Abstract
PAY genes are required for peroxisome assembly in the yeast Yarrowia lipolytica. Here we show that a mutant strain, pay2, is disrupted for the import of proteins targeted by either peroxisomal targeting signal-1 or -2. Electron microscopy of pay2 cells revealed the presence of small peroxisomal "ghosts," similar to the vesicular structures found in fibroblasts of patients with the human peroxisome assembly disorder, Zellweger syndrome. Functional complementation of pay2 with a plasmid library of Y. lipolytica genomic DNA identified a gene, PAY2, that restores growth of pay2 on oleic acid, import of catalase and multifunctional enzyme into peroxisomes, and formation of wild type peroxisomes. The PAY2 gene encodes Pay2p, a hydrophobic polypeptide of 404 amino acids. An antibody raised against Pay2p recognizes a polypeptide of approximately 42-kDa whose synthesis is induced by growth of Y. lipolytica on oleic acid. Pay2p is a peroxisomal integral membrane protein, as it localizes to carbonate-stripped peroxisomal membranes. Pay2p shows no identity to any known protein. Our results suggest that Pay2p is essential for the activity of the peroxisomal import machinery but does not affect the initial steps of peroxisomal membrane proliferation.
Highlights
Peroxisomes are members of the microbody family of organelles, along with the glyoxysomes of plants and the glycosomes of trypanosomes
It has been proposed that Zellweger syndrome arises from a failure to translocate proteins into peroxisomes (Walton et al, 1992; Wendland and Subramani, 1993), causing these proteins to be mislocalized to the cytoplasm where they cannot function correctly and are often rapidly degraded
Peroxisomal targeting signal-1 (PTS-l)l is a tripeptide motif found at the carboxyl termini of many peroxisomal matrix proteins in organisms from yeast to humans (Gould et al, 1989, 1990; Aitchison et al, 1991)
Summary
Vol 270, No., Issue of January 20, pp. 1429-1436, 1995 Printed in U.S.A. The Yarrowia lipolytica Gene PAY2 Encodes a 42-kDa Peroxisomal Integral Membrane Protein Essential for Matrix Protein Import and Peroxisome Enlargement but Not for Peroxisome Membrane Proliferation*. Peroxisomal targeting signal-1 (PTS-l)l is a tripeptide motif found at the carboxyl termini of many peroxisomal matrix proteins in organisms from yeast to humans (Gould et al, 1989, 1990; Aitchison et al, 1991). Functi onal comp lem entation of some of these mutants hav e identified genes encoding a varied set of protein s involved in peroxisom e biogen esis including putative ATP ases (E rdma nn et al , 1991 ; Spong a nd Subramani, 1993; Nuttley et al, 1994 ), tetratricopeptide-r epeat protein s that may be part of the import machinery for PTS-l-targeted protein s (McCollum et al, 1993; va n der Leij et al , 1993 ), a pero xisomal integral membrane protein (Hohfeld et al, 1991 ), and proteins rela ted to ubiquitin-conju gating enzymes (Wiebel and Kunau, 1992 ; Crane et al, 1994 ). Pa y2p is a peroxisomal integr al membrane protein that is unrelated to a ny kn own pr otein
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