Different functions of the C ₃ HC ₄ 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 ΔZn-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-ΔZn-T7 grow like WT in normal atmosphere, contain normal-shaped peroxisomes, but display impaired peroxisomal matrix protein import. PEX12-ΔZn-T7 mutants exhibit unimpaired import of matrix protein and normalshaped 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-ΔZn but not in PEX2-ΔZn-T7 or PEX12-ΔZn-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.Thr Leu Gly Glu Glu Tyr motif in peroxin PEX10 | glyoxysome | metabolomics P eroxisomes perform multiple metabolic processes, including β-oxidation and H 2 O 2 inactivation by catalase. In plants, specialized peroxisomes, the glyoxysomes, contain glyoxylate cycle enzymes for lipid mobilization. Leaf peroxisomes interact with chloroplasts and mitochondria in photorespiration, a metabolic pathway turning two molecules of glycolate in a series of reactions through glyoxylate, glycine (Gly), serine (Ser), and hydroxypyruvate into CO 2 and phosphoglycerate (1-3). When CO 2 is limiting, ribulose-bisphosphate carboxylase/oxygenase functions as an oxygenase and protects the photosynthetic machinery from photodamage by using energy for photorespiration. Mutants lacking enzymes of the photorespiratory cycle are incapable of surviving in ambient air but are able to grow normally in a CO 2 -enriched atmosphere (2). When genes responsible for plant peroxisome biogenesis are impaired, the phenotypes are severe, ranging from sucrose dependency during early seedling development to embryo lethality (4). The biogenesis of peroxisomes relies on a common class of evolutionary conserved genes referred to as "PEX genes" (5, 6).The im...

Metabolic engineering of microorganisms to produce omega-3 very long-chain polyunsaturated fatty acids

“…DHA is an important component of membrane phospholipids 85 of the brain, retina and spermatozoa [14]. EPA can be converted to 86 eicosanoids, a group of biologically active chemicals including pros- 87 taglandin, thromboxane and leucotriene, which play crucial roles in 88 regulation of blood pressure and blood coagulation and participate in 89 many important physiological processes such as inflammatory and 90 immunological reactions [15]. For humans, the most common die- 91 tary intake of essential PUFAs is linoleic acid (LA, 18:2D ) and a-linolenic acid (ALA, 18:3D 9,12,15 ), the precursor 96 of x3-VLCPUFAs, to EPA and DHA.…”

Peroxisome Function, Biogenesis, and Dynamics in Plants

“…The Arabidopsis PEX2, PEX10, and PEX12 RING peroxins all display in vitro ubiquitin-protein ligase activity and are essential for embryogenesis (Hu et al, 2002;Schumann et al, 2003;Sparkes et al, 2003;Fan et al, 2005;Prestele et al, 2010). 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).…”

“…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.…”

Genetic Interactions between PEROXIN12 and Other Peroxisome-Associated Ubiquitination Components

“…Although RING peroxins interact with each other, they have distinct functions (Okumoto et al, 2000;Prestele et al, 2010;Burkhart et al, 2014). S. cerevisiae Pex12, assisted by the Pex4 ubiquitin-conjugating enzyme, is implicated in monoubiquitinating Pex5 to allow Pex5 recycling back to the cytosol for further rounds of cargo delivery (Platta et al, 2009).…”

“…Null mutations in the RING peroxin genes confer embryo lethality in Arabidopsis (Hu et al, 2002;Schumann et al, 2003;Sparkes et al, 2003;Fan et al, 2005;Prestele et al, 2010), necessitating other approaches to study the in vivo functions of these peroxins. Expressing RING peroxins with mutations in the C-terminal zinc-binding RING domains (DZn) confers matrix protein import defects for PEX2-DZn and photorespiration defects for PEX10-DZn but no apparent defects for PEX12-DZn (Prestele et al, 2010). Targeting individual RING peroxins using RNAi confers b-oxidation deficiencies and impairs PTS1 cargo import (Fan et al, 2005;Nito et al, 2007).…”