Development of the yeast Pichia pastoris as a model organism for a genetic and molecular analysis of peroxisome assembly

Abstract

We describe the isolation of mutants of the yeast Pichia pastoris that are deficient in peroxisome assembly (pas). These mutants of P. pastoris can be identified solely by their inability to grow on methanol and oleic acid, the utilization of which requires peroxisomal enzymes, and are defined by the absence of normal peroxisomes as judged by electron microscopy and biochemical fractionation experiments. These mutants are the result of genetic defects at single loci and represent at least eight different complementation groups. The isolation of pas mutants of P. pastoris by a simple screen for mutants unable to use methanol and oleic acid represents a significantly more efficient method for identification of pas mutants than is possible in other organisms. To exploit this advantage fully we also developed new reagents for the genetic and molecular manipulation of P. pastoris. These include a set of auxotrophic strains with an essentially wild-type genetic background, plasmids that act as Escherichia coli-P. pastoris shuttle vectors, and genomic DNA libraries for isolation of P. pastoris genes by functional complementation of mutants or by nucleic acid hybridization. The availability of numerous pas mutants and the reagents necessary for their molecular analysis should lead to the isolation and characterization of genes involved in peroxisome assembly.

Two Mammalian Sec16 Homologues Have Nonredundant Functions in Endoplasmic Reticulum (ER) Export and Transitional ER Organization

“…Unlike the closely related Sacchaormyces cerevisiae, P. pastoris contains discrete tER sites and stacked Golgi organelles similar to those seen in most other eukaryotes (Gould et al, 1992;Rossanese et al, 1999;Mogelsvang et al, 2003). A genetic screen for P. pastoris mutants with disrupted tER organization uncovered a role for the 281-kDa Sec16 protein (Connerly et al, 2005).…”

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

“…Plasmid p82.20 contains the 1.1-kb BamHI fragment of p82.13 in vector pSG560 (Gould et al, 1992). p82.21 contains the 0.7-kb BglII-BamHI fragment, p82.22 the 0.8-kb BamHI-HincII fragment, p82.23 the 0.3-kb HincII fragment, p82.24 the 0.5-kb NarI-BamHI fragment, and p82.25 the 0.9-kb EcoRV-BamHI fragment.…”

“…The five inserts contained an overlapping fragment of 1.1 kb which was isolated from p82.13 as a BamHI fragment and subcloned into the pSG560 vector (Gould et al, 1992) to check for complementation (p82.20; Fig. 1 A).…”

“…cerevisiae and P . pastoris , deletion of PEX4 disrupts targeting via the PTS1 and PTS2 pathway (Wiebel and Kunau, 1992;Gould et al, 1992;our unpublished observation). In Hansenula polymorpha , only the PTS1 pathway was impaired and this defect could be rescued by overexpression of the PTS1 receptor, Pex5p (van der Klei et al, 1998), implying an important role for Pex4p in the import of PTS1-containing proteins in this yeast.…”

Characterization of peroxisome-deficient mutants of Hansenula polymorpha

“…pastoris (Liu et al 1992;Gould et al 1992), and in two genes in Y. Iipolytica (Nuttley et al 1993). In all these peroxisome-deficient yeast mutants, peroxisomalmatrix enzymes are synthesized but, with two notable exceptions, are mislocalized to the cytosol.…”