Rachel K. Szilard

The Yarrowia lipolytica gene PAY5 encodes a peroxisomal integral membrane protein homologous to the mammalian peroxisome assembly factor PAF-1

Pay mutants of the yeast Yarrowia lipolytica fail to assemble functional peroxisomes. One mutant strain, pay5-1, lacks normal peroxisomes and instead contains irregular vesicular structures surrounded by multiple unit membranes. The pay5-1 mutant is not totally deficient in peroxisomal matrix protein targeting, as a subset of matrix proteins continues to localize to a subcellular fraction enriched for peroxisomes. The functionally complementing gene PAY5 encodes a protein, Pay5p, of 380 amino acids (41,720 Da). Pay5p is a peroxisomal integral membrane protein homologous to mammalian PAF-1 proteins, which are essential for peroxisome assembly and whose mutation in humans results in Zellweger syndrome. Pay5p is targeted to mammalian peroxisomes, demonstrating the evolutionary conservation of the targeting mechanism for peroxisomal membrane proteins. Our results suggest that in pay5 mutants, normal peroxisome assembly is blocked, which leads to the accumulation of the membranous vesicular structures observed.

The peroxin Pex17p of the yeast Yarrowia lipolytica is associated peripherally with the peroxisomal membrane and is required for the import of a subset of matrix proteins.

PEX genes encode peroxins, which are required for the biogenesis of peroxisomes. The Yarrowia lipolytica PEX17 gene encodes the peroxin Pex17p, which is 671 amino acids in length and has a predicted molecular mass of 75,588 Da. Pex17p is peripherally associated with the peroxisomal membrane. The carboxyl-terminal tripeptide, Gly-Thr-Leu, of Pex17p is not necessary for its targeting to peroxisomes. Synthesis of Pex17p is low in cells grown in glucose-containing medium and increases after the cells are shifted to oleic acid-containing medium. Cells of the pex17-1 mutant, the original mutant strain, and the pex17-KA mutant, a strain in which most of the PEX17 gene is deleted, fail to form normal peroxisomes but instead contain numerous large, multimembraned structures. The import of peroxisomal matrix proteins in these mutants is selectively impaired. This selective import is not a function of the nature of the peroxisomal targeting signal. We suggest a regulatory role for Pex17p in the import of a subset of matrix proteins into peroxisomes.

The PAH2 gene is required for peroxisome assembly in the methylotrophic yeast Hansenula polymorpha and encodes a member of the tetratricopeptide repeat family of proteins

Peroxisome assembly mutants in the methylotrophic yeast, Hansenula polymorpha, were selected by a novel procedure involving the inability of mutants to use both oleic acid and methanol as carbon sources. These compounds are both metabolized within peroxisomes through two different enzymatic pathways. 15 mutant strains called mut (methanol non-utilizing) were isolated. These strains were assigned to ten genetic complementation groups. Subcellular fractionation analysis showed that peroxisomal matrix enzymes were mislocalized to the cytoplasm in mut strains. Electron microscopy confirmed that the inability of mut strains to grow on oleic acid and methanol was due to defects in peroxisome assembly. Functional complementation of a mutant strain, mut2, with a plasmid library of H. polymorpha genomic DNA sequences has identified a gene, PAH2, that restores growth on methanol and the correct localization of matrix enzymes to the peroxisome. PAH2 encodes Pah2p, a polypeptide of 569 amino acids that is a member of the tetratricopeptide repeat (TPR) family of proteins. Pah2p shows identity with Pas8p and Pas10p, two proteins required for peroxisome assembly in the yeasts Pichia pastoris and Saccharomyces cerevisiae, respectively, and which have been suggested to be receptors that recognize peroxisomal targeting signal-1 (PTS1) motifs.

Peroxisome biogenesis in yeast

Eukaryotic cells have evolved a complex set of intracellular organelles, each of which possesses a specific complement of enzymes and performs unique metabolic functions. This compartmentalization of cellular functions provides a level of metabolic control not available to prokaryotes. However, it presents the eukaryotic cell with the problem of targeting proteins to their specific location (s). Proteins must be efficiently transported from their site of synthesis in the cytosol to their specific organelle (s). Such a process may require translocation across one or more hydrophobic membrane barriers and/or asymmetric integration into specific membranes.