Different functions of the C3HC4 zinc RING finger peroxins PEX10, PEX2, and PEX12 in peroxisome formation and matrix protein import

Abstract

The integral peroxisomal membrane proteins PEX10, PEX2, and PEX12 contain a zinc RING finger close to the C terminus. Loss of function of these peroxins causes embryo lethality at the heart stage in Arabidopsis. Preventing the coordination of Zn(2+) ions by amino acid substitutions in PEX10, PEX2, and PEX12 and overexpressing the resulting conditional sublethal mutations in WT uncovered additional functions of PEX10. Plants overexpressing DeltaZn-mutant PEX10 display deformed peroxisomal shapes causing diminished contact with chloroplasts and possibly with mitochondria. These changes correlated with impaired metabolite transfer and, at high CO(2), recoverable defective photorespiration plus dwarfish phenotype. The N-terminal PEX10 domain is critical for peroxisome biogenesis and plant development. A point mutation in the highly conserved TLGEEY motif results in vermiform peroxisome shape without impairing organelle contact. Addition of an N-terminal T7 tag to WT PEX0 resulted in partially recoverable reduced growth and defective inflorescences persisting under high CO(2). In contrast, plants overexpressing PEX2-DeltaZn-T7 grow like WT in normal atmosphere, contain normal-shaped peroxisomes, but display impaired peroxisomal matrix protein import. PEX12-DeltaZn-T7 mutants exhibit unimpaired import of matrix protein and normal-shaped peroxisomes when grown in normal atmosphere. During seed germination, glyoxysomes form a reticulum around the lipid bodies for mobilization of storage oil. The formation of this glyoxysomal reticulum seemed to be impaired in PEX10-DeltaZn but not in PEX2-DeltaZn-T7 or PEX12-DeltaZn-T7 plants. Both cytosolic PEX10 domains seem essential for peroxisome structure but differ in metabolic function, suggesting a role for this plant peroxin in addition to the import of matrix protein via ubiquitination of PEX5.

Fig. 1.

Phenotype of PEX10, PEX2, and PEX12 mutants grown under 360 or 1,800 ppm CO_2. Plants are shown at an age of 28 d (A, D, G, M, and P) and at 46 d (B, C, E, F, H–L, N, O, and Q). (A, D, G, M) (Upper) 360 ppm CO₂. (Lower) 1,800 ppm CO₂. (B, E, H, I, N, P, Q) 360 ppm CO₂. (C, F, K, L, O) 1,800 ppm CO₂. (A–F and M-–Q) Control plants GFP-PTS1 WT (Left) and PEX10-ΔZn1 (Right). (A–C) Two PEX10-ΔZn1×GFP-PTS1 hybrids : normal growth only under high CO₂, defect in photorespiration. (D–F) Three PEX10-ΔZn-T7-GFP-PTS1 plants: dwarf phenotype under low and high CO₂. (G–L) PEX10-WT-T7-pex10-KO plants show dwarf phenotype under low and high CO₂. (G Upper and H) Two PEX10-WT-T7-pex10-KO plants (Left) and one WT plant (Right). (G Lower and K) PEX10-WT-T7-pex10-KO (Left), PEX10-ΔZn1 (Center), and WT (Right). (I and L) Mutants have smaller siliques and exhibit a nodding inflorescence in comparison to WT showing bigger siliques and an upright inflorescence. (M–O) One PEX2-ΔZn-T7-GFP-PTS1 and one PEX2-WT-T7-GFP-PTS1 plant grow normally under low and high CO₂ with no defect in photosynthetic yield. (P and Q) One WT plant, two PEX12-ΔZn-T7-GFP-PTS1, and one PEX12-WT-T7-GFP-PTS1 transgenic plants grow normally under low and high CO₂ with no defect in photosynthetic yield.

Fig. 2.

Peroxisome morphology and importation of matrix protein in PEX10-ΔZnxGFP-PTS1 hybrids and in PEX10-ΔZn-T7-GFP-PTS1, PEX2-ΔZn-T7-GFP-PTS1, and PEX12-ΔZn-T7-GFP-PTS1 transgenics visualized by GFP-PTS1. (Left) GFP signal from leaf peroxisomes in 14-d-old secondary leaves. (Center) GFP signal merged with chloroplast autofluorescence. (Right) GFP signal from glyoxysomal reticulum of 5-d-old dark-grown cotyledons. (A–C) GFP-PTS1 WT. (D–E) PEX10-ΔZn1xGFP-PTS1 hybrid. (G–I) PEX10-ΔZn-T7-GFP-PTS1 transgenic. (K–M) PEX2-ΔZn-T7-GFP-PTS1 transgenic. (N–P) PEX12-ΔZn-T7-GFP-PTS1 transgenic. (Scale bar, 10 μm.)

Fig. 3.

Focused ion beam (FIB) electron micrographs of 13-d-old leaf tissue of WT (Top row), PEX10-ΔZn1 (Middle row) and PEX10-G93E (Bottom row). The tissue was stained for catalase activity with diaminobenzidine. Images at successive levels of sections exposed by the ion beam are shown. (A–D) Leaf peroxisomes of WT plants are ovoid and in physical contact with chloroplasts. (E–H) Leaf peroxisomes of PEX10-ΔZn1 are worm-like and accumulate at places distant from chloroplasts. (I–M) Leaf peroxisomes of PEX10-G93E plants are elongated but are attached to chloroplasts. Mb, microbody (peroxisome); P, chloroplasts; V, vacuole. (Scale bar, 3 μm.)

Fig. 4.

Model of the photorespiration pathway with link to TCA cycle and glycolysis for the dysfunctional metabolic flux for PEX10-ΔZn1 mutants compared with WT plants. Identified significant reductions or accumulations are marked with red (reduced) or blue (accumulated) arrows. The localization of the measured metabolites is postulated because of the metabolic profiling performed without regard to compartmentalization of the cell.