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

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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The unique degradation pathway of the PTS2 receptor, Pex7, is dependent on the PTS receptor/coreceptor, Pex5 and Pex20

Molecular Biology of the Cell ◽

10.1091/mbc.e13-12-0716 ◽

2014 ◽

Vol 25 (17) ◽

pp. 2634-2643 ◽

Cited By ~ 11

Author(s):

Danielle Hagstrom ◽

Changle Ma ◽

Soumi Guha-Polley ◽

Suresh Subramani

Keyword(s):

Matrix Protein ◽

Protein Import ◽

Degradation Pathway ◽

Matrix Proteins ◽

Receptor Recycling ◽

Wild Type ◽

The Matrix ◽

Peroxisomal Targeting ◽

Targeting Signals ◽

The Stability

Peroxisomal matrix protein import uses two peroxisomal targeting signals (PTSs). Most matrix proteins use the PTS1 pathway and its cargo receptor, Pex5. The PTS2 pathway is dependent on another receptor, Pex7, and its coreceptor, Pex20. We found that during the matrix protein import cycle, the stability and dynamics of Pex7 differ from those of Pex5 and Pex20. In Pichia pastoris, unlike Pex5 and Pex20, Pex7 is constitutively degraded in wild-type cells but is stabilized in pex mutants affecting matrix protein import. Degradation of Pex7 is more prevalent in cells grown in methanol, in which the PTS2 pathway is nonessential, in comparison with oleate, suggesting regulation of Pex7 turnover. Pex7 must shuttle into and out of peroxisomes before it is polyubiquitinated and degraded by the proteasome. The shuttling of Pex7, and consequently its degradation, is dependent on the receptor recycling pathways of Pex5 and Pex20 and relies on an interaction between Pex7 and Pex20. We also found that blocking the export of Pex20 from peroxisomes inhibits PTS1-mediated import, suggesting sharing of limited components in the export of PTS receptors/coreceptors. The shuttling and stability of Pex7 are divergent from those of Pex5 and Pex20, exemplifying a novel interdependence of the PTS1 and PTS2 pathways.

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Pex20p of the Yeast Yarrowia lipolytica Is Required for the Oligomerization of Thiolase in the Cytosol and for Its Targeting to the Peroxisome

The Journal of Cell Biology ◽

10.1083/jcb.142.2.403 ◽

1998 ◽

Vol 142 (2) ◽

pp. 403-420 ◽

Cited By ~ 105

Author(s):

Vladimir I. Titorenko ◽

Jennifer J. Smith ◽

Rachel K. Szilard ◽

Richard A. Rachubinski

Keyword(s):

Yarrowia Lipolytica ◽

High Speed ◽

Matrix Protein ◽

Peroxisomal Membrane ◽

Amino Terminal ◽

Peroxisomal Targeting ◽

In The Wild ◽

Targeting Signal ◽

Polypeptide Chains ◽

Yeast Yarrowia Lipolytica

Pex mutants are defective in peroxisome assembly. In the pex20-1 mutant strain of the yeast Yarrowia lipolytica, the peroxisomal matrix protein thiolase is mislocalized exclusively to the cytosol, whereas the import of other peroxisomal proteins is unaffected. The PEX20 gene was isolated by functional complementation of the pex20-1 strain and encodes a protein, Pex20p, of 424 amino acids (47,274 D). Despite its role in the peroxisomal import of thiolase, which is targeted by an amino-terminal peroxisomal targeting signal-2 (PTS2), Pex20p does not exhibit homology to Pex7p, which acts as the PTS2 receptor. Pex20p is mostly cytosolic, whereas 4–8% is associated with high-speed (200,000 g) pelletable peroxisomes. In the wild-type strain, all newly synthesized thiolase is associated with Pex20p in a heterotetrameric complex composed of two polypeptide chains of each protein. This association is independent of PTS2. Pex20p is required for both the oligomerization of thiolase in the cytosol and its targeting to the peroxisome. Our data suggest that monomeric Pex20p binds newly synthesized monomeric thiolase in the cytosol and promotes the formation of a heterotetrameric complex of these two proteins, which could further bind to the peroxisomal membrane. Translocation of the thiolase homodimer into the peroxisomal matrix would release Pex20p monomers back to the cytosol, thereby permitting a new cycle of binding-oligomerization-targeting-release for Pex20p and thiolase.

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Assembly of alcohol oxidase in peroxisomes of the yeast Hansenula polymorpha requires the cofactor flavin adenine dinucleotide.

Molecular Biology of the Cell ◽

10.1091/mbc.5.8.829 ◽

1994 ◽

Vol 5 (8) ◽

pp. 829-837 ◽

Cited By ~ 27

Author(s):

M E Evers ◽

V I Titorenko ◽

I J van der Klei ◽

W Harder ◽

M Veenhuis

Keyword(s):

Molecular Chaperone ◽

Matrix Protein ◽

Protein Import ◽

Flavin Adenine Dinucleotide ◽

Hansenula Polymorpha ◽

Alcohol Oxidase ◽

Auxotrophic Mutant ◽

Matrix Proteins ◽

Wild Type ◽

Wild Type Control

The peroxisomal flavoprotein alcohol oxidase (AO) is an octamer (600 kDa) consisting of eight identical subunits, each of which contains one flavin adenine dinucleotide molecule as a cofactor. Studies on a riboflavin (Rf) auxotrophic mutant of the yeast Hansenula polymorpha revealed that limitation of the cofactor led to drastic effects on AO import and assembly as well as peroxisome proliferation. Compared to wild-type control cells Rf-limitation led to 1) reduced levels of AO protein, 2) reduced levels of correctly assembled and activated AO inside peroxisomes, 3) a partial inhibition of peroxisomal protein import, leading to the accumulation of precursors of matrix proteins in the cytosol, and 4) a significant increase in peroxisome number. We argue that the inhibition of import may result from the saturation of a peroxisomal molecular chaperone under conditions that normal assembly of a major matrix protein inside the target organelle is prevented.

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Peroxisomal Targeting, Import, and Assembly of Alcohol Oxidase in Pichia pastoris

The Journal of Cell Biology ◽

10.1083/jcb.139.6.1419 ◽

1997 ◽

Vol 139 (6) ◽

pp. 1419-1431 ◽

Cited By ~ 48

Author(s):

Hans R. Waterham ◽

Kimberly A. Russell ◽

Yne de Vries ◽

James M. Cregg

Keyword(s):

Pichia Pastoris ◽

Matrix Protein ◽

Alcohol Oxidase ◽

Mutant Form ◽

Wild Type ◽

Epitope Tag ◽

Peroxisomal Targeting ◽

Targeting Signal ◽

Terminal Amino ◽

Utilization Pathway

Alcohol oxidase (AOX), the first enzyme in the yeast methanol utilization pathway is a homooctameric peroxisomal matrix protein. In peroxisome biogenesis-defective (pex) mutants of the yeast Pichia pastoris, AOX fails to assemble into active octamers and instead forms inactive cytoplasmic aggregates. The apparent inability of AOX to assemble in the cytoplasm contrasts with other peroxisomal proteins that are able to oligomerize before import. To further investigate the import of AOX, we first identified its peroxisomal targeting signal (PTS). We found that sequences essential for targeting AOX are primarily located within the four COOH-terminal amino acids of the protein leucine-alanine-arginine-phenylalanine COOH (LARF). To examine whether AOX can oligomerize before import, we coexpressed AOX without its PTS along with wild-type AOX and determined whether the mutant AOX could be coimported into peroxisomes. To identify the mutant form of AOX, the COOH-terminal LARF sequence of the protein was replaced with a hemagglutinin epitope tag (AOX–HA). Coexpression of AOX–HA with wild-type AOX (AOX-WT) did not result in an increase in the proportion of AOX–HA present in octameric active AOX, suggesting that newly synthesized AOX–HA cannot oligomerize with AOX-WT in the cytoplasm. Thus, AOX cannot initiate oligomerization in the cytoplasm, but must first be targeted to the organelle before assembly begins.

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The Peroxisome Biogenesis Factors Pex4p, Pex22p, Pex1p, and Pex6p Act in the Terminal Steps of Peroxisomal Matrix Protein Import

Molecular and Cellular Biology ◽

10.1128/mcb.20.20.7516-7526.2000 ◽

2000 ◽

Vol 20 (20) ◽

pp. 7516-7526 ◽

Cited By ~ 117

Author(s):

Cynthia S. Collins ◽

Jennifer E. Kalish ◽

James C. Morrell ◽

J. Michael McCaffery ◽

Stephen J. Gould

Keyword(s):

Matrix Protein ◽

Protein Import ◽

Protein Translocation ◽

Matrix Proteins ◽

Peroxisome Biogenesis ◽

Peroxisomal Membrane ◽

The Matrix ◽

Peroxisomal Targeting ◽

Targeting Signal ◽

Mutant Cells

ABSTRACT Peroxisomes are independent organelles found in virtually all eukaryotic cells. Genetic studies have identified more than 20PEX genes that are required for peroxisome biogenesis. The role of most PEX gene products, peroxins, remains to be determined, but a variety of studies have established that Pex5p binds the type 1 peroxisomal targeting signal and is the import receptor for most newly synthesized peroxisomal matrix proteins. The steady-state abundance of Pex5p is unaffected in mostpex mutants of the yeast Pichia pastorisbut is severely reduced in pex4 andpex22 mutants and moderately reduced in pex1and pex6 mutants. We used these subphenotypes to determine the epistatic relationships among several groups ofpex mutants. Our results demonstrate that Pex4p acts after the peroxisome membrane synthesis factor Pex3p, the Pex5p docking factors Pex13p and Pex14p, the matrix protein import factors Pex8p, Pex10p, and Pex12p, and two other peroxins, Pex2p and Pex17p. Pex22p and the interacting AAA ATPases Pex1p and Pex6p were also found to act after Pex10p. Furthermore, Pex1p and Pex6p were found to act upstream of Pex4p and Pex22p. These results suggest that Pex1p, Pex4p, Pex6p, and Pex22p act late in peroxisomal matrix protein import, after matrix protein translocation. This hypothesis is supported by the phenotypes of the corresponding mutant strains. As has been shown previously for P. pastoris pex1,pex6, and pex22 mutant cells, we show here thatpex4Δ mutant cells contain peroxisomal membrane protein-containing peroxisomes that import residual amounts of peroxisomal matrix proteins.

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Characterization of Mmp37p, aSaccharomyces cerevisiaeMitochondrial Matrix Protein with a Role in Mitochondrial Protein Import

Molecular Biology of the Cell ◽

10.1091/mbc.e06-04-0366 ◽

2006 ◽

Vol 17 (9) ◽

pp. 4051-4062 ◽

Cited By ~ 47

Author(s):

Michelle R. Gallas ◽

Mary K. Dienhart ◽

Rosemary A. Stuart ◽

Roy M. Long

Keyword(s):

Matrix Protein ◽

Protein Import ◽

Mitochondrial Protein ◽

Temperature Sensitive ◽

Inner Membrane ◽

Mitochondrial Inner Membrane ◽

Close Proximity ◽

The Matrix ◽

Targeting Signal

Many mitochondrial proteins are encoded by nuclear genes and after translation in the cytoplasm are imported via translocases in the outer and inner membranes, the TOM and TIM complexes, respectively. Here, we report the characterization of the mitochondrial protein, Mmp37p (YGR046w) and demonstrate its involvement in the process of protein import into mitochondria. Haploid cells deleted of MMP37 are viable but display a temperature-sensitive growth phenotype and are inviable in the absence of mitochondrial DNA. Mmp37p is located in the mitochondrial matrix where it is peripherally associated with the inner membrane. We show that Mmp37p has a role in the translocation of proteins across the mitochondrial inner membrane via the TIM23-PAM complex and further demonstrate that substrates containing a tightly folded domain in close proximity to their mitochondrial targeting sequences display a particular dependency on Mmp37p for mitochondrial import. Prior unfolding of the preprotein, or extension of the region between the targeting signal and the tightly folded domain, relieves their dependency for Mmp37p. Furthermore, evidence is presented to show that Mmp37 may affect the assembly state of the TIM23 complex. On the basis of these findings, we hypothesize that the presence of Mmp37p enhances the early stages of the TIM23 matrix import pathway to ensure engagement of incoming preproteins with the mtHsp70p/PAM complex, a step that is necessary to drive the unfolding and complete translocation of the preprotein into the matrix.

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Enlarged Peroxisomes Are Present in Oleic Acid–grown Yarrowia lipolytica Overexpressing the PEX16 Gene Encoding an Intraperoxisomal Peripheral Membrane Peroxin

The Journal of Cell Biology ◽

10.1083/jcb.137.6.1265 ◽

1997 ◽

Vol 137 (6) ◽

pp. 1265-1278 ◽

Cited By ~ 102

Author(s):

Gary A. Eitzen ◽

Rachel K. Szilard ◽

Richard A. Rachubinski

Keyword(s):

Oleic Acid ◽

Yarrowia Lipolytica ◽

Consensus Sequence ◽

Wild Type ◽

Gene Encoding ◽

Peroxisomal Membrane ◽

Peripheral Protein ◽

The Matrix ◽

Peroxisomal Targeting ◽

Targeting Signal

Pex mutants of the yeast Yarrowia lipolytica are defective in peroxisome assembly. The mutant strain pex16-1 lacks morphologically recognizable peroxisomes. Most peroxisomal proteins are mislocalized to a subcellular fraction enriched for cytosol in pex16 strains, but a subset of peroxisomal proteins is localized at, or near, wild-type levels to a fraction typically enriched for peroxisomes. The PEX16 gene was isolated by functional complementation of the pex16-1 strain and encodes a protein, Pex16p, of 391 amino acids (44,479 D). Pex16p has no known homologues. Pex16p is a peripheral protein located at the matrix face of the peroxisomal membrane. Substitution of the carboxylterminal tripeptide Ser-Thr-Leu, which is similar to the consensus sequence of peroxisomal targeting signal 1, does not affect targeting of Pex16p to peroxisomes. Pex16p is synthesized in wild-type cells grown in glucose-containing media, and its levels are modestly increased by growth of cells in oleic acid–containing medium. Overexpression of the PEX16 gene in oleic acid– grown Y. lipolytica leads to the appearance of a small number of enlarged peroxisomes, which contain the normal complement of peroxisomal proteins at levels approaching those of wild-type peroxisomes.

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Cloning and Characterization of theFlavobacterium johnsoniae Gliding Motility GenesgldD and gldE

Journal of Bacteriology ◽

10.1128/jb.183.14.4167-4175.2001 ◽

2001 ◽

Vol 183 (14) ◽

pp. 4167-4175 ◽

Cited By ~ 39

Author(s):

David W. Hunnicutt ◽

Mark J. McBride

Keyword(s):

Membrane Protein ◽

Cytoplasmic Membrane ◽

Sequence Similarity ◽

Gliding Motility ◽

Wild Type ◽

Bacteriophage Infection ◽

Flavobacterium Johnsoniae ◽

Latex Spheres ◽

Serovar Typhimurium

ABSTRACT Cells of Flavobacterium johnsoniae move over surfaces by a process known as gliding motility. The mechanism of this form of motility is not known. Cells of F. johnsoniaepropel latex spheres along their surfaces, which is thought to be a manifestation of the motility machinery. Three of the genes that are required for F. johnsoniae gliding motility,gldA, gldB, and ftsX, have recently been described. Tn4351 mutagenesis was used to identify another gene, gldD, that is needed for gliding. Tn4351-induced gldD mutants formed nonspreading colonies, and cells failed to glide. They also lacked the ability to propel latex spheres and were resistant to bacteriophages that infect wild-type cells. Introduction of wild-type gldD into the mutants restored motility, ability to propel latex spheres, and sensitivity to bacteriophage infection. gldD codes for a cytoplasmic membrane protein that does not exhibit strong sequence similarity to proteins of known function. gldE, which lies immediately upstream ofgldD, encodes another cytoplasmic membrane protein that may be involved in gliding motility. Overexpression ofgldE partially suppressed the motility defects of agldB point mutant, suggesting that GldB and GldE may interact. GldE exhibits sequence similarity to Borrelia burgdorferi TlyC and Salmonella enterica serovar Typhimurium CorC.

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Metabolic control of peroxisome abundance

Journal of Cell Science ◽

10.1242/jcs.112.10.1579 ◽

1999 ◽

Vol 112 (10) ◽

pp. 1579-1590 ◽

Cited By ~ 6

Author(s):

C.C. Chang ◽

S. South ◽

D. Warren ◽

J. Jones ◽

A.B. Moser ◽

...

Keyword(s):

Metabolic Control ◽

Matrix Protein ◽

Protein Import ◽

Zellweger Syndrome ◽

Mutant Gene ◽

Peroxisomal Membrane ◽

Peroxisomal Targeting ◽

Targeting Signal ◽

Complementation Groups ◽

Peroxisomal Targeting Signal

Zellweger syndrome and related disorders represent a group of lethal, genetically heterogeneous diseases. These peroxisome biogenesis disorders (PBDs) are characterized by defective peroxisomal matrix protein import and comprise at least 10 complementation groups. The genes defective in seven of these groups and more than 90% of PBD patients are now known. Here we examine the distribution of peroxisomal membrane proteins in fibroblasts from PBD patients representing the seven complementation groups for which the mutant gene is known. Peroxisomes were detected in all PBD cells, indicating that the ability to form a minimal peroxisomal structure is not blocked in these mutants. We also observed that peroxisome abundance was reduced fivefold in PBD cells that are defective in the PEX1, PEX5, PEX12, PEX6, PEX10, and PEX2 genes. These cell lines all display a defect in the import of proteins with the type-1 peroxisomal targeting signal (PTS1). In contrast, peroxisome abundance was unaffected in cells that are mutated in PEX7 and are defective only in the import of proteins with the type-2 peroxisomal targeting signal. Interestingly, a fivefold reduction in peroxisome abundance was also observed for cells lacking either of two PTS1-targeted peroxisomal beta-oxidation enzymes, acyl-CoA oxidase and 2-enoyl-CoA hydratase/D-3-hydroxyacyl-CoA dehydrogenase. These results indicate that reduced peroxisome abundance in PBD cells may be caused by their inability to import these PTS1-containing enzymes. Furthermore, the fact that peroxisome abundance is influenced by peroxisomal 105-oxidation activities suggests that there may be metabolic control of peroxisome abundance.

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The peroxin Pex17p of the yeast Yarrowia lipolytica is associated peripherally with the peroxisomal membrane and is required for the import of a subset of matrix proteins.

Molecular and Cellular Biology ◽

10.1128/mcb.17.5.2511 ◽

1997 ◽

Vol 17 (5) ◽

pp. 2511-2520 ◽

Cited By ~ 30

Author(s):

J J Smith ◽

R K Szilard ◽

M Marelli ◽

R A Rachubinski

Keyword(s):

Amino Acids ◽

Oleic Acid ◽

Yarrowia Lipolytica ◽

Matrix Proteins ◽

Peroxisomal Membrane ◽

Peroxisomal Targeting ◽

Targeting Signal ◽

Carboxyl Terminal ◽

Peroxisomal Targeting Signal ◽

Yeast Yarrowia Lipolytica

PEX genes encode peroxins, which are required for the biogenesis of peroxisomes. The Yarrowia lipolytica PEX17 gene encodes the peroxin Pex17p, which is 671 amino acids in length and has a predicted molecular mass of 75,588 Da. Pex17p is peripherally associated with the peroxisomal membrane. The carboxyl-terminal tripeptide, Gly-Thr-Leu, of Pex17p is not necessary for its targeting to peroxisomes. Synthesis of Pex17p is low in cells grown in glucose-containing medium and increases after the cells are shifted to oleic acid-containing medium. Cells of the pex17-1 mutant, the original mutant strain, and the pex17-KA mutant, a strain in which most of the PEX17 gene is deleted, fail to form normal peroxisomes but instead contain numerous large, multimembraned structures. The import of peroxisomal matrix proteins in these mutants is selectively impaired. This selective import is not a function of the nature of the peroxisomal targeting signal. We suggest a regulatory role for Pex17p in the import of a subset of matrix proteins into peroxisomes.

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