ISOLATION AND CHARACTERIZATION OF PAS2P, A PEROXISOMAL MEMBRANE-PROTEIN ESSENTIAL FOR PEROXISOME BIOGENESIS IN THE METHYLOTROPHIC YEAST PICHIA-PASTORIS

The pas2 mutant of the methylotrophic yeast Pichia pastoris is charact erized by a deficiency in peroxisome biogenesis. We have cloned the Pp PAS2 gene by functional complementation and show that it encodes a pro tein of 455 amino acids with a molecular mass of 52 kDa. In a Pppas2 n ull mutant, import of both peroxisomal targeting signal 1 (PTS1)- and PTS2-containing proteins is impaired as shown by biochemical fractiona tion and fluorescence microscopy. No morphologically distinguishable p eroxisomal structures could be detected by electron microscopy in Pppa s2 null cells induced on methanol and oleate, suggesting that PpPas2p is involved in the early stages of peroxisome biogenesis. PpPas2p is a peroxisomal membrane protein (PMP) and is resistant to extraction by 1 M NaCl or alkaline sodium carbonate, suggesting that it is a peroxis omal integral membrane protein. Two hydrophobic domains can be disting uished which may be involved in anchoring PpPas2p to the peroxisomal m embrane. PpPas2p is homologous to the Saccharomyces cerevisiae Pas3p. The first 40 amino acids of PpPas2p, devoid of the hydrophobic domains , are sufficient to target a soluble fluorescent reporter protein to t he peroxisomal membrane, with which it associates tightly, A compariso n with the membrane peroxisomal targeting signal of PMP47 of Candida b oidinii revealed a stretch of positively charged amino acids common to both sequences. The role of peroxisomal membrane targeting signals an d transmembrane domains in anchoring PMPs to the peroxisomal membrane is discussed.