scholarly journals Isolation of peroxisome assembly mutants from Saccharomyces cerevisiae with different morphologies using a novel positive selection procedure.

1992 ◽  
Vol 119 (1) ◽  
pp. 153-162 ◽  
Author(s):  
I Van der Leij ◽  
M Van den Berg ◽  
R Boot ◽  
M Franse ◽  
B Distel ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Positive Selection ◽  
Protein Import ◽  
Zellweger Syndrome ◽  
Signal Transduction Pathway ◽  
Selection Procedure ◽  
Complementation Group ◽  
Cell Fractionation ◽  
Wild Type ◽  
Beta Oxidation

We have developed a positive selection system for the isolation of Saccharomyces cerevisiae mutants with disturbed peroxisomal functions. The selection is based on the lethality of hydrogen peroxide (H2O2) that is produced in wild type cells during the peroxisomal beta-oxidation of fatty acids. In total, 17 mutants having a general impairment of peroxisome biogenesis were isolated, as revealed by their inability to grow on oleic acid as the sole carbon source and their aberrant cell fractionation pattern of peroxisomal enzymes. The mutants were shown to have monogenetic defects and to fall into 12 complementation groups. Representative members of each complementation group were morphologically examined by immunocytochemistry using EM. In one mutant the induction and morphology of peroxisomes is normal but import of thiolase is abrogated, while in another the morphology differs from the wild type: stacked peroxisomal membranes are present that are able to import thiolase but not catalase. These mutants suggest the existence of multiple components involved in peroxisomal protein import. Some mutants show the phenotype characteristic of glucose-repressed cells, an indication for the interruption of a signal transduction pathway resulting in organelle proliferation. In the remaining mutants morphologically detectable peroxisomes are absent: this phenotype is also known from fibroblasts of patients suffering from Zellweger syndrome, a disorder resulting from impairment of peroxisomes.

scholarly journals An efficient positive selection procedure for the isolation of peroxisomal import and peroxisome assembly mutants of Saccharomyces cerevisiae.

Genetics ◽  
1993 ◽  
Vol 135 (3) ◽  
pp. 731-740 ◽  
Author(s):  
Y Elgersma ◽  
M van den Berg ◽  
H F Tabak ◽  
B Distel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Oleic Acid ◽  
Protein Import ◽  
Chimeric Protein ◽  
Selection Procedure ◽  
Peroxisome Biogenesis ◽  
Wild Type ◽  
Peroxisomal Protein ◽  
Complementation Groups ◽  
Resistance Protein

Abstract To study peroxisome biogenesis, we developed a procedure to select for Saccharomyces cerevisiae mutants defective in peroxisomal protein import or peroxisome assembly. For this purpose, a chimeric gene was constructed encoding the bleomycin resistance protein linked to the peroxisomal protein luciferase. In wild-type cells this chimeric protein is imported into the peroxisome, which prevents the neutralizing interaction of the chimeric protein with its toxic phleomycin ligand. Peroxisomal import and peroxisome assembly mutants are unable to import this chimeric protein into their peroxisomes. This enables the bleomycin moiety of the chimeric protein to bind phleomycin, thereby preventing its toxicity. The selection is very efficient: upon mutagenesis, 84 (10%) of 800 phleomycin resistant colonies tested were unable to grow on oleic acid. This rate could be increased to 25% using more stringent selection conditions. The selection procedure is very specific; all oleic acid non utilizing (onu) mutants tested were disturbed in peroxisomal import and/or peroxisome assembly. The pas (peroxisome assembly) mutants that have been used for complementation analysis represent 12 complementation groups including three novel ones, designated pas20, pas21 and pas22.

scholarly journals Three peroxisome protein packaging pathways suggested by selective permeabilization of yeast mutants defective in peroxisome biogenesis.

1993 ◽  
Vol 4 (12) ◽  
pp. 1351-1359 ◽  
Author(s):  
J W Zhang ◽  
C Luckey ◽  
P B Lazarow
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Positive Selection ◽  
Selection Procedure ◽  
Cell Fractionation ◽  
Peroxisome Biogenesis ◽  
Fractionation Procedure ◽  
Fractionation Method ◽  
Complementation Groups ◽  
Acyl Coa Oxidase ◽  
Yeast Mutants

We have identified five complementation groups of peroxisome biogenesis (peb) mutants in Saccharomyces cerevisiae by a positive selection procedure. Three of these contained morphologically recognizable peroxisomes, and two appeared to lack the organelle altogether. The packaging of peroxisomal proteins in these mutants has been analyzed with a new gentle cell fractionation procedure. It employs digitonin titration for the selective permeabilization of yeast plasma and intracellular membranes. Proteins were measured by enzymatic assay or by quantitative chemiluminescent immunoblotting. With this gentle fractionation method, it was demonstrated that two mutants are selectively defective in assembling proteins into peroxisomes. Peb1-1 packages catalase and acyl-CoA oxidase within peroxisomes but not thiolase. Peb5-1 packages thiolase and acyl-CoA oxidase within peroxisomes but not catalase. The data suggest that the peroxisome biogenesis machinery contains components that are specific for each of three classes of peroxisomal proteins, represented by catalase, thiolase, and acyl-CoA oxidase. In the two mutants lacking morphologically recognizable peroxisomes, peb2-1 and peb4-1, all three enzymes were mislocalized to the cytosol.

scholarly journals Human peroxisomal targeting signal-1 receptor restores peroxisomal protein import in cells from patients with fatal peroxisomal disorders.

1995 ◽  
Vol 130 (1) ◽  
pp. 51-65 ◽  
Author(s):  
E A Wiemer ◽  
W M Nuttley ◽  
B L Bertolaet ◽  
X Li ◽  
U Francke ◽  
...  
Keyword(s):  
Protein Import ◽  
Zellweger Syndrome ◽  
Complementation Group ◽  
Cell System ◽  
Peroxisomal Disorders ◽  
Peroxisomal Membrane ◽  
Peroxisomal Protein ◽  
Peroxisomal Targeting ◽  
Targeting Signal

Two peroxisomal targeting signals, PTS1 and PTS2, are involved in the import of proteins into the peroxisome matrix. Human patients with fatal generalized peroxisomal deficiency disorders fall into at least nine genetic complementation groups. Cells from many of these patients are deficient in the import of PTS1-containing proteins, but the causes of the protein-import defect in these patients are unknown. We have cloned and sequenced the human cDNA homologue (PTS1R) of the Pichia pastoris PAS8 gene, the PTS1 receptor (McCollum, D., E. Monosov, and S. Subramani. 1993. J. Cell Biol. 121:761-774). The PTS1R mRNA is expressed in all human tissues examined. Antibodies to the human PTS1R recognize this protein in human, monkey, rat, and hamster cells. The protein is localized mainly in the cytosol but is also found to be associated with peroxisomes. Part of the peroxisomal PTS1R protein is tightly bound to the peroxisomal membrane. Antibodies to PTS1R inhibit peroxisomal protein-import of PTS1-containing proteins in a permeabilized CHO cell system. In vitro-translated PTS1R protein specifically binds a serine-lysine-leucine-peptide. A PAS8-PTS1R fusion protein complements the P. pastoris pas8 mutant. The PTS1R cDNA also complements the PTS1 protein-import defect in skin fibroblasts from patients--belonging to complementation group two--diagnosed as having neonatal adrenoleukodystrophy or Zellweger syndrome. The PTS1R gene has been localized to a chromosomal location where no other peroxisomal disorder genes are known to map. Our findings represent the only case in which the molecular basis of the protein-import deficiency in human peroxisomal disorders is understood.

scholarly journals Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae.

1990 ◽  
Vol 10 (12) ◽  
pp. 6742-6754 ◽  
Author(s):  
P K Herman ◽  
S D Emr
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Sorting ◽  
Yeast Cells ◽  
Growth Defect ◽  
Cell Fractionation ◽  
Wild Type ◽  
Vacuolar Protein Sorting ◽  
Vacuolar Compartment ◽  
Dividing Cells ◽  
Vacuolar Protein

VPS34 gene function is required for the efficient localization of a variety of vacuolar proteins. We have cloned and sequenced the wild-type VPS34 gene in order to gain a better understanding of the role of its protein product in this intracellular sorting pathway. Interestingly, disruption of the VPS34 locus resulted in a temperature-sensitive growth defect, indicating that the VPS34 gene is essential for vegetative growth only at elevated growth temperatures. As with the original vps34 alleles, vps34 null mutants exhibited severe vacuolar protein sorting defects and possessed a morphologically normal vacuolar structure. The VPS34 gene DNA sequence identifies an open reading frame that could encode a hydrophilic protein of 875 amino acids. The predicted protein sequence lacks any apparent signal sequence or membrane-spanning domains, suggesting that Vps34p does not enter the secretory pathway. Results from immunoprecipitation experiments with antiserum prepared against a TrpE-Vps34 fusion protein were consistent with this prediction: a rare, unglycosylated protein of approximately 95,000 Da was detected in extracts of wild-type Saccharomyces cerevisiae cells. Cell fractionation studies indicated that a significant portion of the Vps34p is found associated with a particulate fraction of yeast cells. This particulate Vps34p was readily solubilized by treatment with 2 M urea but not with Triton X-100, suggesting that the presence of Vps34p in this pelletable structure is mediated by protein-protein interactions. vp34 mutant cells also exhibited a defect in the normal partitioning of the vacuolar compartment between mother and daughter cells during cell division. In more than 80% of the delta vps34 dividing cells examined, no vacuolar structures were observed in the newly emerging bud, whereas in wild-type dividing cells, more than 95% of the buds had a detectable vacuolar compartment. Our results suggest that the Vps34p may act as a component of a relatively large intracellular structure that functions to facilitate specific steps of the vacuolar protein delivery and inheritance pathways.

scholarly journals Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae

1990 ◽  
Vol 10 (12) ◽  
pp. 6742-6754
Author(s):  
P K Herman ◽  
S D Emr
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Sorting ◽  
Yeast Cells ◽  
Growth Defect ◽  
Cell Fractionation ◽  
Wild Type ◽  
Vacuolar Protein Sorting ◽  
Vacuolar Compartment ◽  
Dividing Cells ◽  
Vacuolar Protein

VPS34 gene function is required for the efficient localization of a variety of vacuolar proteins. We have cloned and sequenced the wild-type VPS34 gene in order to gain a better understanding of the role of its protein product in this intracellular sorting pathway. Interestingly, disruption of the VPS34 locus resulted in a temperature-sensitive growth defect, indicating that the VPS34 gene is essential for vegetative growth only at elevated growth temperatures. As with the original vps34 alleles, vps34 null mutants exhibited severe vacuolar protein sorting defects and possessed a morphologically normal vacuolar structure. The VPS34 gene DNA sequence identifies an open reading frame that could encode a hydrophilic protein of 875 amino acids. The predicted protein sequence lacks any apparent signal sequence or membrane-spanning domains, suggesting that Vps34p does not enter the secretory pathway. Results from immunoprecipitation experiments with antiserum prepared against a TrpE-Vps34 fusion protein were consistent with this prediction: a rare, unglycosylated protein of approximately 95,000 Da was detected in extracts of wild-type Saccharomyces cerevisiae cells. Cell fractionation studies indicated that a significant portion of the Vps34p is found associated with a particulate fraction of yeast cells. This particulate Vps34p was readily solubilized by treatment with 2 M urea but not with Triton X-100, suggesting that the presence of Vps34p in this pelletable structure is mediated by protein-protein interactions. vp34 mutant cells also exhibited a defect in the normal partitioning of the vacuolar compartment between mother and daughter cells during cell division. In more than 80% of the delta vps34 dividing cells examined, no vacuolar structures were observed in the newly emerging bud, whereas in wild-type dividing cells, more than 95% of the buds had a detectable vacuolar compartment. Our results suggest that the Vps34p may act as a component of a relatively large intracellular structure that functions to facilitate specific steps of the vacuolar protein delivery and inheritance pathways.

scholarly journals The influence of depletion of voltage dependent anion selective channel on protein import into the yeast Saccharomyces cerevisiae mitochondria.

2001 ◽  
Vol 48 (3) ◽  
pp. 719-728 ◽  
Author(s):  
A Szczechowicz ◽  
L Hryniewiecka ◽  
H Kmita
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Protein ◽  
Outer Membrane ◽  
Protein Import ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Tom Complex ◽  
Saccharomyces Cerevisiae Mitochondria ◽  
Voltage Dependent ◽  
Selective Channel

The supply of substrates to the respiratory chain as well as of other metabolites (e.g. ATP) into inner compartments of mitochondria is crucial to preprotein import into these organelles. Transport of the compounds across the outer mitochondrial membrane is enabled by mitochondrial porin, also known as the voltage-dependent anion-selective channel (VDAC). Our previous studies led to the conclusion that the transport of metabolites through the outer membrane of the yeast Saccharomyces cerevisiae mitochondria missing VDAC (now termed YVDAC1) is considerably restricted. Therefore we expected that depletion of YVDAC1 should also hamper protein import into the mutant mitochondria. We report here that YVDAC1-depleted mitochondria are able to import a fusion protein termed pSu9-DHFR in the amount comparable to that of wild type mitochondria, although over a considerably longer time. The rate of import of the fusion protein into YVDAC1-depleted mitochondria is dis- tinctly lower than into wild type mitochondria probably due to restricted ATP access to the intermembrane space and is additionally influenced by the way the supporting respiratory substrates are transported through the outer membrane. In the presence of ethanol, diffusing freely through lipid membranes, YVDAC1-depleted mitochondria are able to import the fusion protein at a higher rate than in the presence of external NADH which is, like ATP, transported through the outer membrane by facilitated diffusion. It has been shown that transport of external NADH across the outer membrane of YVDAC1-depleted mitochondria is supported by the protein import machinery, i.e. the TOM complex (Kmita & Budzińska, 2000, Biochim. Biophys. Acta 1509, 86-94.). Since the TOM complex might also contribute to the permeability of the membrane to ATP, it seems possible that external NADH and ATP as well as the imported preprotein could compete with one another for the passage through the outer membrane in YVDAC1-depleted mitochondria.

scholarly journals Novel peroxisome clustering mutants and peroxisome biogenesis mutants of Saccharomyces cerevisiae.

1993 ◽  
Vol 123 (5) ◽  
pp. 1133-1147 ◽  
Author(s):  
J W Zhang ◽  
Y Han ◽  
P B Lazarow
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Zellweger Syndrome ◽  
Selection Procedure ◽  
Peroxisome Biogenesis ◽  
Peroxisomal Membrane ◽  
Genes Encoding ◽  
Complementation Groups ◽  
Intracellular Translocation ◽  
Mutant Collection ◽  
Peroxisomal Function

The goal of this research is to identify and characterize the protein machinery that functions in the intracellular translocation and assembly of peroxisomal proteins in Saccharomyces cerevisiae. Several genes encoding proteins that are essential for this process have been identified previously by Kunau and collaborators, but the mutant collection was incomplete. We have devised a positive selection procedure that identifies new mutants lacking peroxisomes or peroxisomal function. Immunofluorescence procedures for yeast were simplified so that these mutants could be rapidly and efficiently screened for those in which peroxisome biogenesis is impaired. With these tools, we have identified four complementation groups of peroxisome biogenesis mutants, and one group that appears to express reduced amounts of peroxisomal proteins. Two of our mutants lack recognizable peroxisomes, although they might contain peroxisomal membrane ghosts like those found in Zellweger syndrome. Two are selectively defective in packaging peroxisomal proteins and moreover show striking intracellular clustering of the peroxisomes. The distribution of mutants among complementation groups implies that the collection of peroxisome biogenesis mutants is still incomplete. With the procedures described, it should prove straightforward to isolate mutants from additional complementation groups.

scholarly journals Isolation and characterization of a mutant of Saccharomyces cerevisiae with pleiotropic deficiencies in transcriptional activation and repression.

Genetics ◽  
1994 ◽  
Vol 137 (1) ◽  
pp. 55-65 ◽  
Author(s):  
E Lamping ◽  
J Lückl ◽  
F Paltauf ◽  
S A Henry ◽  
S D Kohlwein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Phase ◽  
Transcriptional Activation ◽  
Stationary Growth Phase ◽  
Complementation Group ◽  
Regulatory Function ◽  
Mrna Levels ◽  
Structural Genes ◽  
Wild Type ◽  
Isolation And Characterization

Abstract The isolation of the dep1 mutant of Saccharomyces cerevisiae is reported. The mutant was identified by its disability to regulate expression of structural genes involved in phospholipid biosynthesis, INO1, CHO1 and OPI3, in response to supplementation with soluble lipid precursors. Expression of the INO1, CHO1 and OPI3 genes was not fully derepressed in the absence of soluble lipid precursors, inositol and choline in the dep1 mutant, as compared to wild type. The mutant also exhibited incomplete repression of these same genes in the presence of inositol and choline. Repression by phosphate of the PHO5 gene was reduced in the mutant, as was derepression of this gene in the absence of phosphate. In addition, we show that expression of INO1 and OPI3 structural genes is strongly dependent on the growth phase both in wild-type and dep1 mutant strains. However, in the mutant, elevated basal steady-state mRNA levels were reached in the late stationary growth phase, independent of supplementation conditions. The dep1 mutation represents a new complementation group with respect to phospholipid synthesis and was mapped to a position of about 12 cM distal from the centromere on the left arm of chromosome I. Deficiencies in transcription activation and repression of metabolically unrelated genes, as well as reduced mating efficiency and lack of sporulation of homozygous diploid dep1/dep1 mutants indicate a pleiotropic regulatory function of the DEP1 gene product. Thus, Dep1p appears to be a new member of a class of transcriptional modulators, including Rpd1p/Sin3p/Ume4p/Sdi1p/Gam3p, Rpd3p, Spt10p and Spt21p.

scholarly journals Effects of Mutations of RAD50, RAD51, RAD52, and Related Genes on Illegitimate Recombination in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 383-391 ◽  
Author(s):  
Yasumasa Tsukamoto ◽  
Jun-ichi Kato ◽  
Hideo Ikeda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quantitative Analysis ◽  
Structural Analysis ◽  
Recombination Rate ◽  
Type Strain ◽  
Negative Selection ◽  
Wild Type Strain ◽  
Illegitimate Recombination ◽  
Wild Type ◽  
In The Wild

Abstract To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rud51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.

scholarly journals The GNAQ T96S Mutation Affects Cell Signaling and Enhances the Oncogenic Properties of Hepatocellular Carcinoma

2021 ◽  
Vol 22 (6) ◽  
pp. 3284
Author(s):  
Eugene Choi ◽  
Sung Jean Park ◽  
Gunhee Lee ◽  
Seung Kew Yoon ◽  
Minho Lee ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Expression Vector ◽  
Signal Transduction Pathway ◽  
Inhibitory Effect ◽  
The Cancer Genome Atlas ◽  
Treatment Modalities ◽  
Protein Signaling ◽  
Wild Type ◽  
Anchorage Independent Growth ◽  
Mapk Pathways

Hepatocellular carcinoma (HCC), the most common malignant tumor in the liver, grows and metastasizes rapidly. Despite advances in treatment modalities, the five-year survival rate of HCC remains less than 30%. We sought genetic mutations that may affect the oncogenic properties of HCC, using The Cancer Genome Atlas (TCGA) data analysis. We found that the GNAQ T96S mutation (threonine 96 to serine alteration of the Gαq protein) was present in 12 out of 373 HCC patients (3.2%). To examine the effect of the GNAQ T96S mutation on HCC, we transfected the SK-Hep-1 cell line with the wild-type or the mutant GNAQ T96S expression vector. Transfection with the wild-type GNAQ expression vector enhanced anchorage-independent growth, migration, and the MAPK pathways in the SK-Hep-1 cells compared to control vector transfection. Moreover, cell proliferation, anchorage-independent growth, migration, and the MAPK pathways were further enhanced in the SK-Hep-1 cells transfected with the GNAQ T96S expression vector compared to the wild-type GNAQ-transfected cells. In silico structural analysis shows that the substitution of the GNAQ amino acid threonine 96 with a serine may destabilize the interaction between the regulator of G protein signaling (RGS) protein and GNAQ. This may reduce the inhibitory effect of RGS on GNAQ signaling, enhancing the GNAQ signaling pathway. Single nucleotide polymorphism (SNP) genotyping analysis for Korean HCC patients shows that the GNAQ T96S mutation was found in only one of the 456 patients (0.22%). Our data suggest that the GNAQ T96S hotspot mutation may play an oncogenic role in HCC by potentiating the GNAQ signal transduction pathway.

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