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 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 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 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 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.

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 The newly identified yeast GRD genes are required for retention of late-Golgi membrane proteins.

1996 ◽  
Vol 16 (6) ◽  
pp. 2700-2707 ◽  
Author(s):  
S F Nothwehr ◽  
N J Bryant ◽  
T H Stevens
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Alkaline Phosphatase ◽  
Membrane Proteins ◽  
Golgi Membrane ◽  
Golgi Retention ◽  
Complementation Groups ◽  
Aminopeptidase A ◽  
Dipeptidyl Aminopeptidase ◽  
Yeast Mutants ◽  
Kinetics Of

Processing of A-ALP, a late-Golgi membrane protein constructed by fusing the cytosolic domain of dipeptidyl aminopeptidase A to the transmembrane and lumenal domains of alkaline phosphatase (ALP), serves as a convenient assay for loss of retention of late-Golgi membrane proteins in Saccharomyces cerevisiae. In this study, a large group of novel grd (for Golgi retention defective) yeast mutants, representing 18 complementation groups, were identified on the basis of their mislocalization of A-ALP to the vacuole, where it was proteolytically processed and thus became enzymatically activated. All of the grd mutants exhibited significant mislocalization of A-ALP, as measured by determining the kinetics of A-ALP processing and by analyzing its

scholarly journals Peroxisomes in Saccharomyces cerevisiae: immunofluorescence analysis and import of catalase A into isolated peroxisomes.

1991 ◽  
Vol 11 (1) ◽  
pp. 510-522 ◽  
Author(s):  
R Thieringer ◽  
H Shio ◽  
Y S Han ◽  
G Cohen ◽  
P B Lazarow
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Oleic Acid ◽  
Glucose Repression ◽  
Equilibrium Density ◽  
Fluorescence Signal ◽  
Cell Periphery ◽  
Cell Fractionation ◽  
Peroxisome Biogenesis ◽  
Immunofluorescence Analysis

To isolate peroxisomes from Saccharomyces cerevisiae of a quality sufficient for in vitro import studies, we optimized the conditions for cell growth and for cell fractionation. Stability of the isolated peroxisomes was monitored by catalase latency and sedimentability of marker enzymes. It was improved by (i) using cells that were shifted to oleic acid medium after growth to stationary phase in glucose precultures, (ii) shifting the pH from 7.2 to 6.0 during cell fractionation, and (iii) carrying out equilibrium density centrifugation with Nycodenz containing 0.25 M sucrose throughout the gradient. A concentrated peroxisomal fraction was used for in vitro import of catalase A. After 2 h of incubation, 62% of the catalase was associated with, and 16% was imported into, the organelle in a protease-resistant fashion. We introduced immunofluorescence microscopy for S. cerevisiae peroxisomes, using antibodies against thiolase, which allowed us to identify even the extremely small organelles in glucose-grown cells. Peroxisomes from media containing oleic acid were larger in size, were greater in number, and had a more intense fluorescence signal. The peroxisomes were located, sometimes in clusters, in the cell periphery, often immediately adjacent to the plasma membrane. Systematic immunofluorescence observations of glucose-grown S. cerevisiae demonstrated that all such cells contained at least one and usually several very small peroxisomes despite the glucose repression. This finding fits a central prediction of our model of peroxisome biogenesis: peroxisomes form by division of preexisting peroxisomes; therefore, every cell must have at least one peroxisome if additional organelles are to be induced in that cell.

scholarly journals Multiple classes of yeast mutants are defective in vacuole partitioning yet target vacuole proteins correctly.

1996 ◽  
Vol 7 (9) ◽  
pp. 1375-1389 ◽  
Author(s):  
Y X Wang ◽  
H Zhao ◽  
T M Harding ◽  
D S Gomes de Mesquita ◽  
C L Woldringh ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Basis ◽  
Protein Delivery ◽  
Protein Transport ◽  
Daughter Cells ◽  
A Cell ◽  
Complementation Groups ◽  
Vacuole Inheritance ◽  
Mother Cells ◽  
Yeast Mutants

In Saccharomyces cerevisiae the vacuoles are partitioned from mother cells to daughter cells in a cell-cycle-coordinated process. The molecular basis of this event remains obscure. To date, few yeast mutants had been identified that are defective in vacuole partitioning (vac), and most such mutants are also defective in vacuole protein sorting (vps) from the Golgi to the vacuole. Both the vps mutants and previously identified non-vps vac mutants display an altered vacuolar morphology. Here, we report a new method to monitor vacuole inheritance and the isolation of six new non-vps vac mutants. They define five complementation groups (VAC8-VAC12). Unlike mutants identified previously, three of the complementation groups exhibit normal vacuolar morphology. Zygote studies revealed that these vac mutants are also defective in intervacuole communication. Although at least four pathways of protein delivery to the vacuole are known, only the Vps pathway seems to significantly overlap with vacuole partitioning. Mutants defective in both vacuole partitioning and endocytosis or vacuole partitioning and autophagy were not observed. However, one of the new vac mutants was additionally defective in direct protein transport from the cytoplasm to the vacuole.

scholarly journals Yeast Mutants Deficient in ER-Associated Degradation of the Z Variant of Alpha-1-Protease Inhibitor

Genetics ◽  
1996 ◽  
Vol 144 (4) ◽  
pp. 1355-1362 ◽  
Author(s):  
Ardythe A McCracken ◽  
Igor V Karpichev ◽  
James E Ernaga ◽  
Eric D Werner ◽  
Andrew G Dillin ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Degradation ◽  
Proteinase Inhibitor ◽  
Parent Strain ◽  
Mendelian Inheritance ◽  
Wild Type ◽  
Mutant Strains ◽  
Complementation Groups ◽  
Yeast Mutants ◽  
Wild Type Yeast

Saccharomyces cerevisiae mutants deficient in degradation of alpha-1-proteinase inhibitor Z (A1PiZ) have been isolated and genetically characterized. Wild-type yeast expressing A1PiZ synthesize an ER form of this protein that is rapidly degraded by an intracellular proteolytic process known as ER-associated protein degradation (ERAD). The mutant strains were identified after treatment with EMS using a colony blot immunoassay to detect colonies that accumulated high levels of A1PiZ. A total of 120,000 colonies were screened and 30 putative mutants were identified. The level of A1PiZ accumulation in these mutants, measured by ELISA, ranged from two to 11 times that of A1PiZ in the parent strain. Further studies demonstrated that the increased levels of A1PiZ in most of the mutant strains was not the result of defective secretion or elevated A1PiZ mRNA. Pulse chase experiments indicated that A1PiZ was stabilized in several strains, evidence that these mutants are defective in ER-associated protein degradation. Genetic analyses revealed that most of the mutations were recessive, ∼30% of the mutants characterized conformed to simple Mendelian inheritance, and at least seven complementation groups were identified.

Peroxisomes in Saccharomyces cerevisiae: immunofluorescence analysis and import of catalase A into isolated peroxisomes

1991 ◽  
Vol 11 (1) ◽  
pp. 510-522
Author(s):  
R Thieringer ◽  
H Shio ◽  
Y S Han ◽  
G Cohen ◽  
P B Lazarow
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Oleic Acid ◽  
Glucose Repression ◽  
Equilibrium Density ◽  
Fluorescence Signal ◽  
Cell Periphery ◽  
Cell Fractionation ◽  
Peroxisome Biogenesis ◽  
Immunofluorescence Analysis

To isolate peroxisomes from Saccharomyces cerevisiae of a quality sufficient for in vitro import studies, we optimized the conditions for cell growth and for cell fractionation. Stability of the isolated peroxisomes was monitored by catalase latency and sedimentability of marker enzymes. It was improved by (i) using cells that were shifted to oleic acid medium after growth to stationary phase in glucose precultures, (ii) shifting the pH from 7.2 to 6.0 during cell fractionation, and (iii) carrying out equilibrium density centrifugation with Nycodenz containing 0.25 M sucrose throughout the gradient. A concentrated peroxisomal fraction was used for in vitro import of catalase A. After 2 h of incubation, 62% of the catalase was associated with, and 16% was imported into, the organelle in a protease-resistant fashion. We introduced immunofluorescence microscopy for S. cerevisiae peroxisomes, using antibodies against thiolase, which allowed us to identify even the extremely small organelles in glucose-grown cells. Peroxisomes from media containing oleic acid were larger in size, were greater in number, and had a more intense fluorescence signal. The peroxisomes were located, sometimes in clusters, in the cell periphery, often immediately adjacent to the plasma membrane. Systematic immunofluorescence observations of glucose-grown S. cerevisiae demonstrated that all such cells contained at least one and usually several very small peroxisomes despite the glucose repression. This finding fits a central prediction of our model of peroxisome biogenesis: peroxisomes form by division of preexisting peroxisomes; therefore, every cell must have at least one peroxisome if additional organelles are to be induced in that cell.

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