Novel proteins interacting with peroxisomal protein receptor PEX7 in Arabidopsis thaliana

Abstract

Peroxisomal matrix protein transport relies on 2 cytosolic receptors, PEX5 and PEX7, which import peroxisomal targeting signal type 1 (PTS1) and PTS2-containing proteins, respectively. To better understand the transport mechanism of PEX7, we isolated PEX7 complexes using proteomics. We identified PEX5 as well as PTS1- and PTS2-containing proteins within the complex, thereby confirming the interaction between PEX5 and PEX7 during cargo transport that had been previously characterized by biochemical approaches. In addition, a chaperone T-complex and 2 small Rab GTPases were identified. We recently reported that the RabE1c is involved in the degradation of the PEX7 when abnormal PEX7 is accumulated on the peroxisomal membrane. This study expands our knowledge on the transport machinery via PEX7 by identifying both known and novel PEX7-interacting proteins and thus is helpful for further investigation of the regulation of the peroxisomal protein receptor during its translocation.

Most peroxisomal matrix proteins engaged in the biological processes of peroxisomes contain either PTS1 at their C-terminus or PTS2 near their N-terminus for proper peroxisome targeting.¹ PEX5 and PEX7 are peroxisomal receptors that direct PTS1- and PTS2-containing proteins into peroxisomes, respectively.² Abnormalities in peroxisomal protein transport are associated with Zellweger syndrome, a severe neurological disease in humans, and seed germination arrest in plants.^3,4 In mammalian and plant cells, PEX7-mediated PTS2 protein import is dependent on PEX5 through direct interaction between 2 receptors.^2,5,6 To date, the proteins that directly regulate PEX7 function and translocation behavior, especially during its dislocation, are largely unknown. Therefore, we identified the interacting proteins of PEX7 by isolating GFP-PEX7 protein complexes through immunoprecipitation with an anti-GFP antibody in Arabidopsis.⁷

We conducted 2 immunoprecipitation experiments for the isolation of GFP-PEX7 protein complexes using a homogenization buffer with 2 different types of detergent (DDM or digitonin, respectively) in order to ensure a high probability of identifying unique binding candidates. Furthermore, the proteins with a calculated protein score of lower than 50 after MS/MS and Mascot software analysis were excluded. As a result, a total of 121 proteins were identified, including PEX5, 2 small Rab GTPases, and T-complexes (Table S1). We noticed 1 PTS2-containing protein, 6 PTS1-containing proteins, and 1 protein with both PTS1 and PTS2 signals identified in the complex (Table 1), although 3 of them had a protein score lower than 50. It should be noted that these excluded proteins did not appear in the negative control samples from the GFP-PTS1 and GFP-PEX16 immunoprecipitation experiments.⁷ Based on the fact that PEX5 and PEX7 are receptors for PTS1 and PTS2, respectively,² and that PEX5 and PEX7 can interact with each other in the cytosol,⁶ these results indeed show that peroxisomal receptors and cargo form a large complex in the cell.

Table 1. PTS1- or PTS2-containing proteins that were immunoprecipitated with the GFP-PEX7 protein.

AGI code	Annotation	Score in DDM	Score in Digitonin	type of PTS
AT1G71695.1	PER12 (peroxidase 12)	34	52	1
AT5G27600.1	LACS7 (long-chain acyl-CoA synthetase 7)	51	34	1,2
AT5G47040.1	LON2 (lon protease 2)	530	n.d.	1
AT3G14415.1	GOX2 (glycolate oxidase 2)	117	n.d.	1
AT1G54340.1	ICDH (isocitrate dehydrogenase)	64	n.d.	1
AT2G42490.1	copper amine oxidase	46	n.d.	1
AT5G09660.2	PMDH2 (peroxisomal NAD-malate dehydrogenase 2)	32	n.d.	2
AT1G65520.1	enoyl-CoA hydratase/isomerase family protein	23	n.d.	1

Arabidopsis Genome Initiative (AGI) codes and annotations were obtained from the TAIR database (http://www.arabidopsis.org). Scores were calculated by Mascot (Matrix Science). n.d. indicates not detected.

The T-complex (also called TRiC), which is a cytosolic chaperonin that is composed of 8 different subunits,⁸ was identified as a PEX7-interacting protein. Its mammalian and yeast counterparts were originally found to be involved in the correct folding of actin and tubulin,⁹ while more recent studies have shown that it actually has a broader range of substrate binding and folding capacity.¹⁰ The potential binding between T-complex and PEX7 is also supported by a previous report in a mammalian system showing that T-complex interacts with the WD40 domain,¹¹ which largely constitutes PEX7 within the peptides.¹² Because PEX5 also interacts with the WD40 domain of PEX7 for subsequent cargo import,¹³ it is tempting to hypothesize that T-complex and PEX5 may competitively bind to PEX7 in the cytosol, in which case T-complex may function as a negative regulator for the import of PTS2-containing proteins. Alternatively, T-complex may be simply involved in the correct folding of PEX7, which is also important for peroxisomal biogenesis. In addition, we cannot exclude the possibility that T-complex is required for the recycling of PEX7, during which the quality of the peroxisomal receptor is thought to be important for its recycling.¹⁴ Therefore, our finding suggests that chaperone may play a potential role in peroxisomal protein transport.

RabE1c, which is a small GTPase subfamily member, was also found to be involved in the dislocation of PEX7. Functional characterization of the small GTPase RabE1c showed that it facilitates the degradation of PEX7, rather than PEX7 recycling, when abnormal PEX7 accumulates on the peroxisomal membrane through a direct interaction with PEX7. This might be important during certain stress conditions in which peroxisomal receptors become damaged and accumulate on the peroxisomal membranes (Fig. 1). For example, the H₂O₂ produced inside peroxisomes during development or under certain stress conditions was shown to damage peroxisomal proteins and even those on the membrane of oil bodies when they were in close proximity to peroxisomes during germination of Arabidopsis seedlings.^15,16 Based on these findings, it is reasonable to expect that PEX5 and PEX7 as well as the transport machinery regulating them may also become damaged, resulting in their accumulation on peroxisomal membranes. Removal of abnormal PEX5 was shown to be important for the normal function of peroxisomes, as it provides space on the peroxisomal membrane for the import of newly synthesized PEX5 carrying PTS1-containing protein cargo.¹⁷ This kind of quality control system can also be applied to PEX7, where RabE1c is a key regulator for PEX7 degradation. More studies are needed to reveal the type of stresses, and in particular the damage to PEX7, which necessitate a RabE1c-mediated quality control system of the receptor for normal peroxisomal function and plant growth.

Figure 1. A model for the role of RabE1c during dislocation of PEX7 from the peroxisomal membrane. (A) In wild-type plants, PEX7 is recycled back to the cytosol for the next round of import after the PEX7 receptor-cargo (PTS2-containing protein) complex is imported into the peroxisomes. (B) Under certain stress conditions, such as in the presence of high amounts of H₂O₂ produced within peroxisomes, PEX7 may be damaged and thus accumulate on the peroxisomal membrane, which results in the degradation of PEX7 by its interacting partner RabE1c.

Glossary

Abbreviations:

PTS

peroxisomal targeting signal

DDM

n-dodecyl-D-maltoside

PEX

peroxin

Supplemental Materials

Supplemental materials may be found here: www.landesbioscience.com/journals/psb/article/26829