A pex1 missense mutation improves peroxisome function in a subset of Arabidopsis pex6 mutants without restoring PEX5 recycling

Peroxisomes are eukaryotic organelles critical for plant and human development because they house essential metabolic functions, such as fatty acid β-oxidation. The interacting ATPases PEX1 and PEX6 contribute to peroxisome function by recycling PEX5, a cytosolic receptor needed to import proteins targeted to the peroxisomal matrix. Arabidopsis pex6 mutants exhibit low PEX5 levels and defects in peroxisomal matrix protein import, oil body utilization, peroxisomal metabolism, and seedling growth. These defects are hypothesized to stem from impaired PEX5 retrotranslocation leading to PEX5 polyubiquitination and consequent degradation of PEX5 via the proteasome or of the entire organelle via autophagy. We recovered a pex1 missense mutation in a screen for second-site suppressors that restore growth to the pex6-1 mutant. Surprisingly, this pex1-1 mutation ameliorated the metabolic and physiological defects of pex6-1 without restoring PEX5 levels. Similarly, preventing autophagy by introducing an atg7-null allele partially rescued pex6-1 physiological defects without restoring PEX5 levels. atg7 synergistically improved matrix protein import in pex1-1 pex6-1, implying that pex1-1 improves peroxisome function in pex6-1 without impeding autophagy of peroxisomes (i.e., pexophagy). pex1-1 differentially improved peroxisome function in various pex6 alleles but worsened the physiological and molecular defects of a pex26 mutant, which is defective in the tether anchoring the PEX1–PEX6 hexamer to the peroxisome. Our results support the hypothesis that, beyond PEX5 recycling, PEX1 and PEX6 have additional functions in peroxisome homeostasis and perhaps in oil body utilization.

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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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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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Mitochondrial Lon protease is a gatekeeper for proteins newly imported into the matrix

Communications Biology ◽

10.1038/s42003-021-02498-z ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Yuichi Matsushima ◽

Kazuya Takahashi ◽

Song Yue ◽

Yuki Fujiyoshi ◽

Hideaki Yoshioka ◽

...

Keyword(s):

Protease Activity ◽

Matrix Protein ◽

Protein Import ◽

Atp Hydrolysis ◽

Lon Protease ◽

Mitochondrial Matrix ◽

The Matrix ◽

Specific Proteins ◽

Aggregation Activity ◽

Hydrolysis Activity

AbstractHuman ATP-dependent Lon protease (LONP1) forms homohexameric, ring-shaped complexes. Depletion of LONP1 causes aggregation of a broad range of proteins in the mitochondrial matrix and decreases the levels of their soluble forms. The ATP hydrolysis activity, but not protease activity, of LONP1 is critical for its chaperone-like anti-aggregation activity. LONP1 forms a complex with the import machinery and an incoming protein, and protein aggregation is linked with matrix protein import. LONP1 also contributes to the degradation of imported, aberrant, unprocessed proteins using its protease activity. Taken together, our results show that LONP1 functions as a gatekeeper for specific proteins imported into the mitochondrial matrix.

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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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Interacting proteins identified by genetic interactions: a missense mutation in alpha-tubulin fails to complement alleles of the testis-specific beta-tubulin gene of Drosophila melanogaster.

Molecular and Cellular Biology ◽

10.1128/mcb.9.3.875 ◽

1989 ◽

Vol 9 (3) ◽

pp. 875-884 ◽

Cited By ~ 24

Author(s):

T S Hays ◽

R Deuring ◽

B Robertson ◽

M Prout ◽

M T Fuller

Keyword(s):

Drosophila Melanogaster ◽

Missense Mutation ◽

Null Allele ◽

Genetic Interaction ◽

Structural Proteins ◽

Interacting Proteins ◽

Tubulin Gene ◽

Beta Tubulin ◽

Gene Encoding ◽

Alpha Tubulin

In this paper we demonstrate that failure to complement between mutations at separate loci can be used to identify genes that encode interacting structural proteins. A mutation (nc33) identified because it failed to complement mutant alleles of the gene encoding the testis-specific beta 2-tubulin of Drosophila melanogaster (B2t) did not map to the B2t locus. We show that this second-site noncomplementing mutation is a missense mutation in alpha-tubulin that results in substitution of methionine in place of valine at amino acid 177. Because alpha- and beta-tubulin form a heterodimer, our results suggest that the genetic interaction, failure to complement, is based on the structural interaction between the protein products of the two genes. Although the nc33 mutation failed to complement a null allele of B2t (B2tn), a deletion of the alpha-tubulin gene to which nc33 mapped complemented B2tn. Thus, the failure to complement appears to require the presence of the altered alpha-tubulin encoded by the nc33 allele, which may act as a structural poison when incorporated into either the tubulin heterodimer or microtubules.

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Interacting proteins identified by genetic interactions: a missense mutation in alpha-tubulin fails to complement alleles of the testis-specific beta-tubulin gene of Drosophila melanogaster

Molecular and Cellular Biology ◽

10.1128/mcb.9.3.875-884.1989 ◽

1989 ◽

Vol 9 (3) ◽

pp. 875-884

Author(s):

T S Hays ◽

R Deuring ◽

B Robertson ◽

M Prout ◽

M T Fuller

Keyword(s):

Drosophila Melanogaster ◽

Missense Mutation ◽

Null Allele ◽

Genetic Interaction ◽

Structural Proteins ◽

Interacting Proteins ◽

Tubulin Gene ◽

Beta Tubulin ◽

Gene Encoding ◽

Alpha Tubulin

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A Mechanistic Perspective on PEX1 and PEX6, Two AAA+ Proteins of the Peroxisomal Protein Import Machinery

International Journal of Molecular Sciences ◽

10.3390/ijms20215246 ◽

2019 ◽

Vol 20 (21) ◽

pp. 5246 ◽

Cited By ~ 1

Author(s):

Ana G. Pedrosa ◽

Tânia Francisco ◽

Maria J. Ferreira ◽

Tony A. Rodrigues ◽

Aurora Barros-Barbosa ◽

...

Keyword(s):

Self Assembly ◽

Driving Force ◽

Matrix Protein ◽

Protein Import ◽

Atp Hydrolysis ◽

Transport Proteins ◽

Gtp Hydrolysis ◽

Assembly Mechanism ◽

Dependent Protein ◽

Organelle Membrane

In contrast to many protein translocases that use ATP or GTP hydrolysis as the driving force to transport proteins across biological membranes, the peroxisomal matrix protein import machinery relies on a regulated self-assembly mechanism for this purpose and uses ATP hydrolysis only to reset its components. The ATP-dependent protein complex in charge of resetting this machinery—the Receptor Export Module (REM)—comprises two members of the “ATPases Associated with diverse cellular Activities” (AAA+) family, PEX1 and PEX6, and a membrane protein that anchors the ATPases to the organelle membrane. In recent years, a large amount of data on the structure/function of the REM complex has become available. Here, we discuss the main findings and their mechanistic implications.

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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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