The pas8 mutant of Pichia pastoris exhibits the peroxisomal protein import deficiencies of Zellweger syndrome cells--the PAS8 protein binds to the COOH-terminal tripeptide peroxisomal targeting signal, and is a member of the TPR protein family [published erratum appears in J Cell Biol 1993 Sep;122(5):following 1143]

Abstract

Abstract. We previously described the isolation of mutants of the yeast P~chia pastoris that are deficient in peroxisome assembly (pas mutants). We describe the characterization of one of these mutants, pas8, and the cloning of the PAS8 gene. The pas8 mutant is deficient for growth, but not for division or segregation of peroxisomes, or for induction of peroxisomal proteins. Two distinct peroxisomal targeting signals, PTS1 and PTS2, have been identified that are sufficient to direct proteins to the peroxisomal matrix. We show that the pas8 mutant is deficient in the import of proteins with the PTS1, but not the PTS2, targeting signal. This is the same import deficiency as that found in cells from patients with the lethal human peroxisomal disorder Zellweger syndrome. Cloning and sequencing of the PAS8 gene reveals that it is a novel member of the tetratricopeptide repeat gene family. Antibodies raised against bacterially expressed PAS8 are used to show that PASS is a peroxisomal, membrane-associated protein. Also, we have found that in vitro translated PAS8 protein is capable of binding the PTSl targeting signal specifically, raising the possibility that PAS8 is a PTS1 receptor.T hE compartmentalization of cellular processes into different subcellular compartments is one of the hallmarks of the eukaryotic cell. One such compartment, the peroxisome, is ubiquitously present in virtually all eukaryotic cells. Specialized versions of peroxisome-like compartments (e.g., glycosomes of trypanosomes and glyoxysomes of plants) exist in certain organisms. The peroxisomes, glycosomes, and glyoxysomes have been termed collectively as microbodies. Peroxisomes are characterized by the presence of at least one peroxide-generating oxidase and the enzyme catalase which degrades hydrogen peroxide. Peroxisomes are bounded by a single membrane and vary in size from 0.1 to 1.0 #m in diameter. Like mitochondria, peroxisomes are thought to arise by budding and division of preexisting organelles. Unlike mitochondria, peroxisomes do not contain DNA and must import all constituent proteins.Both peroxisomal matrix and membrane proteins are synthesized in the cytosol on free polysomes and imported posttranslationally into the organdie (for review see Lazarow and Fujiki, 1985).Although the targeting signals for peroxisomal membrane proteins are unknown, recent work has demonstrated the existence of at least two distinct signals that will direct proteins to the peroxisomal matrix (for review see Subramani, 1992).

Routing ofHansenula polymorphaAlcohol Oxidase: An Alternative Peroxisomal Protein-sorting Machinery

“…The extreme carboxyterminal amino acids of AO contains a PTS1 (Waterham et al, 1997;Salomons et al, 2000b) and import of AO into peroxisomes requires the function of the PTS1 receptor, Pex5p (McCollum et al, 1993;van der Klei et al, 1995). However, previous studies revealed that the sorting pathway of AO displays several unusual features.…”

Characterization of peroxisome-deficient mutants of Hansenula polymorpha

“…In all these peroxisome-deficient yeast mutants, peroxisomalmatrix enzymes are synthesized but, with two notable exceptions, are mislocalized to the cytosol. The exceptions are P. pastoris pas8 mutants (S. cerevisiae paslO, H. poIymorpha per3), in which PTS1 enzymes are located in the cytosol but the PTS2-enzyme thiolase is present in small peroxisomes, and S. cerevisiae pas7 mutants, in which PTS1 enzymes, but not thiolase, are imported normally (McCollum et al 1993;van der Leij et al 1993;Marzioch et al 1994;unpublished results). Evidence suggests that the PAS8 product is the receptor for PTS1 proteins while the PAS7 product may be the PTS2-protein receptor.…”

Tetratricopeptide repeat domain of Yarrowia lipolytica Pex5p is essential for recognition of the type 1 peroxisomal targeting signal but does not confer full biological activity on Pex5p

“…We tested the ability of YlPex5p to recognize a PTS1 tripeptide by using a PTS1 binding assay in itro modelled on that of McCollum et al [4]. GST variants ending in a PTS1 (GST-SKL) or the reverse tripeptide LKS (GST-LKS), which does not resemble a PTS1 but maintains its charge density, were immobilized on glutathione-Sepharose beads.…”

“…PTS1 receptors are encoded by the PEX5 gene family [4][5][6][7][8][9][10][11][12][13][14][15][16][17]. These peroxins (proteins involved in peroxisome assembly) are characterized by a tetratricopeptide repeat (TPR) domain in the C-terminal half of the protein.…”

“…Pex5 proteins have been localized to different subcellular compartments, i.e. primarily cytosolic [5,10,11,17,19], associated with the outer side of the peroxisomal membrane [4,9,20], in the peroxisomal matrix [12] and in both the cytosol and peroxisomes [8]. The PEX7 gene family encodes the PTS2 receptor [21][22][23][24][25][26][27].…”