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

We isolated a Pichia pastoris mutant that was unable to grow on the peroxisome-requiring media, methanol and oleate. Cloning the gene by complementation revealed that the encoded protein, Pex22p, is a new peroxin. A Δpex22 strain does not grow on methanol or oleate and is unable to import peroxisomal matrix proteins. However, this strain targets peroxisomal membrane proteins to membranes, most likely peroxisomal remnants, detectable by fluorescence and electron microscopy. Pex22p, composed of 187 amino acids, is an integral peroxisomal membrane protein with its NH2 terminus in the matrix and its COOH terminus in the cytosol. It contains a 25–amino acid peroxisome membrane-targeting signal at its NH2 terminus. Pex22p interacts with the ubiquitin-conjugating enzyme Pex4p, a peripheral peroxisomal membrane protein, in vivo, and in a yeast two-hybrid experiment. Pex22p is required for the peroxisomal localization of Pex4p and in strains lacking Pex22p, the Pex4p is cytosolic and unstable. Therefore, Pex22p anchors Pex4p at the peroxisomal membrane. Strains that do not express Pex4p or Pex22p have similar phenotypes and lack Pex5p, suggesting that Pex4p and Pex22p act at the same step in peroxisome biogenesis. The Saccharomyces cerevisiae hypothetical protein, Yaf5p, is the functional homologue of P. pastoris Pex22p.

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The AAA peroxins Pex1p and Pex6p function as dislocases for the ubiquitinated peroxisomal import receptor Pex5p

Biochemical Society Transactions ◽

10.1042/bst0360099 ◽

2008 ◽

Vol 36 (1) ◽

pp. 99-104 ◽

Cited By ~ 31

Author(s):

Harald W. Platta ◽

Mykhaylo O. Debelyy ◽

Fouzi El Magraoui ◽

Ralf Erdmann

Keyword(s):

Matrix Protein ◽

Protein Import ◽

Matrix Proteins ◽

Genetic Dissection ◽

Cellular Functions ◽

Peroxisomal Membrane ◽

Aaa Atpase ◽

Ubiquitin Conjugating Enzyme ◽

Core Components ◽

Import Receptor

The discovery of the peroxisomal ATPase Pex1p triggered the beginning of the research on AAA (ATPase associated with various cellular activities) proteins and the genetic dissection of peroxisome biogenesis. Peroxisomes are virtually ubiquitous organelles, which are connected to diverse cellular functions. The highly diverse and adaptive character of peroxisomes is accomplished by modulation of their enzyme content, which is mediated by dynamically operating protein-import machineries. The import of matrix proteins into the peroxisomal lumen has been described as the ATP-consuming step, but the corresponding reaction, as well as the ATPase responsible, had been obscure for nearly 15 years. Recent work using yeast and human fibroblast cells has identified the peroxisomal AAA proteins Pex1p and Pex6p as mechano-enzymes and core components of a complex which dislocates the cycling import receptor Pex5p from the peroxisomal membrane back to the cytosol. This AAA-mediated process is regulated by the ubiquitination status of the receptor. Pex4p [Ubc10p (ubiquitin-conjugating enzyme 10)]-catalysed mono-ubiquitination of Pex5p primes the receptor for recycling, thereby enabling further rounds of matrix protein import, whereas Ubc4p-catalysed polyubiquitination targets Pex5p to proteasomal degradation.

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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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Pex24 and Pex32 are required to tether peroxisomes to the ER for organelle biogenesis, positioning and segregation in yeast

Journal of Cell Science ◽

10.1242/jcs.246983 ◽

2020 ◽

Vol 133 (16) ◽

pp. jcs246983 ◽

Cited By ~ 3

Author(s):

Fei Wu ◽

Rinse de Boer ◽

Arjen M. Krikken ◽

Arman Akşit ◽

Nicola Bordin ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Protein ◽

Matrix Protein ◽

Protein Import ◽

Hansenula Polymorpha ◽

Major Effect ◽

Organelle Biogenesis ◽

Peroxisomal Membrane ◽

Contact Sites ◽

Membrane Growth

ABSTRACTThe yeast Hansenula polymorpha contains four members of the Pex23 family of peroxins, which characteristically contain a DysF domain. Here we show that all four H. polymorpha Pex23 family proteins localize to the endoplasmic reticulum (ER). Pex24 and Pex32, but not Pex23 and Pex29, predominantly accumulate at peroxisome–ER contacts. Upon deletion of PEX24 or PEX32 – and to a much lesser extent, of PEX23 or PEX29 – peroxisome–ER contacts are lost, concomitant with defects in peroxisomal matrix protein import, membrane growth, and organelle proliferation, positioning and segregation. These defects are suppressed by the introduction of an artificial peroxisome–ER tether, indicating that Pex24 and Pex32 contribute to tethering of peroxisomes to the ER. Accumulation of Pex32 at these contact sites is lost in cells lacking the peroxisomal membrane protein Pex11, in conjunction with disruption of the contacts. This indicates that Pex11 contributes to Pex32-dependent peroxisome–ER contact formation. The absence of Pex32 has no major effect on pre-peroxisomal vesicles that occur in pex3 atg1 deletion cells.

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Pex5p stabilizes Pex14p: a study using a newly isolated pex5 CHO cell mutant, ZPEG101

Biochemical Journal ◽

10.1042/bj20120911 ◽

2012 ◽

Vol 449 (1) ◽

pp. 195-207 ◽

Cited By ~ 10

Author(s):

Ryuichi Natsuyama ◽

Kanji Okumoto ◽

Yukio Fujiki

Keyword(s):

Matrix Protein ◽

Protein Import ◽

Chinese Hamster ◽

Ovary Cell ◽

Cell Mutant ◽

Peroxisomal Membrane ◽

Multiple Binding Sites ◽

Multiple Binding ◽

Signal Type ◽

Mutant Phenotypes

Pex5p [PTS (peroxisome-targeting signal) type 1 receptor] plays an essential role in peroxisomal matrix protein import. In the present study, we isolated a novel PEX5-deficient CHO (Chinese-hamster ovary) cell mutant, termed ZPEG101, showing typical peroxisomal import defects of both PTS1 and PTS2 proteins. ZPEG101 is distinct from other known pex5 CHO mutants in its Pex5p expression. An undetectable level of Pex5p in ZPEG101 results in unstable Pex14p, which is due to inefficient translocation to the peroxisomal membrane. All of the mutant phenotypes of ZPEG101 are restored by expression of wild-type Pex5pL, a longer form of Pex5p, suggesting a role for Pex5p in sustaining the levels of Pex14p in addition to peroxisomal matrix protein import. Complementation analysis using various Pex5p mutants revealed that in the seven pentapeptide WXXXF/Y motifs in Pex5pL, known as the multiple binding sites for Pex14p, the fifth motif is an auxiliary binding site for Pex14p and is required for Pex14p stability. Furthermore, we found that Pex5p–Pex13p interaction is essential for the import of PTS1 proteins as well as catalase, but not for that of PTS2 proteins. Therefore ZPEG101 with no Pex5p would be a useful tool for investigating Pex5p function and delineating the mechanisms underlying peroxisomal matrix protein import.

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PEX3 functions as a PEX19 docking factor in the import of class I peroxisomal membrane proteins

The Journal of Cell Biology ◽

10.1083/jcb.200311131 ◽

2004 ◽

Vol 164 (6) ◽

pp. 863-875 ◽

Cited By ~ 154

Author(s):

Yi Fang ◽

James C. Morrell ◽

Jacob M. Jones ◽

Stephen J. Gould

Keyword(s):

Membrane Proteins ◽

Matrix Protein ◽

Protein Import ◽

Class Ii ◽

Class I ◽

Peroxisomal Membrane ◽

Direct Role ◽

Conserved Motif ◽

Peroxisome Membrane ◽

Import Receptor

PEX19 is a chaperone and import receptor for newly synthesized, class I peroxisomal membrane proteins (PMPs). PEX19 binds these PMPs in the cytoplasm and delivers them to the peroxisome for subsequent insertion into the peroxisome membrane, indicating that there may be a PEX19 docking factor in the peroxisome membrane. Here we show that PEX3 is required for PEX19 to dock at peroxisomes, interacts specifically with the docking domain of PEX19, and is required for recruitment of the PEX19 docking domain to peroxisomes. PEX3 is also sufficient to dock PEX19 at heterologous organelles and binds PEX19 via a conserved motif that is essential for this docking activity and for PEX3 function in general. Not surprisingly, transient inhibition of PEX3 abrogates class I PMP import but has no effect on class II PMP import or peroxisomal matrix protein import. Taken together, these results suggest that PEX3 plays a selective, essential, and direct role in PMP import as a docking factor for PEX19.

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Pex19 Binds Multiple Peroxisomal Membrane Proteins, Is Predominantly Cytoplasmic, and Is Required for Peroxisome Membrane Synthesis

The Journal of Cell Biology ◽

10.1083/jcb.148.5.931 ◽

2000 ◽

Vol 148 (5) ◽

pp. 931-944 ◽

Cited By ~ 208

Author(s):

Katherine A. Sacksteder ◽

Jacob M. Jones ◽

Sarah T. South ◽

Xiaoling Li ◽

Yifei Liu ◽

...

Keyword(s):

Steady State ◽

Membrane Proteins ◽

Broad Spectrum ◽

Matrix Protein ◽

Protein Import ◽

Nuclear Accumulation ◽

Eukaryotic Cells ◽

Membrane Synthesis ◽

Peroxisomal Membrane ◽

Peroxisome Membrane

Peroxisomes are components of virtually all eukaryotic cells. While much is known about peroxisomal matrix protein import, our understanding of how peroxisomal membrane proteins (PMPs) are targeted and inserted into the peroxisome membrane is extremely limited. Here, we show that PEX19 binds a broad spectrum of PMPs, displays saturable PMP binding, and interacts with regions of PMPs required for their targeting to peroxisomes. Furthermore, mislocalization of PEX19 to the nucleus leads to nuclear accumulation of newly synthesized PMPs. At steady state, PEX19 is bimodally distributed between the cytoplasm and peroxisome, with most of the protein in the cytoplasm. We propose that PEX19 may bind newly synthesized PMPs and facilitate their insertion into the peroxisome membrane. This hypothesis is supported by the observation that the loss of PEX19 results in degradation of PMPs and/or mislocalization of PMPs to the mitochondrion.

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Reevaluation of the role of Pex1 and dynamin-related proteins in peroxisome membrane biogenesis

The Journal of Cell Biology ◽

10.1083/jcb.201412066 ◽

2015 ◽

Vol 211 (5) ◽

pp. 1041-1056 ◽

Cited By ~ 31

Author(s):

Alison M. Motley ◽

Paul C. Galvin ◽

Lakhan Ekal ◽

James M. Nuttall ◽

Ewald H. Hettema

Keyword(s):

Protein Delivery ◽

Matrix Protein ◽

Protein Import ◽

De Novo ◽

Primary Role ◽

Peroxisome Biogenesis ◽

Peroxisomal Membrane ◽

Related Proteins ◽

Peroxisome Membrane

A recent model for peroxisome biogenesis postulates that peroxisomes form de novo continuously in wild-type cells by heterotypic fusion of endoplasmic reticulum–derived vesicles containing distinct sets of peroxisomal membrane proteins. This model proposes a role in vesicle fusion for the Pex1/Pex6 complex, which has an established role in matrix protein import. The growth and division model proposes that peroxisomes derive from existing peroxisomes. We tested these models by reexamining the role of Pex1/Pex6 and dynamin-related proteins in peroxisome biogenesis. We found that induced depletion of Pex1 blocks the import of matrix proteins but does not affect membrane protein delivery to peroxisomes; markers for the previously reported distinct vesicles colocalize in pex1 and pex6 cells; peroxisomes undergo continued growth if fission is blocked. Our data are compatible with the established primary role of the Pex1/Pex6 complex in matrix protein import and show that peroxisomes in Saccharomyces cerevisiae multiply mainly by growth and division.

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Ubiquitination of the peroxisomal import receptor Pex5p

Biochemical Journal ◽

10.1042/bj20040572 ◽

2004 ◽

Vol 384 (1) ◽

pp. 37-45 ◽

Cited By ~ 116

Author(s):

Harald W. PLATTA ◽

Wolfgang GIRZALSKY ◽

Ralf ERDMANN

Keyword(s):

Protein Import ◽

Ring Finger ◽

Proteasomal Degradation ◽

Peroxisomal Membrane ◽

Peroxisomal Biogenesis ◽

Peroxisomal Targeting ◽

Ubiquitin Conjugating Enzyme ◽

Targeting Signal ◽

Import Receptor ◽

In Cells

Proteins harbouring a peroxisomal targeting signal of type 1 (PTS1) are recognized by the import receptor Pex5p in the cytosol which directs them to a docking and translocation complex at the peroxisomal membrane. We demonstrate the ubiquitination of Pex5p in cells lacking components of the peroxisomal AAA (ATPases associated with various cellular activities) or Pex4p–Pex22p complexes of the peroxisomal protein import machinery and in cells affected in proteasomal degradation. In cells lacking components of the Pex4p–Pex22p complex, mono-ubiquitinated Pex5p represents the major modification, while in cells lacking components of the AAA complex polyubiquitinated forms are most prominent. Ubiquitination of Pex5p is shown to take place exclusively at the peroxisomal membrane after the docking step, and requires the presence of the RING-finger peroxin Pex10p. Mono- and poly-ubiquitination are demonstrated to depend on the ubiquitin-conjugating enzyme Ubc4p, suggesting that the ubiquitinated forms of Pex5p are targeted for proteasomal degradation. Accumulation of ubiquitinated Pex5p in proteasomal mutants demonstrates that the ubiquitination of Pex5p also takes place in strains which are not affected in peroxisomal biogenesis, indicating that the ubiquitination of Pex5p represents a genuine stage in the Pex5p receptor cycle.

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Peroxisomes exhibit compromised structure and matrix protein content in SARS-CoV-2-infected cells

Molecular Biology of the Cell ◽

10.1091/mbc.e21-02-0074 ◽

2021 ◽

pp. mbc.E21-02-0074

Author(s):

Barbara Knoblach ◽

Ray Ishida ◽

Tom C. Hobman ◽

Richard A. Rachubinski

Keyword(s):

Protein Content ◽

Matrix Protein ◽

Protein Import ◽

Structural Integrity ◽

Matrix Proteins ◽

Peroxisome Biogenesis ◽

Peroxisomal Membrane ◽

Antiviral Signaling ◽

Infected Cells ◽

Novel Coronavirus

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that has triggered global health and economic crises. Here we report the effects of SARS-CoV-2 infection on peroxisomes of human cell lines, Huh-7 and SK-N-SH. Peroxisomes undergo dramatic changes in morphology in SARS-CoV-2-infected cells. Rearrangement of peroxisomal membranes is followed by redistribution of peroxisomal matrix proteins to the cytosol, resulting in a dramatic decrease in the numbers of mature peroxisomes. The SARS-CoV-2 ORF14 protein was shown to interact physically with human PEX14, a peroxisomal membrane protein required for matrix protein import and peroxisome biogenesis. Given the important roles of peroxisomes in innate immunity, SARS-CoV-2 may directly target peroxisomes, resulting in loss of peroxisome structural integrity, matrix protein content and ability to function in antiviral signaling. [Media: see text]

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