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

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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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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Autophagy Inhibitors Do Not Restore Peroxisomal Functions in Cells With the Most Common Peroxisome Biogenesis Defect

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.661298 ◽

2021 ◽

Vol 9 ◽

Author(s):

Femke C. C. Klouwer ◽

Kim D. Falkenberg ◽

Rob Ofman ◽

Janet Koster ◽

Démi van Gent ◽

...

Keyword(s):

Matrix Protein ◽

Protein Import ◽

Therapeutic Option ◽

Cell Types ◽

Matrix Proteins ◽

Peroxisome Biogenesis ◽

Metabolic Functions ◽

Minimal Improvement ◽

Different Cell Types ◽

Autophagy Inhibition

Peroxisome biogenesis disorders within the Zellweger spectrum (PBD-ZSDs) are most frequently associated with the c.2528G>A (p.G843D) mutation in the PEX1 gene (PEX1-G843D), which results in impaired import of peroxisomal matrix proteins and, consequently, defective peroxisomal functions. A recent study suggested that treatment with autophagy inhibitors, in particular hydroxychloroquine, would be a potential therapeutic option for PBD-ZSD patients carrying the PEX1-G843D mutation. Here, we studied whether autophagy inhibition by chloroquine, hydroxychloroquine and 3-methyladenine indeed can improve peroxisomal functions in four different cell types with the PEX1-G843D mutation, including primary patient cells. Furthermore, we studied whether autophagy inhibition may be the mechanism underlying the previously reported improvement of peroxisomal functions by L-arginine in PEX1-G843D cells. In contrast to L-arginine, we observed no improvement but a worsening of peroxisomal metabolic functions and peroxisomal matrix protein import by the autophagy inhibitors, while genetic knock-down of ATG5 and NBR1 in primary patient cells resulted in only a minimal improvement. Our results do not support the use of autophagy inhibitors as potential treatment for PBD-ZSD patients, whereas L-arginine remains a therapeutically promising compound.

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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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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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Yeast pex1 cells contain peroxisomal ghosts that import matrix proteins upon reintroduction of Pex1

The Journal of Cell Biology ◽

10.1083/jcb.201506059 ◽

2015 ◽

Vol 211 (5) ◽

pp. 955-962 ◽

Cited By ~ 21

Author(s):

Kèvin Knoops ◽

Rinse de Boer ◽

Anita Kram ◽

Ida J. van der Klei

Keyword(s):

Cross Sections ◽

Matrix Protein ◽

Protein Import ◽

Electron Tomography ◽

High Resolution Electron Microscopy ◽

Matrix Proteins ◽

Peroxisomal Membrane ◽

Resolution Electron ◽

Microscopy Techniques ◽

Aaa Atpases

Pex1 and Pex6 are two AAA-ATPases that play a crucial role in peroxisome biogenesis. We have characterized the ultrastructure of the Saccharomyces cerevisiae peroxisome-deficient mutants pex1 and pex6 by various high-resolution electron microscopy techniques. We observed that the cells contained peroxisomal membrane remnants, which in ultrathin cross sections generally appeared as double membrane rings. Electron tomography revealed that these structures consisted of one continuous membrane, representing an empty, flattened vesicle, which folds into a cup shape. Immunocytochemistry revealed that these structures lack peroxisomal matrix proteins but are the sole sites of the major peroxisomal membrane proteins Pex2, Pex10, Pex11, Pex13, and Pex14. Upon reintroduction of Pex1 in Pex1-deficient cells, these peroxisomal membrane remnants (ghosts) rapidly incorporated peroxisomal matrix proteins and developed into peroxisomes. Our data support earlier views that Pex1 and Pex6 play a role in peroxisomal matrix protein import.

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Peroxisome Synthesis in the Absence of Preexisting Peroxisomes

The Journal of Cell Biology ◽

10.1083/jcb.144.2.255 ◽

1999 ◽

Vol 144 (2) ◽

pp. 255-266 ◽

Cited By ~ 173

Author(s):

Sarah T. South ◽

Stephen J. Gould

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Matrix Protein ◽

Protein Import ◽

Zellweger Syndrome ◽

Matrix Proteins ◽

Peroxisomal Membrane ◽

Peroxisomal Membrane Protein ◽

The Matrix ◽

Inactivating Mutations

Zellweger syndrome and related diseases are caused by defective import of peroxisomal matrix proteins. In all previously reported Zellweger syndrome cell lines the defect could be assigned to the matrix protein import pathway since peroxisome membranes were present, and import of integral peroxisomal membrane proteins was normal. However, we report here a Zellweger syndrome patient (PBD061) with an unusual cellular phenotype, an inability to import peroxisomal membrane proteins. We also identified human PEX16, a novel integral peroxisomal membrane protein, and found that PBD061 had inactivating mutations in the PEX16 gene. Previous studies have suggested that peroxisomes arise from preexisting peroxisomes but we find that expression of PEX16 restores the formation of new peroxisomes in PBD061 cells. Peroxisome synthesis and peroxisomal membrane protein import could be detected within 2–3 h of PEX16 injection and was followed by matrix protein import. These results demonstrate that peroxisomes do not necessarily arise from division of preexisting peroxisomes. We propose that peroxisomes may form by either of two pathways: one that involves PEX11-mediated division of preexisting peroxisomes, and another that involves PEX16-mediated formation of peroxisomes in the absence of preexisting peroxisomes.

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Two Independent Pathways Traffic the Intraperoxisomal Peroxin PpPex8p into Peroxisomes: Mechanism and Evolutionary Implications

Molecular Biology of the Cell ◽

10.1091/mbc.e05-08-0758 ◽

2006 ◽

Vol 17 (2) ◽

pp. 690-699 ◽

Cited By ~ 38

Author(s):

Lan Zhang ◽

Sébastien Léon ◽

Suresh Subramani

Keyword(s):

Matrix Protein ◽

Protein Import ◽

Alternative Pathway ◽

Direct Interaction ◽

Accessory Protein ◽

Peroxisome Biogenesis ◽

Peroxisomal Membrane ◽

Independent Manner ◽

The Matrix ◽

Shed Light

Among peroxins involved in peroxisome biogenesis, only Pex8p is predominantly intraperoxisomal at steady state. Pex8p is necessary for peroxisomal matrix protein import via the PTS1 and PTS2 pathways. It is proposed to bridge two peroxisomal membrane subcomplexes comprised of the docking (Pex13p, Pex14p, Pex17p) and RING (Pex2p, Pex10p, Pex12p) peroxins and is also implicated in cargo release of PTS1 proteins in the matrix. We show that Pichia pastoris Pex8p (PpPex8p) enters the peroxisome matrix using two redundant pathways in a Pex14p-dependent, but Pex2p-independent, manner, showing that the intact importomer and RING subcomplex are not required for its import. One pathway depends on the TPR motifs in Pex5p, the C-terminal PTS1 sequence (AKL) in PpPex8p, and the intraperoxisomal presence of this peroxin. The alternative pathway uses the PTS2 receptor, Pex7p, its accessory protein, Pex20p, and a putative PTS2 motif in PpPex8p, but does not require intraperoxisomal PpPex8p. Pex20p interaction with PpPex8p is independent of Pex7p, but the interaction of PpPex8p with Pex7p requires Pex20p. These data suggest a direct interaction between PpPex8p and Pex20p. Our studies shed light on the mechanism and evolution of the dual import pathways for PpPex8p.

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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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