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© The Rockefeller University Press, 0021-9525/1999//99 $5.00
The Journal of Cell Biology, Volume 146, Number 1, , 1999 99-112

Original Article

Pex22p of Pichia pastoris, Essential for Peroxisomal Matrix Protein Import, Anchors the Ubiquitin-Conjugating Enzyme, Pex4p, on the Peroxisomal Membrane



Antonius Kollera, William B. Snydera, Klaas Nico Fabera, Thibaut J. Wenzela, Linda Rangellb, Gilbert A. Kellerb, and Suresh Subramania

a Department of Biology, University of California San Diego, La Jolla, California 92093-0322
b Pharmacological Science, Genentech, South San Francisco, California 94080
Department of Biology, University of California San Diego, Bonner Hall, Room 3230, 9500 Gilman Drive, La Jolla, CA 92093-0322.(619) 534-0053(619) 534-2327

ssubramani{at}ucsd.edu

We isolated a Pichia pastoris mutant that was unable to grow on the peroxisome-requiring media, methanol and oleate. Cloning the gene by complementation revealed that the encoded protein, Pex22p, is a new peroxin. A {Delta}pex22 strain does not grow on methanol or oleate and is unable to import peroxisomal matrix proteins. However, this strain targets peroxisomal membrane proteins to membranes, most likely peroxisomal remnants, detectable by fluorescence and electron microscopy. Pex22p, composed of 187 amino acids, is an integral peroxisomal membrane protein with its NH2 terminus in the matrix and its COOH terminus in the cytosol. It contains a 25–amino acid peroxisome membrane-targeting signal at its NH2 terminus. Pex22p interacts with the ubiquitin-conjugating enzyme Pex4p, a peripheral peroxisomal membrane protein, in vivo, and in a yeast two-hybrid experiment. Pex22p is required for the peroxisomal localization of Pex4p and in strains lacking Pex22p, the Pex4p is cytosolic and unstable. Therefore, Pex22p anchors Pex4p at the peroxisomal membrane. Strains that do not express Pex4p or Pex22p have similar phenotypes and lack Pex5p, suggesting that Pex4p and Pex22p act at the same step in peroxisome biogenesis. The Saccharomyces cerevisiae hypothetical protein, Yaf5p, is the functional homologue of P. pastoris Pex22p.

Key Words: organelle • peroxin • peroxisome • protein transport • yeast

© 1999 The Rockefeller University Press

1.used in this paper: AD, activation domain; AOX, alcohol oxidase; DB, DNA-binding domain; DSP, dithiobis(succinimidylpropionate); G6PDH, glucose-6-phosphate dehydrogenase; GFP, green fluorescent protein; IPTG, isopropyl β-d-thiogalactopyranoside; mPTS, membrane peroxisome targeting signal; Pex, peroxin; PNS, post nuclear supernatant; PTS, peroxisome-targeting signal; UBC, ubiquitin-conjugating enzyme

Klaas Nico Faber's present address is University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, Eukaryotic Microbiology, Kerklaan 30, 9751 NN Haren, The Netherlands.

Thibaut J. Wenzel's present address is Gist-Brocades, Food Specialities Division, Wateringseweg 1, 2600 MA Delft, The Netherlands.


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