Three peroxisome protein packaging pathways suggested by selective permeabilization of yeast mutants defective in peroxisome biogenesis.
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.
Zhang
J W
JW
Department of Cell Biology and Anatomy, Mount Sinai School of Medicine, New York, New York 10029.
Luckey
C
C
Lazarow
P B
PB
D000101:Acetyl-CoA C-Acetyltransferase / Q000378:metabolism*; D042962:Acyl-CoA Oxidase; D000595:Amino Acid Sequence; D015153:Blotting, Western / Q000379:methods; D002374:Catalase / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects*; D003600:Cytosol / Q000378:metabolism; D004072:Digitonin / Q000494:pharmacology*; D005656:Fungal Proteins / Q000378:metabolism*; D007425:Intracellular Membranes / Q000187:drug effects*; D008163:Luminescent Measurements; D008830:Microbodies / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins; D012641:Selection, Genetic
D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D010088:Oxidoreductases; D002374:Catalase; D042962:Acyl-CoA Oxidase; C467991:POX1 protein, S cerevisiae; D000101:Acetyl-CoA C-Acetyltransferase; D004072:Digitonin