Pexophagy and peroxisomal protein turnover in plants

Abstract

Peroxisomes are dynamic, vital organelles that sequester a variety of oxidative reactions and their toxic byproducts from the remainder of the cell. The oxidative nature of peroxisomal metabolism predisposes the organelle to self-inflicted damage, highlighting the need for a mechanism to dispose of damaged peroxisomes. In addition, the metabolic requirements of plant peroxisomes change during development, and obsolete peroxisomal proteins are degraded. Although pexophagy, the selective autophagy of peroxisomes, is an obvious mechanism for executing such degradation, pexophagy has only recently been described in plants. Several recent studies in the reference plant Arabidopsis thaliana implicate pexophagy in the turnover of peroxisomal proteins, both for quality control and during functional transitions of peroxisomal content. In this review, we describe our current understanding of the occurrence, roles, and mechanisms of pexophagy in plants.

Keywords: Autophagy; LON protease; Organelle quality control; Peroxisome; Pexophagy; Protein degradation.

Fig. 1. Working model for Arabidopsis peroxisome biogenesis and destruction via pexophagy

A) Peroxisomes can be generated de novo from the ER. PEX3, PEX16, and PEX19 facilitate insertion of peroxisomal membrane proteins (PMPs) into membranes, which is necessary for budding of pre-peroxisomes from the ER. B) Peroxisome matrix proteins are imported using a suite of peroxins (numbered ovals). Proteins bearing either a C-terminal peroxisomal-targeting sequence 1 (PTS1) or an N-terminal PTS2 bind to PEX5 or PEX7, respectively. The resulting complex docks with PEX13 and PEX14, allowing cargo import. Following import, membrane-associated PEX5 is ubiquitinated via the PEX4 ubiquitin (Ub)-conjugating enzyme and the complex of Ub-protein ligases (PEX2, PEX10, and PEX12). Ubiquitinated PEX5 is removed from the peroxisomal membrane by a heterohexameric AAA-ATPase complex of PEX1 and PEX6, and de-ubiquitinated PEX5 can facilitate further rounds of import. Alternatively, polyubiquitinated PEX5 undergoes proteasomal degradation. Inside the peroxisome, the protease DEG15 removes the N-terminal PTS2 region of PTS2 proteins, and the protease LON2 is positioned to degrade obsolete or damaged matrix proteins (marked with a white asterisk). C) During early stages of autophagy, a double membrane referred to as an isolation membrane forms. The ubiquitin-like protein ATG8 is conjugated to phosphatidylethanolamine, allowing its localization to the isolation membrane. D) During pexophagy, condemned peroxisomes are selectively recruited to expanding isolation membranes. A selective autophagy receptor (brown ovals) is postulated to bind to the peroxisome and to ATG8, connecting the condemned organelle to the autophagy machinery. The signal on plant peroxisomes that is recognized by the selective autophagy receptor to mark the peroxisome for degradation has not been identified, but candidates include ubiquitinated proteins, such as PEX5 or a matrix protein; PMPs, such as PEX3 and PEX14; and oxidized or aggregated matrix proteins (marked with white X). E) The isolation membrane completely encloses its cargo, forming an autophagosome. F) The autophagosome merges with the vacuole, forming an autophagic body, and the contents of the autophagic body are degraded into their constitutive nutrients (e.g., amino acids and lipids) and released into the cytosol for reuse.

Fig. 2. The peroxisomal protease LON2 inhibits pexophagy in Arabidopsis

A) Preventing autophagy suppresses protein import defects and the large puncta phenotype of lon2-2. Cotyledon mesophyll cells in 8-d-old seedlings of the indicated genotypes expressing 35S:PTS2-GFP [81] were imaged for fluorescence using confocal microscopy. PTS2-GFP bears an N-terminal PTS2 that localizes GFP (green) to peroxisomes in wild-type Columbia-0 (Wt), atg2-4 and lon2-2 atg2-3. PTS2-GFP is partially cytosolic (diffuse fluorescence) in lon2-2. Peroxisomes are enlarged in lon2-2 and clustered in atg2-4 and lon2-2 atg2-3. Chlorophyll autofluorescence (magenta) marks chloroplasts. Scale bar = 10 μm. B) Preventing autophagy suppresses the IBA resistance of lon2-2. Wild-type Columbia-0 (Wt), lon2-2 [38], atg2-4 [28], and lon2-2 atg2-3 [28] seeds were plated on medium containing 0.5% sucrose (mock) and grown for 4 days. Half of the seedlings were then transferred to medium supplemented with 0.5% sucrose and 10 μM IBA, and seedlings were grown for an additional 4 days before measuring lateral root formation. Error bars represent standard deviation of the means (n ≥ 10). C) Preventing autophagy suppresses the PTS2-processing defect of lon2-2, and obsolete matrix proteins are stabilized in lon2-2 atg2-3 double mutants. Extracts from 6-day-old light-grown seedlings of the indicated genotypes were processed for immunoblotting in duplicate, and membranes were serially probed with antibodies recognizing the peroxisome matrix proteins thiolase [13] or malate dehydrogenase [PMDH; 82], shown in the upper panel, or malate synthase [MLS; 83], shown in the lower panel. Thiolase and PMDH are PTS2 proteins synthesized as precursors (p) in the cytosol and cleaved to mature forms (m) lacking the PTS2 region in the peroxisome. Protein loading was monitored by probing with an antibody recognizing HSC70 (SPA-817; StressGen Bioreagents).