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

doi:10.1074/jbc.271.31.18973

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

From the ^‡ Department of Biology, University of California at San Diego, La Jolla, California 92093-0322, and the
^¶ Laboratory for Electron Microscopy, University of Groningen, Biological Center, 9751 NN Haren, The Netherlands

^∥ To whom correspondence should be addressed:
Dept. of Biology, UCSD, Rm. 4314, Bonner Hall, 9500 Gilman Dr., La Jolla, CA 92093-0322.
Tel.: 619-534-2327; Fax: 619-534-0053; E-mail: subra{at}jeeves.ucsd.edu

↵^§ Present address: Institute for Hematology, Erasmus University, Rotterdam, The Netherlands.

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.

Footnotes

↵* This work was supported in part by a grant from the Human Frontier Science Program Organization (HFSPO) and the American Heart Association (to E. A. C. W.), an HFSPO fellowship (to K. N. F.), a fellowship from the Deutsche Forschungsgemeinshaft (to G. L.), a fellowship from the European Molecular Biology Organization (to T. W.), and by National Institutes of Health Grant NIHDK41737 and National Science Foundation Grant MCB9316018 (to S. S.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) Z72390[GenBank].
↵¹ The abbreviations used are:

PTS(s)

peroxisomal targeting signal(s)

PMP

peroxisomal membrane protein

mPTS

peroxisomal membrane targeting signal

PCR

polymerase chain reaction

kb

kilobase

GFP

green fluorescent protein

PAGE

polyacrylamide gel electrophoresis

TMD

transmembrane domain

PAS

peroxisome assembly.
- Received April 5, 1996.