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 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.

Metabolic control of peroxisome abundance

1999 ◽  
Vol 112 (10) ◽  
pp. 1579-1590 ◽  
Author(s):  
C.C. Chang ◽  
S. South ◽  
D. Warren ◽  
J. Jones ◽  
A.B. Moser ◽  
...  
Keyword(s):  
Metabolic Control ◽  
Matrix Protein ◽  
Protein Import ◽  
Zellweger Syndrome ◽  
Mutant Gene ◽  
Peroxisomal Membrane ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Complementation Groups ◽  
Peroxisomal Targeting Signal

Zellweger syndrome and related disorders represent a group of lethal, genetically heterogeneous diseases. These peroxisome biogenesis disorders (PBDs) are characterized by defective peroxisomal matrix protein import and comprise at least 10 complementation groups. The genes defective in seven of these groups and more than 90% of PBD patients are now known. Here we examine the distribution of peroxisomal membrane proteins in fibroblasts from PBD patients representing the seven complementation groups for which the mutant gene is known. Peroxisomes were detected in all PBD cells, indicating that the ability to form a minimal peroxisomal structure is not blocked in these mutants. We also observed that peroxisome abundance was reduced fivefold in PBD cells that are defective in the PEX1, PEX5, PEX12, PEX6, PEX10, and PEX2 genes. These cell lines all display a defect in the import of proteins with the type-1 peroxisomal targeting signal (PTS1). In contrast, peroxisome abundance was unaffected in cells that are mutated in PEX7 and are defective only in the import of proteins with the type-2 peroxisomal targeting signal. Interestingly, a fivefold reduction in peroxisome abundance was also observed for cells lacking either of two PTS1-targeted peroxisomal beta-oxidation enzymes, acyl-CoA oxidase and 2-enoyl-CoA hydratase/D-3-hydroxyacyl-CoA dehydrogenase. These results indicate that reduced peroxisome abundance in PBD cells may be caused by their inability to import these PTS1-containing enzymes. Furthermore, the fact that peroxisome abundance is influenced by peroxisomal 105-oxidation activities suggests that there may be metabolic control of peroxisome abundance.

scholarly journals Diphtheria toxin-resistant mutants of Saccharomyces cerevisiae.

1985 ◽  
Vol 5 (12) ◽  
pp. 3357-3360 ◽  
Author(s):  
J Y Chen ◽  
J W Bodley ◽  
D M Livingston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Diphtheria Toxin ◽  
Elongation Factor ◽  
Selection Procedure ◽  
Prolonged Storage ◽  
Elongation Factor 2 ◽  
Resistant Phenotype ◽  
Complementation Groups ◽  
Mendelian Character

We developed a selection procedure based on the observation that diphtheria toxin kills spheroplasts of Saccharomyces cerevisiae (Murakami et al., Mol. Cell. Biol. 2:588-592, 1982); this procedure yielded mutants resistant to the in vitro action of the toxin. Spheroplasts of mutagenized S. cerevisiae were transformed in the presence of diphtheria toxin, and the transformed survivors were screened in vitro for toxin-resistant elongation factor 2. Thirty-one haploid ADP ribosylation-negative mutants comprising five complementation groups were obtained by this procedure. The mutants grew normally and were stable to prolonged storage. Heterozygous diploids produced by mating wild-type sensitive cells with the mutants revealed that in each case the resistant phenotype was recessive to the sensitive phenotype. Sporulation of these diploids yielded tetrads in which the resistant phenotype segregated as a single Mendelian character. From these observations, we concluded that these mutants are defective in the enzymatic steps responsible for the posttranslational modification of elongation factor 2 which is necessary for recognition by diphtheria toxin.

scholarly journals Inhibitors of Copi and Copii Do Not Block PEX3-Mediated Peroxisome Synthesis

2000 ◽  
Vol 149 (7) ◽  
pp. 1345-1360 ◽  
Author(s):  
Sarah T. South ◽  
Katherine A. Sacksteder ◽  
Xiaoling Li ◽  
Yifei Liu ◽  
Stephen J. Gould
Keyword(s):  
Membrane Proteins ◽  
Zellweger Syndrome ◽  
Brefeldin A ◽  
Membrane Traffic ◽  
Dominant Negative ◽  
Peroxisome Biogenesis ◽  
Membrane Synthesis ◽  
Peroxisomal Membrane ◽  
Peroxisome Membrane ◽  
Inactivating Mutations

In humans, defects in peroxisome biogenesis are the cause of lethal diseases typified by Zellweger syndrome. Here, we show that inactivating mutations in human PEX3 cause Zellweger syndrome, abrogate peroxisome membrane synthesis, and result in reduced abundance of peroxisomal membrane proteins (PMPs) and/or mislocalization of PMPs to the mitochondria. Previous studies have suggested that PEX3 may traffic through the ER en route to the peroxisome, that the COPI inhibitor, brefeldin A, leads to accumulation of PEX3 in the ER, and that PEX3 overexpression alters the morphology of the ER. However, we were unable to detect PEX3 in the ER at early times after expression. Furthermore, we find that inhibition of COPI function by brefeldin A has no effect on trafficking of PEX3 to peroxisomes and does not inhibit PEX3-mediated peroxisome biogenesis. We also find that inhibition of COPII-dependent membrane traffic by a dominant negative SAR1 mutant fails to block PEX3 transport to peroxisomes and PEX3-mediated peroxisome synthesis. Based on these results, we propose that PEX3 targeting to peroxisomes and PEX3-mediated peroxisome membrane synthesis may occur independently of COPI- and COPII-dependent membrane traffic.

Diphtheria toxin-resistant mutants of Saccharomyces cerevisiae

1985 ◽  
Vol 5 (12) ◽  
pp. 3357-3360
Author(s):  
J Y Chen ◽  
J W Bodley ◽  
D M Livingston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Diphtheria Toxin ◽  
Elongation Factor ◽  
Selection Procedure ◽  
Prolonged Storage ◽  
Elongation Factor 2 ◽  
Resistant Phenotype ◽  
Complementation Groups ◽  
Mendelian Character

We developed a selection procedure based on the observation that diphtheria toxin kills spheroplasts of Saccharomyces cerevisiae (Murakami et al., Mol. Cell. Biol. 2:588-592, 1982); this procedure yielded mutants resistant to the in vitro action of the toxin. Spheroplasts of mutagenized S. cerevisiae were transformed in the presence of diphtheria toxin, and the transformed survivors were screened in vitro for toxin-resistant elongation factor 2. Thirty-one haploid ADP ribosylation-negative mutants comprising five complementation groups were obtained by this procedure. The mutants grew normally and were stable to prolonged storage. Heterozygous diploids produced by mating wild-type sensitive cells with the mutants revealed that in each case the resistant phenotype was recessive to the sensitive phenotype. Sporulation of these diploids yielded tetrads in which the resistant phenotype segregated as a single Mendelian character. From these observations, we concluded that these mutants are defective in the enzymatic steps responsible for the posttranslational modification of elongation factor 2 which is necessary for recognition by diphtheria toxin.

scholarly journals Pex13 Inactivation in the Mouse Disrupts Peroxisome Biogenesis and Leads to a Zellweger Syndrome Phenotype

2003 ◽  
Vol 23 (16) ◽  
pp. 5947-5957 ◽  
Author(s):  
Megan Maxwell ◽  
Jonas Bjorkman ◽  
Tam Nguyen ◽  
Peter Sharp ◽  
John Finnie ◽  
...  
Keyword(s):  
Protein Import ◽  
Zellweger Syndrome ◽  
Cre Recombinase ◽  
Acid Oxidation ◽  
Metabolic Dysfunction ◽  
Peroxisome Biogenesis ◽  
Peroxisomal Membrane ◽  
Severe Impairment ◽  
Biochemical Analyses ◽  
Neuronal Migration Defect

ABSTRACT Zellweger syndrome is the archetypical peroxisome biogenesis disorder and is characterized by defective import of proteins into the peroxisome, leading to peroxisomal metabolic dysfunction and widespread tissue pathology. In humans, mutations in the PEX13 gene, which encodes a peroxisomal membrane protein necessary for peroxisomal protein import, can lead to a Zellweger phenotype. To develop mouse models for this disorder, we have generated a targeted mouse with a loxP-modified Pex13 gene to enable conditional Cre recombinase-mediated inactivation of Pex13. In the studies reported here, we crossed these mice with transgenic mice that express Cre recombinase in all cells to generate progeny with ubiquitous disruption of Pex13. The mutant pups exhibited many of the clinical features of Zellweger syndrome patients, including intrauterine growth retardation, severe hypotonia, failure to feed, and neonatal death. These animals lacked morphologically intact peroxisomes and showed deficient import of matrix proteins containing either type 1 or type 2 targeting signals. Biochemical analyses of tissue and cultured skin fibroblasts from these animals indicated severe impairment of peroxisomal fatty acid oxidation and plasmalogen synthesis. The brains of these animals showed disordered lamination in the cerebral cortex, consistent with a neuronal migration defect. Thus, Pex13−/− mice reproduce many of the features of Zellweger syndrome and PEX13 deficiency in humans.

scholarly journals PAS3, a Saccharomyces cerevisiae gene encoding a peroxisomal integral membrane protein essential for peroxisome biogenesis.

1991 ◽  
Vol 114 (6) ◽  
pp. 1167-1178 ◽  
Author(s):  
J Höhfeld ◽  
M Veenhuis ◽  
W H Kunau
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Protein ◽  
Integral Membrane Protein ◽  
Amino Terminus ◽  
Peroxisome Biogenesis ◽  
Coding Region ◽  
Gene Encoding ◽  
Peroxisomal Membrane ◽  
E Coli ◽  
Protease Digestion

Saccharomyces cerevisiae pas3-mutants are described which conform the pas-phenotype recently reported for the peroxisomal assembly mutants pas1-1 and pas2 (Erdmann, R., M. Veenhuis, D. Mertens, and W.-H Kunau, 1989, Proc. Natl. Acad. Sci. USA. 86:5419-5423). The isolation of pas3-mutants enabled us to clone the PAS3 gene by functional complementation. DNA sequence analysis revealed a 50.6-kD protein with at least one domain of sufficient length and hydrophobicity to span a lipid bilayer. To verify these predictions antibodies were raised against a truncated portion of the PAS3 coding region overexpressed in E. coli. Pas3p was identified as a 48 kD peroxisomal integral membrane protein. It is shown that a lack of this protein causes the peroxisome-deficient phenotype and the cytosolic mislocalization of peroxisomal matrix enzymes. Based on protease digestion experiments Pas3p is discussed to be anchored in the peroxisomal membrane by its amino-terminus while the bulk of the molecule is exposed to the cytosol. These findings are consistent with the possibility that Pas3p is one component of the peroxisomal import machinery.

scholarly journals Phosphorylation-dependent Pex11p and Fis1p interaction regulates peroxisome division

2012 ◽  
Vol 23 (7) ◽  
pp. 1307-1315 ◽  
Author(s):  
Saurabh Joshi ◽  
Gaurav Agrawal ◽  
Suresh Subramani
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Pichia Pastoris ◽  
Peroxisome Biogenesis ◽  
Peroxisomal Membrane ◽  
Protein Levels ◽  
Protein Biogenesis ◽  
Peroxisome Division ◽  
Dependent Interaction

Peroxisome division is regulated by the conserved peroxin Pex11p. In Saccharomyces cerevisiae (Sc), induction of the phosphoprotein ScPex11p coincides with peroxisome biogenesis. We show that the ScPex11p homologue in Pichia pastoris (PpPex11p) is phosphorylated at serine 173. PpPex11p expression and phosphorylation are induced in oleate and coordinated with peroxisome biogenesis. PpPex11p transits to peroxisomes via the endoplasmic reticulum (ER). PpPex11p is unstable and ER restricted gin pex3Δ and pex19Δ cells, which are impaired in peroxisomal membrane protein biogenesis. In oleate medium, the P. pastoris mutants pex11A (constitutively unphosphorylated; S173A) and pex11D (constitutively phosphorylated; S173D) exhibit juxtaposed elongated peroxisomes (JEPs) and hyperdivided forms, respectively, although protein levels remain unchanged. In contrast with ScPex11p, the ER-to-peroxisome translocation in P. pastoris is phosphorylation independent, and the phosphorylation occurs at the peroxisome. We show that PpPex11p interacts with the peroxisome fission machinery via PpFis1p and is regulated by phosphorylation because PpPex11p and PpPex11Dp interact more strongly with PpFis1p than PpPex11Ap. Neither PpPex11p nor PpFis1p is necessary for peroxisome division in methanol medium. We propose a model for the role of PpPex11p in the regulation of peroxisome division through a phosphorylation-dependent interaction with the fission machinery, providing novel insights into peroxisome morphogenesis.

scholarly journals A dual function for Pex3p in peroxisome formation and inheritance

2009 ◽  
Vol 187 (4) ◽  
pp. 463-471 ◽  
Author(s):  
Joanne M. Munck ◽  
Alison M. Motley ◽  
James M. Nuttall ◽  
Ewald H. Hettema
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Steady State ◽  
De Novo ◽  
Retention Factor ◽  
Dual Function ◽  
Peroxisome Biogenesis ◽  
Peroxisomal Membrane

Saccharomyces cerevisiae Pex3p has been shown to act at the ER during de novo peroxisome formation. However, its steady state is at the peroxisomal membrane, where its role is debated. Here we show that Pex3p has a dual function: one in peroxisome formation and one in peroxisome segregation. We show that the peroxisome retention factor Inp1p interacts physically with Pex3p in vitro and in vivo, and split-GFP analysis shows that the site of interaction is the peroxisomal membrane. Furthermore, we have generated PEX3 alleles that support peroxisome formation but fail to support recruitment of Inp1p to peroxisomes, and as a consequence are affected in peroxisome segregation. We conclude that Pex3p functions as an anchor for Inp1p at the peroxisomal membrane, and that this function is independent of its role at the ER in peroxisome biogenesis.

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