Isolation and Characterization of Pas2p, a Peroxisomal Membrane Protein Essential for Peroxisome Biogenesis in the Methylotrophic Yeast Pichia pastoris

Georg H Lüers,Marten Veenhuis,Suresh Subramani,Klaas Nico Faber,Erik A.C Wiemer,Thibaut Wenzel

doi:10.1074/jbc.271.31.18973

Georg H Lüers, Marten Veenhuis + Show 4 more

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https://doi.org/10.1074/jbc.271.31.18973

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Abstract

The pas2 mutant of the methylotrophic yeast Pichia pastoris is characterized by a deficiency in peroxisome biogenesis. We have cloned the PpPAS2 gene by functional complementation and show that it encodes a protein of 455 amino acids with a molecular mass of 52 kDa. In a Pppas2 null mutant, import of both peroxisomal targeting signal 1 (PTS1)- and PTS2-containing proteins is impaired as shown by biochemical fractionation and fluorescence microscopy. No morphologically distinguishable peroxisomal structures could be detected by electron microscopy in Pppas2 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 peroxisomal integral membrane protein. Two hydrophobic domains can be distinguished which may be involved in anchoring PpPas2p to the peroxisomal membrane. 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 the peroxisomal membrane, with which it associates tightly. A comparison with the membrane peroxisomal targeting signal of PMP47 of Candida boidinii revealed a stretch of positively charged amino acids common to both sequences. The role of peroxisomal membrane targeting signals and transmembrane domains in anchoring PMPs to the peroxisomal membrane is discussed.

Highlights

Involvement in H2O2 metabolism and the ␤-oxidation of fatty acids (Van den Bosch et al, 1992; Wiemer and Subramani, 1994)
Yeast strains were grown at 30 °C in YPD (1% w/v yeast extract, 2% w/v Bacto-peptone, 2% w/v dextrose), YPM (1% w/v yeast extract, 2% w/v Bacto-peptone, 0.5% v/v methanol), YPOT (1% w/v yeast extract, 2% w/v Bacto-peptone, 0.2% v/v oleate, 0.02% v/v Tween 40), or in synthetic medium consisting of 0.67% w/v yeast nitrogen base, supplemented with 50 ␮g/ml of the appropriate amino acids and with one of the following carbon sources: 2% w/v dextrose (SD), 0.5% v/v methanol (SM), or 0.2% v/v oleate and 0.02% v/v Tween 40 (SOT)
In a genetic cross between the PpPas2 null mutant and the PpPas2 mutant, no complementation was observed for growth on methanol or oleate

Summary

Introduction

Involvement in H2O2 metabolism and the ␤-oxidation of fatty acids (Van den Bosch et al, 1992; Wiemer and Subramani, 1994). Much progress has been made in recent years delineating the molecular requirements for import of peroxisomal matrix proteins (metabolic enzymes). Which show a general defect in peroxisome assembly (pas, per pay, or peb mutants; Erdmann et al, 1989; Cregg et al, 1990; Gould et al, 1992; Liu et al, 1992; Van der Leij et al, 1992; Elgersma et al, 1993; Nuttley et al, 1993; Zhang and Lazarow, 1993). In these strains the bulk of the peroxisomal matrix proteins is found in the cytosol. Some contain peroxisomes with an aberrant morphology reminiscent of the peroxisomal ghost structures found in fibroblast cell lines derived from patients with peroxisomal disorders (Wiemer et al, 1989)

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