when folding intermediates are destabilized by the addition of urea. Glycerol could offset the urea effect, suggesting that the polyol acts by stabilizing crucial folding intermediates and not by increasing solvent viscosity. The stabilization effect of glycerol on tailspike folding intermediates was confirmed in experiments using a temperature-sensitive folding mutant protein, by fluorescence measurements of subunit folding kinetics, and by temperature up-shift experiments. Our results suggest that the chemical chaperone effect of polyols observed in the folding of large proteins is due to preferential hydration favoring structure formation in folding intermediates.

Phenotype–genotype relationships in peroxisome biogenesis disorders of PEX1-defective complementation group 1 are defined by Pex1p–Pex6p interaction

2001 ◽  
Vol 357 (2) ◽  
pp. 417-426 ◽  
Author(s):  
Shigehiko TAMURA ◽  
Naomi MATSUMOTO ◽  
Atsushi IMAMURA ◽  
Nobuyuki SHIMOZAWA ◽  
Yasuyuki SUZUKI ◽  
...  
Keyword(s):  
Zellweger Syndrome ◽  
Complementation Group ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Peroxisome Biogenesis ◽  
Causative Gene ◽  
Aaa Atpase ◽  
Peroxisome Biogenesis Disorders ◽  
The Stability

The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS), neonatal adrenoleucodystrophy (NALD) and infantile Refsum disease (IRD), are fatal autosomal recessive diseases caused by impaired peroxisome biogenesis, of which 12 genotypes have been reported. ZS patients manifest the severest clinical and biochemical abnormalities, whereas those with NALD and IRD show less severity and the mildest features respectively. We have reported previously that temperature-sensitive peroxisome assembly is responsible for the mildness of the clinical features of IRD. PEX1 is the causative gene for PBDs of complementation group E (CG-E, CG1 in the U.S.A. and Europe), the PBDs of highest incidence, encoding the peroxin Pex1p of the AAA ATPase family. It has been also reported that Pex1p and Pex6p interact with each other. In the present study we investigated phenotype–genotype relationships of CG1 PBDs. Pex1p from IRD such as Pex1p with the most frequently identified mutation at G843D was largely degraded in vivo at 37°C, whereas a normal level of Pex1p was detectable at the permissive temperature. In contrast, PEX1 proteins derived from ZS patients, including proteins with a mutation at L664P or the deletion of residues 634–690, were stably present at both temperatures. Pex1p-G843D interacted with Pex6p at approx. 50% of the level of normal Pex1p, whereas Pex1p from ZS patients mostly showing non-temperature-sensitive peroxisome biogenesis hardly bound to Pex6p. Taking these results together, we consider it most likely that the stability of Pex1p reflects temperature-sensitive peroxisome assembly in IRD fibroblasts. Failure in Pex1p–Pex6p interaction gives rise to more severe abnormalities, such as those manifested by patients with ZS.

Temperature-sensitive mutants in rfaI and rfaJ, genes for galactosyltransferase I and glucosyltransferase II, for synthesis of lipopolysaccharide in Salmonella typhimurium

1985 ◽  
Vol 31 (9) ◽  
pp. 861-869 ◽  
Author(s):  
Sunil K. Kadam ◽  
Mark S. Peppler ◽  
Kenneth E. Sanderson
Keyword(s):  
Salmonella Typhimurium ◽  
Temperature Sensitive ◽  
Side Chains ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Temperature Sensitive Mutants ◽  
Specific Phage ◽  
Phage P22 ◽  
Phage Sensitivity ◽  
Sensitivity Data

Certain rough mutants of Salmonella typhimurium LT2 were shown to be temperature sensitive for the production of lipopolysaccharide (LPS). When grown at the restrictive temperature (42 or 45 °C), the cells contained LPS deficient in O (somatic) side chains, based on phage-sensitivity data and gel electrophoresis of the LPS. Cells grown at the permissive temperature, 30 °C, made LPS resembling that of smooth cells. The mobility of the LPS in gels, the phage sensitivity patterns, and gas chromatographic analysis indicate that LPS of 45 °C-grown cells of SA126 (rfaJ3012) is of chemotype Rb2, with one glucose and two galactose units (and thus inferred to be due to a mutation in rfaJ), and LPS of 45 °C-grown cells of SA134 (rfaI3020) is of chemotype Rb3, with one glucose and one galactose unit (inferred to be rfaI). These inferences were confirmed, for pKZ26 (pBR322-rfaGBIJ) and pKZ27 (pBR322-rfaGBI) both complement rfaI3020, but only pKZ26 complemented rfaJ3012. In addition, pKZ26 carrying a Tn5 insertion resulting in loss of complementation of a known rfaJ mutation, but not of rfaG, B, or I, also resulted in loss of rfaJ3012 complementation. Based on gel analysis, there is a small amount of the LPS containing smooth side chains in cells of SA126 grown at 45 °C; following a switch to 30 °C, the amount of LPS with O side chains gradually increased, and the amount of core LPS was reduced, though even after 3 h the LPS does not fully resemble that of smooth strains. Cells grown at 42 °C show limited capacity to adsorb a smooth-specific phage, P22, but the capacity to adsorb the phage increased fourfold after 5 min growth at 30 °C, and by 60 min at 30 °C adsorption resembled that seen by smooth cells. These data indicate that synthesis of the smooth LPS begins less than 5 min after shift to the permissive temperature, and that phage adsorption is a more sensitive test for the appearance of smooth side chains than the use of polyacrylamide gels with silver stains.

Mutations that Stabilize Folding Intermediates of Phage P22 Tailspike Protein: Foldingin Vivoandin Vitro, Stability, and Structural Context

1995 ◽  
Vol 249 (1) ◽  
pp. 185-194 ◽  
Author(s):  
Martina Beißinger ◽  
Sang Chul Lee ◽  
Stefan Steinbacher ◽  
Peter Reinemer ◽  
Robert Huber ◽  
...  
Keyword(s):  
Folding Intermediates ◽  
Structural Context ◽  
Phage P22 ◽  
P22 Tailspike ◽  
Tailspike Protein

scholarly journals Intragenic suppressors of folding defects in the P22 tailspike protein.

Genetics ◽  
1991 ◽  
Vol 127 (2) ◽  
pp. 263-277 ◽  
Author(s):  
B Fane ◽  
J King
Keyword(s):  
Amino Acid Sequences ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Chain Folding ◽  
Phage P22 ◽  
Nonsense Codons ◽  
P22 Tailspike ◽  
Polypeptide Chains ◽  
Tailspike Protein

Abstract Within the amino acid sequences of polypeptide chains little is known of the distribution of sites and sequences critical for directing chain folding and assembly. Temperature-sensitive folding (tsf) mutations identifying such sites have been previously isolated and characterized in gene 9 of phage P22 encoding the tailspike endorhamnosidase. We report here the isolation of a set of second-site conformational suppressors which alleviate the defect in such folding mutants. The suppressors were selected for their ability to correct the defects of missense tailspike polypeptide chains, generated by growth of gene 9 amber mutants on Salmonella host strains inserting either tyrosine, serine, glutamine or leucine at the nonsense codons. Second-site suppressors were recovered for 13 of 22 starting sites. The suppressors of defects at six sites mapped within gene 9. (Suppressors for seven other sites were extragenic and distant from gene 9.) The missense polypeptide chains generated from all six suppressible sites displayed ts phenotypes. Temperature-sensitive alleles were isolated at these amber sites by pseudoreversion. The intragenic suppressors restored growth at the restrictive temperature of these presumptive tsf alleles. Characterization of protein maturation in cells infected with mutant phages carrying the intragenic suppressors indicates that the suppression is acting at the level of polypeptide chain folding and assembly.

scholarly journals A Yeast taf17 Mutant Requires the Swi6 Transcriptional Activator for Viability and Shows Defects in Cell Cycle-Regulated Transcription

Genetics ◽  
2000 ◽  
Vol 154 (4) ◽  
pp. 1561-1576
Author(s):  
Neil Macpherson ◽  
Vivien Measday ◽  
Lynda Moore ◽  
Brenda Andrews
Keyword(s):  
Cell Cycle ◽  
Transcriptional Activation ◽  
Essential Gene ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Synthetic Lethal ◽  
Cycle Arrest ◽  
A Cell ◽  
Allelism Tests ◽  
Complementation Groups

Abstract In Saccharomyces cerevisiae, the Swi6 protein is a component of two transcription factors, SBF and MBF, that promote expression of a large group of genes in the late G1 phase of the cell cycle. Although SBF is required for cell viability, SWI6 is not an essential gene. We performed a synthetic lethal screen to identify genes required for viability in the absence of SWI6 and identified 10 complementation groups of swi6-dependent lethal mutants, designated SLM1 through SLM10. We were most interested in mutants showing a cell cycle arrest phenotype; both slm7-1 swi6Δ and slm8-1 swi6Δ double mutants accumulated as large, unbudded cells with increased 1N DNA content and showed a temperature-sensitive growth arrest in the presence of Swi6. Analysis of the transcript levels of cell cycle-regulated genes in slm7-1 SWI6 mutant strains at the permissive temperature revealed defects in regulation of a subset of cyclin-encoding genes. Complementation and allelism tests showed that SLM7 is allelic with the TAF17 gene, which encodes a histone-like component of the general transcription factor TFIID and the SAGA histone acetyltransferase complex. Sequencing showed that the slm7-1 allele of TAF17 is predicted to encode a version of Taf17 that is truncated within a highly conserved region. The cell cycle and transcriptional defects caused by taf17slm7-1 are consistent with the role of TAFIIs as modulators of transcriptional activation and may reflect a role for TAF17 in regulating activation by SBF and MBF.

scholarly journals A Screen for Genes Involved in the Anaphase Proteolytic Pathway Identifies tsm1+, a Novel Schizosaccharomyces pombe Gene Important for Microtubule Integrity

Genetics ◽  
1998 ◽  
Vol 149 (3) ◽  
pp. 1251-1264
Author(s):  
Ekaterina L Grishchuk ◽  
James L Howe ◽  
J Richard McIntosh
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Nuclear Division ◽  
Mitotic Division ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Anaphase Promoting Complex ◽  
Chloromethyl Ketone ◽  
Proteolytic Pathway ◽  
Functional Involvement ◽  
Spindle Elongation

Abstract The growth of several mitotic mutants of Schizosaccharomyces pombe, including nuc2-663, is inhibited by the protease inhibitor N-Tosyl-L-Phenylalanine Chloromethyl Ketone (TPCK). Because nuc2+ encodes a presumptive component of the Anaphase Promoting Complex, which is required for the ubiquitin-dependent proteolysis of certain proteins during exit from mitosis, we have used sensitivity to TPCK as a criterion by which to search for novel S. pombe mutants defective in the anaphase-promoting pathway. In a genetic screen for temperature-sensitive mitotic mutants that were also sensitive to TPCK at a permissive temperature, we isolated three tsm (TPCK-sensitive mitotic) strains. Two of these are alleles of cut1+, but tsm1-512 maps to a novel genetic location. The tsm1-512 mutation leads to delayed nuclear division at restrictive temperatures, apparently as a result of an impaired ability to form a metaphase spindle. After shift of early G2 cells to 36°, tsm1-512 arrests transiently in the second mitotic division and then exits mitosis, as judged by spindle elongation and septation. The chromosomes, however, often fail to segregate properly. Genetic interactions between tsm1-512 and components of the anaphase proteolytic pathway suggest a functional involvement of the Tsm1 protein in this pathway.

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.

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