Peroxisomal Ubiquitin-Protein Ligases Peroxin2 and Peroxin10 Have Distinct But Synergistic Roles in Matrix Protein Import and Peroxin5 Retrotranslocation in Arabidopsis    

Abstract

Peroxisomal matrix proteins carry peroxisomal targeting signals (PTSs), PTS1 or PTS2, and are imported into the organelle with the assistance of peroxin (PEX) proteins. From a microscopy-based screen to identify Arabidopsis (Arabidopsis thaliana) mutants defective in matrix protein degradation, we isolated unique mutations in PEX2 and PEX10, which encode ubiquitin-protein ligases anchored in the peroxisomal membrane. In yeast (Saccharomyces cerevisiae), PEX2, PEX10, and a third ligase, PEX12, ubiquitinate a peroxisome matrix protein receptor, PEX5, allowing the PEX1 and PEX6 ATP-hydrolyzing enzymes to retrotranslocate PEX5 out of the membrane after cargo delivery. We found that the pex2-1 and pex10-2 Arabidopsis mutants exhibited defects in peroxisomal physiology and matrix protein import. Moreover, the pex2-1 pex10-2 double mutant exhibited severely impaired growth and synergistic physiological defects, suggesting that PEX2 and PEX10 function cooperatively in the wild type. The pex2-1 lesion restored the unusually low PEX5 levels in the pex6-1 mutant, implicating PEX2 in PEX5 degradation when retrotranslocation is impaired. PEX5 overexpression altered pex10-2 but not pex2-1 defects, suggesting that PEX10 facilitates PEX5 retrotranslocation from the peroxisomal membrane. Although the pex2-1 pex10-2 double mutant displayed severe import defects of both PTS1 and PTS2 proteins into peroxisomes, both pex2-1 and pex10-2 single mutants exhibited clear import defects of PTS1 proteins but apparently normal PTS2 import. A similar PTS1-specific pattern was observed in the pex4-1 ubiquitin-conjugating enzyme mutant. Our results indicate that Arabidopsis PEX2 and PEX10 cooperate to support import of matrix proteins into plant peroxisomes and suggest that some PTS2 import can still occur when PEX5 retrotranslocation is slowed.

Genetic Interactions between PEROXIN12 and Other Peroxisome-Associated Ubiquitination Components

“…Mutants with defective peroxisomes that inefficiently metabolize fatty acids display reduced growth that can be partially ameliorated by providing Suc (Hayashi et al, 1998;Zolman et al, 2000Zolman et al, , 2001. As previously reported (Zolman and Bartel, 2004;Zolman et al, 2005;Burkhart et al, 2014), some mutants defective in peroxisomal ubiquitindependent machinery, pex4-1 and pex6-1, displayed growth defects without Suc whereas pex2-1 and pex10-2 resembled wild type in this assay (Fig. 2, A and B).…”

“…Because DEG15 is a PTS1 protein (Reumann, 2004) and because the two cargo receptors are interdependent in plants (Hayashi et al, 2005;Woodward and Bartel, 2005;Ramón and Bartel, 2010), robust PTS2 processing requires both PTS1 and PTS2 import. Unlike wild-type seedlings, which fully process thiolase and peroxisomal malate dehydrogenase (PMDH), mutants defective in components of the peroxisomal ubiquitin-dependent machinery display various degrees of impaired processing (Zolman et al, 2005;Mano et al, 2006;Ratzel et al, 2011;Burkhart et al, 2014); pex2-1 and pex6-1 accumulated high levels of PMDH precursor and detectable thiolase precursor whereas pex10-2 and pex4-1 displayed minor defects in this assay (Fig. 3A).…”

“…In contrast, pex10-2 and pex4-1 roots were moderately IBA resistant, and pex2-1 showed only slight IBA resistance (Fig. 2, A and B; Zolman et al, 2005;Burkhart et al, 2014). IBA-derived IAA also stimulates lateral root formation in wild type (Zolman et al, 2000;.…”

“…We compared the phenotypes of pex12-1 to mutants defective in other peroxins implicated in recycling or degrading the PTS1 cargo receptor, PEX5. PEX12 is part of a peroxisomal membrane complex of three RINGfamily ubiquitin-protein ligases (PEX2, PEX10, PEX12; Fan et al, 2005;Mano et al, 2006;Kaur et al, 2013;Burkhart et al, 2014) that together with the PEX4 ubiquitin-conjugating enzyme are thought to be involved in the PEX5 ubiquitination that allows retrotranslocation by the PEX1 and PEX6 ATPases. Mutations in PEX2, PEX4, PEX6, PEX10, or PEX12 can cause peroxisomal defects ascribed to impaired PEX5 recycling and consequent defects in matrix protein import (Zolman and Bartel, 2004;Zolman et al, 2005;Mano et al, 2006;Burkhart et al, 2013).…”

Peroxisome Function, Biogenesis, and Dynamics in Plants

“…Expressing truncated RING peroxins without the C-terminal catalytic zincbinding RING domains (DZn) in wild type confers dominant-negative matrix protein import defects for PEX2-DZn and photorespiration defects attributed to decreased peroxisome-chloroplast interactions for PEX10-DZn (Prestele et al, 2010). RNAi lines targeting RING peroxin genes (Nito et al, 2007) and several viable RING peroxin mutants (Mano et al, 2006;Burkhart et al, 2014;Kao et al, 2016) show typical peroxisomal defects, including impaired b-oxidation and matrix protein import. Moreover, PTS1 and PTS2 receptor levels are increased in RING peroxin mutants (Kao et al, 2016), and PEX5 is excessively membrane-associated in a pex12 mutant (Mano et al, 2006), suggesting that the RING peroxins facilitate PEX5 and PEX7 retrotranslocation.…”