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 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.
Wiemer
E A
EA
Department of Biology, University of California at San Diego, La Jolla, California 92093-0322, USA.
Lüers
G H
GH
Faber
K N
KN
Wenzel
T
T
Veenhuis
M
M
Subramani
S
S
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017353:Gene Deletion; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008830:Microbodies / Q000378:metabolism*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009154:Mutation; D000074401:Peroxins; D010843:Pichia / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D021382:Protein Sorting Signals / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid
D018528:ATP-Binding Cassette Transporters; D004271:DNA, Fungal; D005656:Fungal Proteins; D008565:Membrane Proteins; C070895:PEX3 protein, S cerevisiae; C489627:Pas2 protein, Pichia pastoris; D000074401:Peroxins; D021382:Protein Sorting Signals; C406793:SPF1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins