Pex19p Interacts with Pex3p and Pex10p and Is Essential for Peroxisome Biogenesis in Pichia pastoris

We report the cloning and characterization of Pichia pastoris PEX19 by complementation of a peroxisome-deficient mutant strain. Import of peroxisomal targeting signal 1- and 2-containing peroxisomal matrix proteins is defective inpex19 mutants. PEX19 encodes a hydrophilic 299-amino acid protein with sequence similarity toSaccharomyces cerevisiae Pex19p and human and Chinese hamster PxF, all farnesylated proteins, as well as hypothetical proteins from Caenorhabditis elegans andSchizosaccharomyces pombe. The farnesylation consensus is conserved in PpPex19p but dispensable for function and appears unmodified under the conditions tested. Pex19p localizes predominantly to the cytosolic fraction. Biochemical and two-hybrid analyses confirmed that Pex19p interacts with Pex3p, as seen in S. cerevisiae, but unexpectedly also with Pex10p. Two-hybrid analysis demonstrated that the amino-terminal 42 amino acids of Pex19p interact with the carboxyl-terminal 335 amino acids of Pex3p. In addition, the extreme carboxyl terminus of Pex19p (67 amino acids) is required for interaction with the amino-terminal 380 amino acids of Pex10p. Biochemical and immunofluorescence microscopy analyses ofpex19Δ cells identified the membrane protein Pex3p in peroxisome remnants that were not previously observed in S. cerevisiae. These small vesicular and tubular (early) remnants are morphologically distinct from other Pppex mutant (late) remnants, suggesting that Pex19p functions at an early stage of peroxisome biogenesis.

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The Pichia pastoris PER6 gene product is a peroxisomal integral membrane protein essential for peroxisome biogenesis and has sequence similarity to the Zellweger syndrome protein PAF-1.

Molecular and Cellular Biology ◽

10.1128/mcb.16.5.2527 ◽

1996 ◽

Vol 16 (5) ◽

pp. 2527-2536 ◽

Cited By ~ 41

Author(s):

H R Waterham ◽

Y de Vries ◽

K A Russel ◽

W Xie ◽

M Veenhuis ◽

...

Keyword(s):

Pichia Pastoris ◽

Membrane Protein ◽

Sequence Similarity ◽

Zellweger Syndrome ◽

Methylotrophic Yeast ◽

Integral Membrane Protein ◽

Biochemical Properties ◽

Podospora Anserina ◽

Peroxisome Biogenesis ◽

Membrane Spanning

We report the cloning of PER6, a gene essential for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris. The PER6 sequence predicts that its product Per6p is a 52-kDa polypeptide with the cysteine-rich C3HC4 motif. Per6p has significant overall sequence similarity with the human peroxisome assembly factor PAF-1, a protein that is defective in certain patients suffering from the peroxisomal disorder Zellweger syndrome, and with car1, a protein required for peroxisome biogenesis and caryogamy in the filamentous fungus Podospora anserina. In addition, the C3HC4 motif and two of the three membrane-spanning segments predicted for Per6p align with the C3HC4 motifs and the two membrane-spanning segments predicted for PAF-1 and car1. Like PAF-1, Per6p is a peroxisomal integral membrane protein. In methanol- or oleic acid-induced cells of per6 mutants, morphologically recognizable peroxisomes are absent. Instead, peroxisomal remnants are observed. In addition, peroxisomal matrix proteins are synthesized but located in the cytosol. The similarities between Per6p and PAF-1 in amino acid sequence and biochemical properties, and between mutants defective in their respective genes, suggest that Per6p is the putative yeast homolog of PAF-1.

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The Drosophila Polycomb-group gene Enhancer of zeste contains a region with sequence similarity to trithorax

Molecular and Cellular Biology ◽

10.1128/mcb.13.10.6357-6366.1993 ◽

1993 ◽

Vol 13 (10) ◽

pp. 6357-6366

Author(s):

R S Jones ◽

W M Gelbart

Keyword(s):

Amino Acids ◽

Sequence Similarity ◽

Protein Product ◽

Polycomb Group ◽

Acute Leukemias ◽

Trithorax Group ◽

Acid Protein ◽

Enhancer Of Zeste ◽

Gene Enhancer ◽

Carboxy Terminal

As is typical of Polycomb-group loci, the Enhancer of zeste [E(z)] gene negatively regulates the segment identity genes of the Antennapedia (ANT-C) and Bithorax (BX-C) gene complexes. A second class of loci, collectively known as the trithorax group, plays an antagonistic role as positive regulators of the ANT-C and BX-C genes. Molecular analysis of the E(z) gene predicts a 760-amino-acid protein product. A region of 116 amino acids near the E(z) carboxy terminus is 41.2% identical (68.4% similar) with a carboxy-terminal region of the trithorax protein. This portion of the trithorax protein is part of a larger region previously shown to share extensive homology with a human protein (ALL-1/Hrx) that is implicated in acute leukemias. Over this same 116 amino acids, E(z) and ALL-1/Hrx are 43.9% identical (68.4% similar). Otherwise, E(z) is not significantly similar to any previously described proteins. As this region of sequence similarity is shared by two proteins with antagonistic functions, we suggest that it may comprise a domain that interacts with a common target, either nucleic acid or protein. Opposite effects on transcription might then be determined by other portions of the two proteins.

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Signal peptide for peroxisomal targeting: Replacement of an essential histidine residue by certain amino acids converts the amino-terminal presequence of peroxisomal 3-ketoacyl-CoA thiolase to a mitochondrial signal peptide

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(92)90818-6 ◽

1992 ◽

Vol 186 (2) ◽

pp. 811-818 ◽

Cited By ~ 21

Author(s):

Takashi Osumi ◽

Toshiro Tsukamoto ◽

Shingo Hata

Keyword(s):

Amino Acids ◽

Signal Peptide ◽

Histidine Residue ◽

Amino Terminal ◽

Peroxisomal Targeting

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An internal region of the peroxisomal membrane protein PMP47 is essential for sorting to peroxisomes

The Journal of Cell Biology ◽

10.1083/jcb.124.6.915 ◽

1994 ◽

Vol 124 (6) ◽

pp. 915-925 ◽

Cited By ~ 51

Author(s):

MT McCammon ◽

JA McNew ◽

PJ Willy ◽

JM Goodman

Keyword(s):

Amino Acids ◽

Sequence Similarity ◽

Candida Boidinii ◽

Carrier Proteins ◽

Peroxisomal Membrane ◽

Lower Efficiency ◽

Mitochondrial Carrier ◽

Peroxisomal Targeting ◽

Membrane Spanning ◽

Mitochondrial Carrier Proteins

Targeting sequences on peroxisomal membrane proteins have not yet been identified. We have attempted to find such a sequence within PMP47, a protein of the methylotrophic yeast, Candida boidinii. This protein of 423 amino acids shows sequence similarity with proteins in the family of mitochondrial carrier proteins. As such, it is predicted to have six membrane-spanning domains. Protease susceptibility experiments are consistent with a six-membrane-spanning model for PMP47, although the topology for the peroxisomal protein is inverted compared with the mitochondrial carrier proteins. PMP47 contains two potential peroxisomal targeting sequences (PTS1), an internal SKL (residues 320-322) and a carboxy terminal AKE (residues 421-423). Using a heterologous in vivo sorting system, we show that efficient sorting occurs in the absence of both sequences. Analysis of PMP47-dihydrofolate reductase (DHFR) fusion proteins revealed that amino acids 1-199 of PMP47, which contain the first three putative membrane spans, do not contain the necessary targeting information, whereas a fusion with amino acids 1-267, which contains five spans, is fully competent for sorting to peroxisomes. Similarly, a DHFR fusion construct containing residues 268-423 did not target to peroxisomes while residues 203-420 appeared to sort to that organelle, albeit at lower efficiency than the 1-267 construct. However, DHFR constructs containing only amino acids 185-267 or 203-267 of PMP47 were not found to be associated with peroxisomes. We conclude that amino acids 199-267 are necessary for peroxisomal targeting, although additional sequences may be required for efficient sorting to, or retention by, the organelles.

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Role of the PAS1 gene of Pichia pastoris in peroxisome biogenesis.

The Journal of Cell Biology ◽

10.1083/jcb.127.5.1259 ◽

1994 ◽

Vol 127 (5) ◽

pp. 1259-1273 ◽

Cited By ~ 47

Author(s):

J A Heyman ◽

E Monosov ◽

S Subramani

Keyword(s):

Pichia Pastoris ◽

Biochemical Analysis ◽

Sequence Similarity ◽

Carbon Sources ◽

Peroxisome Biogenesis ◽

Daughter Cells ◽

New Information ◽

Membrane Bound ◽

High Level

Several groups have reported the cloning and sequencing of genes involved in the biogenesis of yeast peroxisomes. Yeast strains bearing mutations in these genes are unable to grow on carbon sources whose metabolism requires peroxisomes, and these strains lack morphologically normal peroxisomes. We report the cloning of Pichia pastoris PAS1, the homologue (based on a high level of protein sequence similarity) of the Saccharomyces cerevisiae PAS1. We also describe the creation and characterization of P. pastoris pas1 strains. Electron microscopy on the P. pastoris pas1 cells revealed that they lack morphologically normal peroxisomes, and instead contain membrane-bound structures that appear to be small, mutant peroxisomes, or "peroxisome ghosts." These "ghosts" proliferated in response to induction on peroxisome-requiring carbon sources (oleic acid and methanol), and they were distributed to daughter cells. Biochemical analysis of cell lysates revealed that peroxisomal proteins are induced normally in pas1 cells. Peroxisome ghosts from pas1 cells were purified on sucrose gradients, and biochemical analysis showed that these ghosts, while lacking several peroxisomal proteins, did import varying amounts of several other peroxisomal proteins. The existence of detectable peroxisome ghosts in P. pastoris pas1 cells, and their ability to import some proteins, stands in contrast with the results reported by Erdmann et al. (1991) for the S. cerevisiae pas1 mutant, in which they were unable to detect peroxisome-like structures. We discuss the role of PAS1 in peroxisome biogenesis in light of the new information regarding peroxisome ghosts in pas1 cells.

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Pex18p and Pex21p, a Novel Pair of Related Peroxins Essential for Peroxisomal Targeting by the PTS2 Pathway

The Journal of Cell Biology ◽

10.1083/jcb.143.7.1859 ◽

1998 ◽

Vol 143 (7) ◽

pp. 1859-1869 ◽

Cited By ~ 117

Author(s):

P. Edward Purdue ◽

Xudong Yang ◽

Paul B. Lazarow

Keyword(s):

Subcellular Localization ◽

Protein Targeting ◽

Chondrodysplasia Punctata ◽

Peroxisome Biogenesis ◽

Rhizomelic Chondrodysplasia Punctata ◽

Peroxisomal Biogenesis ◽

Peroxisomal Targeting ◽

Two Hybrid ◽

Import Receptor

We have identified ScPex18p and ScPex21p, two novel S. cerevisiae peroxins required for protein targeting via the PTS2 branch of peroxisomal biogenesis. Targeting by this pathway is known to involve the interaction of oligopeptide PTS2 signals with Pex7p, the PTS2 receptor. Pex7p function is conserved between yeasts and humans, with defects in the human protein causing rhizomelic chondrodysplasia punctata (RCDP), a severe, lethal peroxisome biogenesis disorder characterized by aberrant targeting of several PTS2 peroxisomal proteins, but uncertainty remains about the subcellular localization of this receptor. Previously, we have reported that ScPex7p resides predominantly in the peroxisomal matrix, suggesting that it may function as a highly unusual intraorganellar import receptor, and the data presented in this paper identify Pex18p and Pex21p as key components in the targeting of Pex7p to peroxisomes. They each interact specifically with Pex7p both in two-hybrid analyses and in vitro. In cells lacking both Pex18p and Pex21p, Pex7p remains cytosolic and PTS2 targeting is completely abolished. Pex18p and Pex21p are weakly homologous to each other and display partial functional redundancy, indicating that they constitute a two-member peroxin family specifically required for Pex7p and PTS2 targeting.

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The Hansenula polymorpha PER1 gene is essential for peroxisome biogenesis and encodes a peroxisomal matrix protein with both carboxy- and amino-terminal targeting signals.

The Journal of Cell Biology ◽

10.1083/jcb.127.3.737 ◽

1994 ◽

Vol 127 (3) ◽

pp. 737-749 ◽

Cited By ~ 145

Author(s):

H R Waterham ◽

V I Titorenko ◽

P Haima ◽

J M Cregg ◽

W Harder ◽

...

Keyword(s):

Matrix Protein ◽

Sequence Similarity ◽

Hansenula Polymorpha ◽

Matrix Proteins ◽

Regulatory Function ◽

Wild Type ◽

Amino Terminal ◽

Peroxisomal Targeting ◽

Targeting Signal

We describe the cloning of the Hansenula polymorpha PER1 gene and the characterization of the gene and its product, PER1p. The gene was cloned by functional complementation of a per1 mutant of H. polymorpha, which was impaired in the import of peroxisomal matrix proteins (Pim- phenotype). The DNA sequence of PER1 predicts that PER1p is a polypeptide of 650 amino acids with no significant sequence similarity to other known proteins. PER1 expression was low but significant in wild-type H. polymorpha growing on glucose and increased during growth on any one of a number of substrates which induce peroxisome proliferation. PER1p contains both a carboxy- (PTS1) and an amino-terminal (PTS2) peroxisomal targeting signal which both were demonstrated to be capable of directing bacterial beta-lactamase to the organelle. In wild-type H. polymorpha PER1p is a protein of low abundance which was demonstrated to be localized in the peroxisomal matrix. Our results suggest that the import of PER1p into peroxisomes is a prerequisite for the import of additional matrix proteins and we suggest a regulatory function of PER1p on peroxisomal protein support.

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Peroxisome Targeting Signal Type 1 (PTS1) Receptor Is Involved in Import of Both PTS1 and PTS2: Studies withPEX5-Defective CHO Cell Mutants

Molecular and Cellular Biology ◽

10.1128/mcb.18.1.388 ◽

1998 ◽

Vol 18 (1) ◽

pp. 388-399 ◽

Cited By ~ 148

Author(s):

Hidenori Otera ◽

Kanji Okumoto ◽

Keita Tateishi ◽

Yuka Ikoma ◽

Eiko Matsuda ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Protein Translocation ◽

Single Gene ◽

Chinese Hamster ◽

Cho Cell ◽

Amino Terminal ◽

Signal Type ◽

Targeting Signal

ABSTRACT To investigate the mechanisms of peroxisome assembly and the molecular basis of peroxisome assembly disorders, we isolated and characterized a peroxisome-deficient CHO cell mutant, ZP139, which was found to belong to human complementation group II, the same group as that of our earlier mutant, ZP105. These mutants had a phenotypic deficiency in the import of peroxisomal targeting signal type 1 (PTS1) proteins. Amino-terminal extension signal (PTS2)-mediated transport, including that of 3-ketoacyl coenzyme A thiolase, was also defective in ZP105 but not in ZP139. PEX5 cDNA, encoding the PTS1 receptor (PTS1R), was isolated from wild-type CHO-K1 cells. PTS1R’s deduced primary sequence comprised 595 amino acids, 7 amino acids less than the human homolog, and contained seven tetratricopeptide repeat (TPR) motifs at the C-terminal region. Chinese hamster PTS1R showed 94, 28, and 24% amino acid identity with PTS1Rs from humans, Pichia pastoris, and Saccharomyces cerevisiae, respectively. A PTS1R isoform (PTS1RL) with 632 amino acid residues was identified in CHO cells; for PTS1R, 37 amino acids were inserted between residues at positions 215 and 216 of a shorter isoform (PTS1RS). Southern blot analysis of CHO cell genomic DNA suggested that these two isoforms are derived from a single gene. Both types of PEX5 complemented impaired import of PTS1 in mutants ZP105 and ZP139. PTS2 import in ZP105 was rescued only by PTS1RL. This finding strongly suggests that PTS1RL is also involved in the transport of PTS2. Mutations inPEX5 were determined by reverse transcription-PCR: a G-to-A transition resulted in one amino acid substitution: Gly298Glu of PTS1RS (G335E of PTS1RL) in ZP105 and Gly485Glu of PTS1RS (G522E of PTS1RL) in ZP139. Both mutations were in the TPR domains (TPR1 and TPR6), suggesting the functional consequence of these domains in protein translocation. The implications of these mutations are discussed.

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Ropporin, a sperm-specific binding protein of rhophilin, that is localized in the fibrous sheath of sperm flagella

Journal of Cell Science ◽

10.1242/jcs.113.1.103 ◽

2000 ◽

Vol 113 (1) ◽

pp. 103-112 ◽

Cited By ~ 3

Author(s):

A. Fujita ◽

K. Nakamura ◽

T. Kato ◽

N. Watanabe ◽

T. Ishizaki ◽

...

Keyword(s):

Amino Acids ◽

Specific Binding ◽

Pdz Domain ◽

Small Gtpase ◽

Regulatory Subunit ◽

Yeast Two Hybrid ◽

Fibrous Sheath ◽

Amino Terminal ◽

Two Hybrid

The small GTPase Rho; functions as a molecular switch that regulates various cellular processes such as cell adhesion, motility, gene expression and cytokinesis. We previously isolated several putative Rho; targets including rhophilin which bound selectively to the GTP-bound form of Rho;. Rhophilin is expressed highly in testis and is localized specifically in sperm flagella. The presence of a PDZ domain at the carboxy terminus of rhophilin suggested that rhophilin works as an adaptor molecule. To test this hypothesis, we employed a yeast two hybrid system using the rhophilin PDZ domain as a bait, and screened a mouse testis cDNA library. We isolated several positive clones containing the same insert. The open reading frame of the cDNA encoded a novel protein of 212 amino acids designated as ropporin from a Japanese word ‘oppo’ (the tail). The amino-terminal 40 amino acid sequence of ropporin showed high homology to that of the regulatory subunit of type II cAMP-dependent protein kinase, which is involved in dimerization and binding to A-kinase anchoring proteins. Consistently, a yeast two hybrid assay and gel filtration of recombinant ropporin indicated that ropporin dimerizes through this domain. Deletion analysis indicated that the carboxy-terminal four amino acids are essential for binding of ropporin to rhophilin, and ropporin and RhoV14 coprecipitated in the presence of rhophilin in vitro. Northern blot analysis showed that ropporin is exclusively expressed in testis, and induced at the late stage of spermatogenesis. This induction paralleled that of rhophilin. Immunocytochemistry using anti-ropporin antibody showed that ropporin is localized in the principal piece and the end piece of sperm flagella. Electronmicroscopy revealed that ropporin is mostly localized in the inner surface of the fibrous sheath while rhophilin is present in the outer surface of the outer dense fiber. These results suggest that rhophilin and ropporin may form a complex in sperm flagella.

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