THE PAH2 GENE IS REQUIRED FOR PEROXISOME ASSEMBLY IN THE METHYLOTROPHIC YEAST HANSENULA-POLYMORPHA AND ENCODES A MEMBER OF THE TETRATRICOPEPTIDE REPEAT FAMILY OF PROTEINS

Peroxisome assembly mutants in the methylotrophic yeast, Hansenula pol ymorpha, were selected by a novel procedure involving the inability of mutants to use both oleic acid and methanol as carbon sources. These compounds are both metabolized within peroxisomes through two differen t enzymatic pathways. 15 mutant strains called mut (methanol non-utili zing) were isolated. These strains were assigned to ten genetic comple mentation groups. Subcellular fractionation analysis showed that perox isomal matrix enzymes were mislocalized to the cytoplasm in mut strain s. Electron microscopy confirmed that the inability of mut strains to grow on oleic acid and methanol was due to defects in peroxisome assem bly, Functional complementation of a mutant strain, mut2, with a plasm id library of H. polymorpha genomic DNA sequences has identified a gen e, PAH2, that restores growth on methanol and the correct localization of matrix enzymes to the peroxisome, PAH2 encodes Pah2p, a polypeptid e of 569 amino acids that is a member of the tetratricopetide repeat ( TPR) family of proteins. Pah2p shows identity with Pas8p and Pas10p, t wo proteins required for peroxisome assembly in the yeasts Pichia past or is and Saccharomyces cei evisiae, respectively, and which have been suggested to be receptors that recognize peroxisomal targeting signal -1 (PTS1) motifs.