A Framework to Investigate Peroxisomal Protein Phosphorylation in Arabidopsis

Review

A Framework to Investigate Peroxisomal Protein Phosphorylation in Arabidopsis Amr R.A. Kataya,1,2,3,*,@ Douglas G. Muench,2 and Greg B. Moorhead2 Peroxisomes perform essential roles in a range of cellular processes, highlighted by lipid metabolism, reactive species detoxification, and response to a variety of stimuli. The ability of peroxisomes to grow, divide, respond to changing cellular needs, interact with other organelles, and adjust their proteome as required, suggest that, like other organelles, their specialized roles are highly regulated. Similar to most other cellular processes, there is an emerging role for protein phosphorylation to regulate these events. In this review, we establish a knowledge framework of key players that control protein phosphorylation events in the plant peroxisome (i.e., the protein kinases and phosphatases), and highlight a vastly expanded set of (phospho)substrates. Insights into the Regulation of Peroxisome-Related Processes Peroxisomes (see Glossary) are single-membraned (one bilayer) organelles, first identified in 1966 by Christian de Duve, that exist in all eukaryotes. They were named upon recognition of their ability to dispose of H2O2. Peroxisomes are adaptable and have evolved specialized metabolic roles across eukaryotic species, including plant tissues. In plants they are best known for their role in photorespiration, the glyoxylate cycle, and as the sole site for fatty acid b-oxidation. Specialized roles in several plant tissues have led to the recognition of four peroxisome types in plants [1,2]. To perform their specialized functions, peroxisomes are known to cooperate with, and physically interact with, endoplasmic reticulum (ER), mitochondria, lysosomes, chloroplasts, and lipid droplets [3–6]. Due to the fragile nature of peroxisomes and their association with other organelles, it has been difficult to characterize their proteome by mass spectrometry regardless of the cellular condition. That said, several studies have successfully performed peroxisome proteomics and identified multiple new (low abundance) peroxisomal proteins [7–15], confirming and identifying potential new peroxisomal functions, pathways, and pathway regulators (Box 1). The methods and strategies used in peroxisomal proteome studies were reviewed by Bussell et al. [7]. However, it is difficult to interpret solely by proteomic studies whether the newly identified proteins are indeed peroxisomal or their identification resulted from cross-contamination during the organelle isolation steps. Bioinformatics and experimental validation have complemented proteomics and helped to verify the identity of many newly identified putative peroxisomal proteins. Using the knowledge of peroxisomal targeting signals (PTS) (Box 1), algorithms have been developed and used to predict many putative low-abundance peroxisomal proteins [16–18]. This approach has helped to identify several key players involved in peroxisomal protein phosphorylation, a major post-translational modification (PTM) that, current evidence suggests, is not excluded from this organelle [7,19–22]. It is important to recognize that many of the events that produce peroxisomes and allow them to adapt, remodel, regulate their metabolic events, and divide, occur outside the peroxisome and thus the protein kinases and phosphatases that regulate these events may reside in the cytosol. 366

Trends in Plant Science, April 2019, Vol. 24, No. 4 © 2018 Elsevier Ltd. All rights reserved.

https://doi.org/10.1016/j.tplants.2018.12.002

Highlights We have cataloged over 100 polypeptides as peroxisomal phospho-proteins using available phosphoproteomic datasets, fully establishing protein phosphorylation as a regulatory mechanism in peroxisomes. Numerous peroxisome-targeted protein phosphatases and kinases have been identified, establishing a framework of key players that control peroxisomal phosphorylation. Phospho-proteins have been identified as components of most peroxisomal processes, many functioning in the fatty acid b-oxidation pathway.

1 Department of Chemistry, Bioscience, and Environmental Engineering, University of Stavanger, Stavanger, 4036, Norway 2 Department of Biological Sciences, University of Calgary, Calgary, T2N 1N4, Canada 3 www.katayaproject.com @ Twitter: @AmrKataya1

*Correspondence: [email protected], [email protected] (A.R.A. Kataya).

Box 1. Peroxisome Functions, Biogenesis, and Protein Import Plant peroxisomes are involved in the biosynthesis of membrane phospholipids, fatty acid (FA) b-oxidation, photorespiration, and synthesis of auxins and vitamins, such as phylloquinone and biotin. Peroxisome roles also extend to catabolism of branched amino acids, polyamines, sulfur-containing compounds, and purines [61,66]. Peroxisomes function in responses to environmental stresses, consistent with their role in detoxification of reactive oxygen species (ROS), reactive nitrogen species, and reactive sulfur species (reviewed in [86,91]). All peroxisomal proteins are nuclear-encoded and assemble in the peroxisome with the aid of a group of proteins called peroxins (PEXs). Peroxisome biogenesis is initiated at the endoplasmic reticulum (ER), where insertion of a subset of peroxisomal membrane proteins (PMPs) by peroxins PEX3, PEX16, and PEX19 allow pre-peroxisomes to bud from ER subdomains (Group I PMPs). Insertion of additional PMPs (Group II PMPs) into the budded pre-peroxisome also utilizes PEX3, PEX16, and PEX19. Peroxisomal matrix proteins are synthesized in the cytosol and contain surface exposed import signals called peroxisomal targeting signals (PTS) type 1 (PTS1) and PTS type 2 (PTS2). PTS1 signals reside in the C terminal three amino acids of the protein and are designated with the ‘greater than’ symbol (>; see Table 1). The nine amino acid PTS2 signal sequences (R[L/I]X5HL) are near the N terminus of the protein and are cleaved in the peroxisomal matrix [17,92]. The PTS1- or PTS2-containing matrix proteins are recognized in the cytosol by soluble receptors PEX5 and PEX7, respectively, that guide them to a docking site at the peroxisome membrane, allowing the import of fully folded proteins. Several non-PTS peroxisomal proteins have been identified and were shown to enter via a piggyback mechanism [19,29]. Peroxisome number is controlled by division, a process that involves three steps: elongation, membrane constriction, and final fission via fission and dynamin-related proteins (Figure 2, reviewed in [61,93]).

In this review, we shed light on the identification of the peroxisomal phosphorylation key players: the protein kinases and protein phosphatases of the model plant Arabidopsis thaliana. We also compile a vast number of peroxisome-related, ‘experimentally detected’ phospho-proteins, that cover multiple peroxisomal processes.

An Inventory of Peroxisomal Protein Phosphatases and Kinases Reversible protein phosphorylation is known to control protein activity, protein–protein interactions, protein degradation, and subcellular protein targeting [23,24]. Protein kinases transfer a phosphoryl group from ATP predominately to Ser (S), Thr (T), and Tyr (Y) residues on proteins, a reaction that can be reversed by protein phosphatases by hydrolyzing the phosphoester bond. The number of predicted protein kinases and phosphatases in Arabidopsis is approximately 942 and 150, respectively [25,26]. Kinome phylogeny in Arabidopsis shows two major clades of 561 predicted membrane-bound receptor kinases and 381 soluble kinases [25]. Protein phosphatases are divided into four families: PPP (serine/threonine-specific phosphoprotein phosphatases), PPM/PP2C (Mg2+-dependent protein phosphatases), Asp-based protein phosphatases, and PTP (phospho-tyrosine phosphatases). The PPP family further divides into subgroups: PP1, PP2/PP2A, PP3/PP2B, PP4, PP5, PP6, PP7, PPKL/Kelch, and Shewanella-like phosphatase (SLP), RLPH, ALPH (reviewed in [24,27]). Peroxisomal Protein Phosphatases Three subunits [catalytic (C), scaffolding (A), regulatory (B)] form the PP2A heterotrimeric complex. Arabidopsis has five C, three A, and 17 B subunits that could form 255 possible PP2A holoenzyme combinations [28]. The B subunits are responsible for localization and substrate specificity and are classified into B, B0 , and B00 families. The B0 subunits that are most closely related to B0 h (i.e., B0 u, B0 g, and B0 z) were studied, as B0 h is targeted to various subcellular locations [22]. B0 u harbours a functional PTS1 (Box 1 and Table 1) and is localized to peroxisomes [22]. Because the PP2A C and A subunits lack clear PTSs (Table 1), it was important to provide evidence for the targeting of the PP2A heterotrimeric complex to peroxisomes. Kataya and coauthors investigated the interaction of B0 u with the C and A subunits and demonstrated interactions with either C2 or C5, and A2 [19]. Importantly, the trimer has been detected in peroxisomes, but remains cytosolic upon deletion of the PTS1 on the B0 u polypeptide. It was concluded that B0 u could carry its interacting partners into peroxisomes in a

Glossary Catalase: degrades H2O2 to water and oxygen. Glyoxysomes: specialized peroxisomes that predominate in germinating seeds and also perform fatty acid oxidation and the glyoxylate cycle, converting lipids to carbohydrate. LON2: major ATP-dependent protease of peroxisomes. MAP kinase phosphatase 1 (MKP1): dual specificity protein phosphatase that targets both the phospho-T and -Y in the MAP activation loop to inactive MAPKs. Peroxins (PEXs): proteins implicated in peroxisome de novo biogenesis, peroxisome proliferation, protein import, and pexophagy. Peroxisomal membrane proteins (PMPs): proteins that reside in the peroxisomal membrane. Peroxisomal targeting domain (PTD): the C terminal three amino acids of a protein that constitutes a PTS1, plus the preceeding seven amino acids (X7-PTS1). Peroxisomal targeting signal (PTS): a short amino acid motif that targets proteins to the peroxisome. Peroxisome remodeling: specific changes in the peroxisomal proteome to reflect current cellular needs through turnover and import of specific proteins. Peroxisome unusual positioning 1 and 2: mutants that showed accumulation of aggregated peroxisomes and represent ATG2 and ATG18a, respectively. Peroxisomes: named after their involvement in oxidative reactions that produce hydrogen peroxide (H2O2), which is degraded by catalase into molecular oxygen and water. Pexophagy: autophagy-dependent peroxisome degradation. POL-like phosphatases 2 and 3: PP2C type protein phosphatases that localize to the peroxisome. Post-translational modifications (PTMs): covalent modification of protein sidechains that confer specific properties to the protein (e. g., phosphorylation). PTS type 1 (PTS1): conserved tripeptide (e.g., SKL>) located at the protein C terminus that destines a protein to the peroxisome.

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process coined piggybacking [19,29]. PP2A has important functions in eukaryotes [27], and its role in fatty acid (FA) b-oxidation in plants was demonstrated when b0 u-1 and b0 u-2 mutant seedlings were found partially impaired in FA mobilization after seed germination [19]. This was the first report showing a role for a phospho-PTM on peroxisome metabolism. Additionally, a comparative phospho-proteomic study using b0 u-1 and wild type (WT) seedlings implicated 3ketoacyl-CoA thiolase 1 (KAT1) and long-chain acyl-CoA synthetase 5 (LACS5) as potential substrates for PP2A [19]. Multiple protein phosphatase-related proteins were identified as possessing a putative PTS by employing prediction algorithms for PTS identification [16] and by searching the Arabidopsis proteome for predicted PTS sequences [20,21]. Initially, seven new candidates were identified and a novel PTS1 was reported for MAP kinase phosphatase 1 (MKP1). In this study, eYFP was fused with the peroxisomal targeting domain (PTD) [20,21] from MKP1 and confirmed the ability of the fusion to target peroxisomes in two transient expression systems [20]. Interestingly, fusion of eYFP to the N terminus of full-length MKP1 appeared to change targeting from cytosol to peroxisomes under oxidative and salt stress [20]. Two PP2C family members, POL-like phosphatases 2 and 3 (PLL2 and PLL3), were also predicted to harbour similar putative minor PTS1 signals [30]. Full-length Arabidopsis PLL2 and PLL3 eYFP fusions targeted to peroxisomes, and in the case of PLL2, localized to the nucleus as well [21]. Homozygous mutants for PLL3, represented by the pll3-7 mutant allele, showed a mild developmental phenotype that was found to be sugar dependent, whereas PLL2 mutants lacked an obvious phenotype [21]. SLP1 is a PPP serine/threonine phosphatase (Table 1) previously identified in the proteome of isolated peroxisomes from Arabidopsis greening cotyledons [13,31]. This may reflect chloroplast contamination in this study as SLP1 is targeted to chloroplasts [21,32], although the latter study did not rule out SLP1 localization to the peroxisome. Notably, several algal homologs of SLP1 harbour a well-recognized PTS1 [SKL>, SRL>, and SNL>] that is not present in higher plants [21]. SLP2, the closest homologue of SLP1, is activated by the mitochondrial intermembrane space import and assembly protein 40 (Mia40). Mia40 is an oxidoreductase that docks target proteins in the mitochondrial intermembrane space, accepting electrons from these proteins and forming disulfide bonds on pairs of cysteines. SLP2 activity is increased 35-fold after oxidation by Mia40 [31]. Mia40 demonstrated dual localization to the mitochondrial intermembrane space and to peroxisomes in Arabidopsis [31,33]. In addition, a recent study identified two novel PTS1s, including the sequence PSL> that was found in another PP2C member (At3g05640) [34]. Peroxisomal Protein Kinases Identification of peroxisomal protein kinases is crucial for understanding the phospho-PTM events that take place in this organelle. In 2002, Fukao et al. performed a proteomic analysis of isolated Arabidopsis leaf peroxisomal proteins and identified three protein kinases: At4g31220, At5g18910, and At3g46410 [13]. The designated silver stained two-dimensional gel spot for At4g31220 (30 kDa) was later determined to be a degradation product of At4g31230 (85 kDa) and named protein kinase 2 (PK2) [35], a member of the 1.4.1 (receptor-like cytoplasmic kinase IX) family [36]. The authors demonstrated that the fusion protein targeted peroxisomes in vivo, and verified the functionality of PK2-PTS1 (PKL>). Further studies on the other two putative peroxisomal protein kinases have not been performed. Glyoxysomes are specialized plant peroxisomes found in germinating seeds and function to convert fatty acids to acetyl-CoA. A glyoxysomal protein kinase (GPK1, also known as PK7) 368

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PTS type 2 (PTS2): a motif that resides near the protein N terminus that targets a protein to the peroxisome. PTS2 has RLX5HL as the prototype sequence that is cleaved after import. Reactive oxygen species (ROS), reactive nitrogen species, and reactive sulfur species: ROS [e.g.,  H2O2 and superoxide radicals (O2 )] are produced from metabolic processes in peroxisomes that include FA b-oxidation, photorespiration, purine, and polyamine metabolism. ROS are involved in inter- and intracellular signaling. An equilibrium between the synthesis rate and degradation rate is normally established. However, unusual biotic and abiotic stresses can change this balance and lead to a stronger signaling cascade, cellular damage, or programmed cell death. Sucrose-dependent 1 (SDP1): a major TAG lipase in plants that is delivered to oil bodies from peroxisomes through physical interaction of the organelles. Superoxide dismutase 3: catalyzes  the superoxide (O2 ) radical into oxygen (O2) or hydrogen peroxide (H2O2). Unsaturated FAs: fatty acids with at least one double bond in the fatty acid chain.

Table 1. Investigated Peroxisomal Phospho-Regulatory Key Players in Arabidopsis thalianaa AGI codea

Protein

Acronym

PTS1-like

Proteomics study

Prediction scoreb/PTD localization

Localization-related study

Protein kinase-like

–

SKD>

Fukao et al. [13]

0.195/N.D.

N.D.

At3g46410

Protein kinase family

–

N.D.

Fukao et al. [13]

1.249/N.D.

N.D.

At3g17420

Glyoxysomal protein kinase 1

AtPK7/GPK1

AKI>

Fukao et al. [12]

0.324/Cytosol

Fukao et al. [12]; Ma and Reumann [35]

At5g04870

Calcium-dep. protein kinase 1

CDPK1

LKL>

N.D.

0.321/ Peroxisome

Coca and San Segundo [39]; Dammann et al. [38]; Lingner et al. [16]

At3g20530

Protein kinase 1

AtPK1

SKL>

N.D.

0.890/ Peroxisome

Lingner et al. [16]; Wang et al. [18]

At4g31230

Protein kinase 2

AtPK2

PKL>

Fukao et al. [13]

0.650/ Peroxisome

Ma and Reumann [35]

At4g18950

Protein kinase 3

AtPK3/RPK1

SHL>

N.D.

0.330/Cytosol

Ma and Reumann [35]

At3g61960

Ser/Thr-protein kinase-like

AtPK4/ATG1a

SHL>

N.D.

0.312/ Peroxisome

Ma and Reumann [35]

At1g69270

Protein kinase 5

AtPK5

SRL>

N.D.

0.996/ Peroxisome

Ma and Reumann [35]

At3g08720

Protein kinase 6

AtPK6

SNL>

N.D.

0.527/ Peroxisome

Ma and Reumann [35]

At5g03730

Constitutive triple response 1

CTR1

SDL>

N.D.

0.336/ Peroxisome

Chowdhary et al. [41]

At1g13460

PP2A regulatory subunit B0 u

B0 u

SSL>

N.D.

0.368/N.D.

Matre et al. [22]

At3g25800

PP2A scaffolding 2

A2

At1g10430

PP2A catalytic subunit 2

C2

Piggy- backing B0 u

At1g69960

PP2A catalytic subunit 5

C5

N.D. N.D. N.D.

1.240/N.D. 0.189/N.D. 0.279/N.D.

At1g07010

Shewanella-like phosphatase 1

SLP1

N.D.

Fukao et al. [13]

0.757/ Cytosolic

Uhrig and Moorhead [32]; Kataya et al. [21]

At3g09400

PP2C family protein

PLL3

SSM>

N.D.

0.515/ Peroxisome

Kataya et al. [21]

AT5g02400

PP2C family protein

PLL2

SSM>

N.D.

0.515/ Peroxisome

Kataya et al. [21]

Protein kinases At5g18910

Protein phosphatases Trends in Plant Science, April 2019, Vol. 24, No. 4

Kataya et al. [19]

369

370

Table 1. (continued)

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AGI codea

Protein

Acronym

PTS1-like

Proteomics study

Prediction scoreb/PTD localization

Localization-related study

At2g01880

Purple acid phosphatase7

PAP7

AHL>

N.D.

0.130/ Peroxisome

Kataya et al. [21]; Lingner et al. [16]

At3g55270

MAP kinase phosphatase 1

MKP1

SAL>

N.D.

0.325/ Peroxisome

Kataya et al. [20]

At2g39970

NAD carrier

PMP38/PXN

N.D.

Fukao et al. [89]

1.2/N.D.

Linka et al. [90]

At3g05290

Adenine nucleotide carrier 1

PNC1

N.D.

N.D.

1.251/N.D.

Linka et al. [90]

At5g27520

Adenine nucleotide carrier 2

PNC2

N.D.

Eubel et al. [15]

1.162/N.D.

Linka et al. [90]

ATP importers

N.D., Unidentified/unstudied. a The bold and underlined AGIs are the confirmed full-length peroxisomal proteins. PTD refers to peroxisomal targeting domains that contain a PTS1 and comprise the C terminal 10 amino acids. Localization-related studies were performed for some of the PTD sequences presented here and were typically fused at their N terminus to protein reporters and expressed in vivo for targeting signal functionality. b Prediction score was determined according to PredPlantPTS1, http://ppp.gobics.de/submission (threshold score: 0.412; min/max score: 1.966/1.188; [16]).

was identified in an Arabidopsis glyoxysomal proteome (Table 1); it possessed a putative PTS1 and targeted to peroxisomes in immunochemical assays against subfractionated glyoxysomes [12]. Suborganellar localization and protease sensitivity analyses have defined GPK1 as a glyoxysomal peripheral membrane protein with its putative kinase domain located inside the glyoxysome [12]. In a later study, GPK1 and GPK1DPTS1 showed targeting to punctate structures, which did not coincide with peroxisomes in vivo [35]. It is postulated that this was due to the GPK1 localizing to intermediate vesicles that could not fuse with peroxisomes in onion epidermal cells, reflecting the specific transport machinery of glyoxysomes. Calcium-dependent protein kinases (CDPKs) are specific to plants and protists and are activated by calcium signals. Many Arabidopsis CDPKs are membrane-associated, consistent with the presence of N terminal acylation sites [37,38]. In addition to targeting to the peroxisome membrane when fused with a reporter at the C terminus of CDPK1 [38,39], strong biochemical and additional imaging data supported CDPK1 targeting to the peroxisome membrane in an N myristoylation-dependent fashion [37–40]. In support of its peroxisomal association, CDPK1 was also shown to have a weak functional PTS1 [16]. Interestingly, CDPK1 was found to concomitantly target lipid droplets, in an N myristoylation-dependent manner [39]. This implies a mechanism for calcium regulation of peroxisome and lipid body processes (Box 2). Ma and Reumann [35] investigated putative PTS1s belonging to seven protein kinases (Table 1) and of these, four kinase-PTDs targeted peroxisomes. Although possessing functional PTDs, the four full-length protein kinases (PK2, PK4/ATG1a, PK5/RPK1, PK6) failed to target peroxisomes. The authors suggested that these kinases target peroxisomes, perhaps transiently, under unknown conditions [35]. Experimental PTD validations, and more recently bioinformatic data from different groups, suggest that PK1 (as defined by Ma and Reumann [35]) localizes to the peroxisome [16,18]. The protein kinase constitutive triple response 1 (CTR1; At5g03730) Box 2. A Model for Regulation of FA b-oxidation During germination, FAs are hydrolyzed from triacylglycerol (TAG) stored in the lipid droplet/oil body by lipases, with the major TAG lipase in Arabidopsis being the membrane-associated phospho-protein sucrose-dependent 1 (SDP1) (Figure 2; also see online supplemental information Table S1) [57,87,94]. FA transport to peroxisomes is facilitated by the direct association of lipid droplets with peroxisomes where FA b-oxidation occurs [87]. Defects in peroxisomal FA b-oxidation are thought to provide negative feedback on TAG mobilization. For instance, the b-oxidation dysfunction mutants kat2, pxa1/ped1, and sdp1 (Figure 1) all display the accumulation of lipids in oil bodies due to hampered TAG hydrolysis. This is similar to the effect of diphenyl methylsulphonate, which blocks TAG degradation and leads to accumulation of peroxisomes around oil bodies [95]. Notably, eight enzymes related to TAG metabolism and FA b-oxidation were shown to be phosphorylated in protein phosphatase (PP2A) b0 u-1 knockout lines, but not in WT seedlings [19]. Among these enzymes, two lipid droplet lipases were found to be phosphorylated [19] and implicate a phosphorylation-dependent inactivation for lipid mobilization in oil bodies. We present a model for protein phosphorylation for the peroxisome-lipid droplet interaction during germination (Figure I) based on the following evidence: (i) finding a partial defect in FA b-oxidation in the phosphatase (PP2A b0 u-1) mutant implicates a role of phosphorylation in controlling b-oxidation enzyme activity [19,21]; (ii) lipase phosphorylation in the PP2A b0 u-1 mutant implicates a putative feedback loop to suppress TAG breakdown [19]; (iii) peroxisomes are suggested to perform negative feedback under conditions of excess energy (high sucrose), and accumulation of FAs during FA b-oxidation defects as in kat1 and sdp1 mutants [57,87,96]; (iv) in addition to targeting the peroxisomal membrane, CDPK1 has been observed at the oil body surface [39] (myristoylation of CDPK1 and its association with the lipid droplet and peroxisome surface, implicated that CDPK1 localization is on the cytosolic side of these membranes); (v) the specific physical interaction of peroxisomes and lipid bodies during seedling germination [57,87]. Additionally, we propose positive feedback effect for TAG breakdown (Figure I) during depletion of energy (low sucrose), initiating the mobilization of cell reserves (TAG). This is supported by the protrusion-dependent transfer of SDP1 lipase to lipid droplets in a sucrose-dependent fashion [87]. This compilation of data supports a model whereby a phospho-regulatory network is controlling TAG hydrolysis and FA b-oxidation that involves crosstalk between peroxisomes and oil bodies.

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– Feedback

+ Feedback

Oil body

–TAG mobilizaƟon • FA accumulaƟon • β-oxidaƟon defect • High sucrose

CDPK1

TAG

P E: InacƟve ?

+TAG mobilizaƟon • Early germinaƟon • Low sucrose

E: AcƟve

FAs P SD

CDPK1

1

PKs

P E: InacƟve

FA β-oxidaion

PP2A E: AcƟve

Succinate

Peroxisome

Sucrose, energy

Figure I. Model for Phospho-Regulation of Triacylglyceride (TAG) Mobilization and Fatty Acid (FA) b-Oxidation. The physical interaction of oil bodies with peroxisomes is facilitated by peroxisomal protrusions that allow the movement of the TAG lipase, sucrose-dependent 1 (SDP1), to oil bodies [87,88]. TAG-derived FAs are transported to peroxisomes for b-oxidation. Loss of the PP2A B0 u-1 subunit in b0 u-1 mutant and subsequent absence of PP2A trimer targeting to peroxisomes, suggest that dephosphorylation of b-oxidation enzymes is necessary for FA breakdown. Conversely, this observation supports the notion that phosphorylation by undefined protein kinases (PKs) inactivates multiple b-oxidation enzymes. Peroxisomal membrane-associated calcium dependent kinase 1 (CDPK1) plays a yet-to-be defined role in TAG mobilization. E, enzyme.

harbors a unique PTS1 and experimentally validated PTD [41]. CTR1 regulates the movement of ETHYLENE INSENSITIVE 2 (EIN2) into the nucleus and is involved in ethylene signalling suppression [42]. Whether full-length CTR1 can target to peroxisomes by its functional PTS1 has not yet been investigated. However, one report has shown that PEX1 transcript is upregulated in the ethylene overproducer eto1-1 and this increase was additive in the presence of salt [43]. As outlined here, a growing list of protein kinases and phosphatases is being assembling for plant peroxisomes (Table 1). This information allows us to be confident that the molecular machinery exists in this organelle to regulate protein function by phosphorylation.

Phosphorylation of Peroxisomal Targeting Sequences Based on studies in other organisms, it was postulated that phosphorylation of plant peroxisomal proteins controls shuttling of proteins between peroxisomes, the cytosol, and other organelles under varying conditions. Yeast glycerol-3-phosphate dehydrogenase 1 (GPD1) can change its localization from the nucleus or cytosol to peroxisomes via phosphorylation of two serine residues located downstream of the N terminal PTS2 signal (Box 1) [44]. This finding supports the role of protein phosphorylation in regulating peroxisome biogenesis, as well as crosstalk between peroxisomes and other cellular compartments. Similarly, Arabidopsis histidine triad nucleotide-binding 1 (HIT3) harbors a PTS2 signal that is flanked downstream by three phosphorylated residues (see the online supplemental information Figure S1). It will be of 372

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P

SDP1

Oil body TAG

FaƩy acids

P

P

PXA1

1 DP

CDPK1

S

A

P

1 DP

S

PXN NAD+

CoA + faƩy acids

B

ACT1

P

NAD+

P

LACS2,5,6

P

AMP

P

AMP

C

Acyl-CoA C18:1 Δ9cis

P

CDPK1

2 cycles of β-oxidaƟon

ACX3,4

ECI3

3trans,5cis-Dienoyl-CoA

C18:1 Δ6cis

2 cycles of β-oxidaƟon

2trans-Enoyl-CoA

2trans,5cis-Dienoyl-CoA P

P

MFP2/AIM1

P

2cis-Enoyl-CoA

MFP2/AIM1

P

3R-Hydroxyacyl-CoA

P

ECH2

DCI1 3-Ketoacyl-CoA 2trans,4trans-Dienoyl-CoA P KAT1 Acetyl-CoA Red 3trans-Enoyl-CoA ECI3

P

MFP2/AIM1

3S-Hydroxyacyl-CoA

2trans-Enoyl-CoA

Acyl-CoA (n-2)

P

2trans-Enoyl-CoA

CSY2

P

Oxaloacetate MDH

Citrate

ACO

Glyoxylate cycle

Malate

Isocitrate P

ICL Succinate

Glyoxylate MS

Figure 1. Representative Scheme of Peroxisomal b-Oxidation, Glyoxylate Cycle, and Interaction with Lipid Bodies. Experimentally validated phosphorylated proteins (see online supplemental information Table S1) are marked with a circle. (A) b-oxidation pathway, (B and C) b-oxidation of unsaturated fatty acids (see broken red arrows). Details about the detected phosphopeptides, modified residue identity and position, and their resources are available in online supplemental information Table S1. In glyoxysomes, the acetyl-CoA produced by fatty acid b-oxidation is used as a substrate for the glyoxylate cycle, where succinate is produced and transferred to mitochondria for further metabolism. Moreover, the peroxisomal ATP-binding cassette transporter (PXA1) and NAD+ transporter (PXN) were found to be phosphorylated (see online supplemental information Table S1). Representation of transport of the lipase sucrose-dependent 1 (SDP1) to oil bodies through peroxisomal protrusions is also shown [57,87]. Protein kinase CDPK1 is shown myristoylated and at oil body and peroxisome surfaces. The pathway of fatty acid b-oxidation is modified from [53]. ACX, Acyl-CoA oxidase; AIM, abnormal inflorescence meristem (i.e., MFP1); CSY, citrate synthase; DCl1, delta(3,5),delta(2,4)dienoyl-CoA isomerase 1; ECl3, delta (3), delta (2)-enoyl CoA isomerase; ICL, isocitrate lyase; KAT1, 3-ketoacyl-CoA thiolase; LACS, long-chain acyl-CoA synthetase; MDH, malate dehydrogenase; MFP, multifunctional protein; MS, malate synthase; TAG, triacylglycerol.

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interest to investigate the impact of phosphorylation on HIT3 localization, especially since the protein was shown to dually target the chloroplast periphery and peroxisomes [10]. Online supplemental information Figure S1 contains six proteins that have phospho-sites in the PTS1like sequence or upstream in the targeting enhancing elements region (Box 1). Given that the targeting enhancing elements are thought to improve peroxisomal targeting efficiency [16], studying the impact of these phospho-residues may provide insight into protein shuttling under different cellular conditions.

Arabidopsis Peroxisomal Phospho-proteome Direct identification of phosphorylated peroxisomal proteins is difficult because of the challenges associated with peroxisome purification. An early report used titanium-dioxide for phosphopeptide enrichment for proteins derived from isolated Arabidopsis cell culture peroxisomes, and detected only a single significant phosphorylation event (S155) for the peroxisomal membrane-localized NAD+ transporter (PMP38/PXN) [15]. Focused studies investigating the Arabidopsis peroxisome phospho-proteome are absent and only 14 phosphorylated peroxisomal proteins have been identified and collated into a single article prior to this review [7,19,21]. Isolation of proteins, optimizing phospho-enrichment techniques, and the development of mass spectrometry methods resulted in an explosion of identified phosphorylation sites for the global Arabidopsis phospho-proteome. Van Wijk and coauthors performed a meta-analysis of 27 published reports and datasets, in combination with their in-house phospho-proteomes, and assembled more than 60 000 phosphopeptides matching 8141 nonredundant proteins from different Arabidopsis tissues and growth conditions [45]. We have gathered information about validated peroxisomal and peroxisome-related proteins (identified by subcellular localization and/or proteomics, with most indexed at the SUBA database [46]) and matched them with the files generated from van Wijk’s analysis [45]. From this, we have compiled a list of 60 peroxisomal proteins that were phosphorylated. We further catalogued the peroxisomal phospho-proteome to more than 100 proteins when the database PhosPhAt 4.0 was searched for proteins linked to peroxisomes (see online supplemental information Table S1) [47]. This wealth of new information related to phosphorylation sites (or any other covalent modification) provides, as our article title suggests, a framework for many more studies that explore the function of a covalent modification at a given site. It is possible that some or even many covalent modifications play no functional role in the cell and that many of these modifications likely work in conjunction with modification of other sites on the protein [48]. These datasets are a starting point to unravel protein regulation by covalent modifications. In addition, caution must be taken when using assembled phospho-proteomic datasets from multiple studies, because most of these studies do not report false discovery rates for peptide identification, which can lead to incorrect phospho-site assignment. We refer readers to the following articles for an indepth discussion of interpreting phospho-proteomic datasets [49–51]. Here, we will briefly discuss several peroxisomal processes where we have catalogued phosphorylated protein(s) (see online supplemental information Tables S1 and S2), and then propose some putative roles for the phospho-PTM. b-Oxidation Triacylglycerides (TAG) are broken down into FAs in lipid droplets. The b-oxidation pathway successively removes two carbon units from FAs in each b-oxidation cycle [52,53]. In plants and yeast, b-oxidation is a peroxisomal process that leads to chain shortening of acyl-CoA esters to yield chain-shortened acyl-CoA and acetyl-CoA or enoyl-CoA, depending on the 374

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substrates (for details see [53]). In oilseed plants, FA b-oxidation is essential to supply energy and carbon metabolites after seed germination. This energy pathway involves FA b-oxidation, the glyoxylate cycle, and gluconeogenesis. Peroxisomes have been shown to physically interact with lipid droplets in a sucrose-dependent fashion (Box 2). Lipid droplet-derived FAs are transported into peroxisomes, putatively through the peroxisomal ABC transporter 1 (PXA1), also named peroxisome-deficient 3 (PED3) and comatose (CTS) [53–56]. Phosphorylation of PXA1 has been observed (Figures 1 and 2; also see online supplemental information Table S1), and is thought to fine-tune the transport of FAs into the peroxisome [19,55]. It is fascinating that the anchor on peroxisomes for association with lipid droplets is in fact PXA1/PED3 [57]. After transport into peroxisomes, FAs are activated into their CoA form with the help of long-chain acyl-CoA synthetases [58,59]. Three members of long-chain acylCoA synthetases (isoform 2, 5, and 6) are catalogued as phospho-proteins. Remarkably, select isoforms of all the b-oxidation enzymes are known to be phosphorylated, as are several glyoxylate cycle enzymes (Figure 1; also see online supplemental information Table S1). Arabidopsis MFP2 and AIM1 catalyze the second step of b-oxidation and additional activities for some unsaturated FAs (reviewed in [53]), and are experimentally validated to be phosphorylated (Figure 1; also see online supplemental information Table S1). The 2trans-enoyl-CoA hydratase motif of MFP2 was reported to be only active against C18:0 substrates, whereas the L3-hydroxyacyl-CoA dehydrogenase domain is active against C6:0, C12:0, and C18:0 substrates [53,60]. Additionally, representative candidates such as delta (3) and delta (2)-enoyl CoA isomerase (ECIs) proposed to activate unsaturated FAs have shown phosphorylation status (Figure 1B,C; also see online supplemental information Table S1). Here, shortened FA-CoA and acetyl-CoA are the outcomes, which fed into another b-oxidation cycle and the glyoxylate cycle, respectively (for review see [53,61]). Acetyl-CoA can be further metabolized in seedlings via the glyoxylate cycle, which has two phosphorylated enzymes (citrate synthase and isocitrate lyase) (Figure 1; also see online supplemental information Table S1). Although not yet detected as phospho-proteins in Arabidopsis, malate synthase and malate dehydrogenase have been reported to be phosphorylated in castor bean glyoxysomes and pea leaf peroxisomes (D.P. Mantilla, University of Granada, 2009; [62]). The high degree of protein phosphorylation now observed for the steps of FA b-oxidation in peroxisomes hints that phosphorylation may be a key regulatory event for this process and is worthy of focused studies to explore this possibility. To this end, we propose a model for regulation of these processes and crosstalk between peroxisomes and lipid droplets (see Figure I in Box 2). ROS-detoxification The Arabidopsis peroxisomal antioxidant system includes three catalase isoforms, each of which are found to be phosphorylated (Figure 2; also see online supplemental information Table S1). Monodehydroascorbate reductase 1 and 4 are involved in H2O2 detoxification through the ascorbate-glutathione cycle [63] and are known phospho-proteins (Figure 2; also see online supplemental information Table S1). The role of phosphorylation in regulating peroxisomal antioxidant system is also supported by the observed increase in phosphorylation of superoxide dismutase 3 (Figure 2; also see online supplemental information Table S1) upon 2,4D-treated pea leaves, a condition that promotes ROS and nitric oxide accumulation (D.P. Mantilla, University of Granada, 2009). Peroxisome Biogenesis A recent study of protein kinase and phosphatase deletion mutants in Saccharomyces cerevisiae revealed a fundamental role for protein phosphorylation in peroxisome formation, Trends in Plant Science, April 2019, Vol. 24, No. 4

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PUFAs Ribulose-1,5bisphosphate

18:3/16:3 P 13-LOX 13-HPOT/11-HPHT

P AOS 12,13-EOT/10,11-EHT P AOC

Glycerate

Mitochondrion

Chloroplast

P

PM P pe Pr PM ro eP xis om e

cis/dnOPDA

Glycine

Pexophagy PE UP 1,2 P

Serine

IBA

P

Benzoyl-CoA

H2O2

P SP1

Ub Ub

H2O + O2 CAT1,2,3

P X5

P PEX2

2 PEX4

PE

ub

PEX1 2 PEX1 0 PEX2

Ub

P Ascorbate- MDAR1,4 glutathione cycle

P

P

Cargo

SA

P

Ub

Ub

Peroxisome

P

P DRP3A,B

PMD1

MPK17

O2–

PhotorespiraƟon Purine metabolism SulfitedetoxificaƟon Polyamine metabolism

FIS1

Fission

Benzoate P BZO1

IAA

β-oxidaƟon

Cargo

P

Cinnamoic acid

IAA-CoA ACH P

(+)-7-iso-JA

LON2 P

P

β-oxid.

(+)-7-iso-JA-CoA ACH P

SAGT1 P

Ub Ub Ub

Pex19 P PMP

2-trans-IBA-CoA

β-oxid.

9

PE

P 4CLL2/6/9 OPC8/6-CoA

P

IBA-CoA IBR3 P

OPC8:0/6:0 OPCL

x1

PE X1 3 X1 4

cis/dnOPDA OPR3 P

CSD3 P

4 P X1 PE

2 glycolate GOX1,2 P 2 glyoxylate SAGT1 P Glycerate GGT1 P 2 glycine P HPR1 Hydroxypyruvate

Pe

X5 PE

PEX1

P

Ub Ub Ub

Pe x1 9 PM P P

P

NH4+ Serine

P

PXA1

CO2 NAD+

PE X PE 16 X3

NADH2

ER

X5

Glycolate

PM

P ca TS1 rg o

3-phosphoglycerate

PE

Calvin cycle

2-phosphoglycolate

P P

P

Perox

ule

Figure 2. Overview of Phospho-proteins Implicated in Various Peroxisomal-Related Processes. Experimentally validated phosphorylated proteins (see online supplemental information Tables S1 and S2) are marked with a circle. The photorespiration scheme, including the crosstalk between chloroplast, mitochondria, and peroxisomes, are presented (details available at Hodges et al. [85]). Scheme of jasmonic acid (JA) biosynthesis displays the phosphorylation of most of the key enzymes involved from chloroplast and peroxisomes (see online supplemental information Table S1). Salicylic acid (SA), and indole-acetic acid (IAA) peroxisomal biosynthetic pathways are also represented (see online supplemental information Table S1). A brief overview of reactive oxygen species (ROS) biosynthesis and detoxification of key enzymes and their phosphorylation status are also shown, as are biogenesis factors affecting peroxisome abundance and content (see online supplemental information Table S1). This includes: (i) proteins involved in transporting of peroxisomal membrane proteins (PMPs) and matrix proteins; (ii) recycling of transporters (PEX5), induction of peroxule formation and proliferation; (iii) degradation of matrix proteins (ex. LON2); and (iv) pexophagy. Details about abbreviations, the detected phosphopeptides, modified residue identity and position, and their resources are available in online supplemental information Table S1.

size, and fission [64]. Our observations, as detailed below, suggest an equally important role in plant peroxisome biogenesis. Peroxisomes originate from the ER and mature by subsequent membrane and matrix protein import [65]. Mature peroxisomes can then undergo elongation and constriction, and ultimately fission to yield daughter peroxisomes (Figure 2). 376

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Peroxins (PEXs) or peroxisome biogenesis factors, are required for peroxisome assembly, which includes both the insertion of peroxisomal membrane proteins (PMPs) and the import of folded matrix destined proteins. PEX3, PEX16, and PEX19 are responsible for inserting PMPs into peroxisomal membranes (reviewed in [61,66]) and all display phosphorylation (Figure 2; also see online supplemental information Table S1). PEX5, which recognizes and binds PTS1 cargo motifs and the docking machinery (PEX13 and PEX14) on the peroxisomal membrane, is phosphorylated (Figure 2; also see online supplemental information Table S1), implicating a phospho-regulatory role during transport and import. Of interest is the presence of two phosphorylated serine residues (308, 328) located between a series of nine WXXXF/Y repeats in PEX5, which are responsible for binding with PEX14 [67]. Indeed, a single mutation of Ser (318) to Leu in the mutant PEX5-1 affected PEX5 binding with PEX7 (the PTS2 receptor). This impacted the sucrose dependence phenotype in the pex5-1 mutant seedlings and affected PTS2-mediated import into peroxisomes [68,69]. As demonstrated in yeast, after importing PTS1-cargo into peroxisomes, PEX5 is recycled back to the cytosol by mono-ubiquitination with the help of PEX4 that is bound to PEX22 (also a known phospho-protein) (Figure 2; also see online supplemental information Table S1). In addition, the peroxisomal E3 ubiquitin ligase SP1, that is reported to interact with the peroxisome docking complex (PEX13-PEX14) and the (RING)-finger PEX2 [70–72], is also reported as a phosphorylated protein (Figure 2; also see online supplemental information Table S1). Regulation of peroxisome biogenesis by protein phosphorylation has been demonstrated in yeast, where the phosphorylation of PEX11 is essential for stimulating organelle fission [73,74]. Similarly, in Arabidopsis, a role has been shown for MAP kinase 17 (MPK17) that affects the actin-binding protein PMD1 and peroxisome proliferation under stress [75]. PMD1 is one of three known proteins involved in constriction and fission [61,66]. PEX11, dynamin-related proteins 3A and 3B, as well as MPK17 show phospho-PTMs (Figure 2; also see online supplemental information Table S1). It is notable that many of these phosphorylation events occur in the cytosol of the cell and the kinases and phosphatases that control these events undoubtedly reside in that compartment. Pexophagy Pexophagy is a term used to define autophagy of peroxisomes [76–78]. Autophagy is a conserved mechanism that allows organelle turnover strategies to survive starvation, and allows turnover of protein aggregates and aberrant organelles. By contrast, the degradation of individual proteins occurs to maintain a proteome that reflects current cellular (or peroxisome) needs and, in coordination with protein import, is termed peroxisome remodeling [79]. The major peroxisomal protease, LON2, is a known phospho-protein (Figure 2; also see online supplemental Table S1) and implicates phospho-regulation during peroxisome remodeling. LON2 is interpreted to prevent pexophagy because lon2 seedlings showed a defect in peroxisome metabolism, and excessive pexophagy [80]. However, coordination of complementary functions between LON2 and pexophagy has also been implicated [66]. In relation to pexophagy, two mutants named peroxisome unusual positioning 1 and 2 [mutated in autophagy-related genes (ATG2 and ATG18A, respectively)] (Figure 2; also see online supplemental information Table S1) displayed accumulation of aggregated peroxisomes, catalase inactivation, and oxidative damage highlighting a role for selective pexophagy as a quality control mechanism in the cell. From mammalian studies, ubiquitinated PEX14p was reported to bind to the ATG8p-homolog, LC3, and was proposed to support recognition of ubiquitinated PMPs by autophagy receptors (reviewed in [81]). In the methylotrophic yeast, Hansenula polymorpha, only phosphorylated PEX14p is needed for pexophagy, while the nonphosphorylated version was devoted only for protein import [82]. Trends in Plant Science, April 2019, Vol. 24, No. 4

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Signalling Molecule Biosynthesis Jasmonic acid (JA) has pivotal roles in plant development and defence mechanisms [83]. JA is derived from C18:3 and C16:3 unsaturated FAs. The JA biosynthesis pathway starts in plastids and finishes in peroxisomes (Figure 2). The regulation of JA biosynthesis is expected to be dynamic and highly regulated for rapid responses against herbivores and necrotrophic pathogens. Oxophytodienoate-reductase 3, which catalyzes step one of the peroxisomal portion of the JA biosynthesis pathway, is phosphorylated, as are three 4-coumarate-CoA ligases (2, 6, and 9) and this is predicted to activate step two (Figure 2; also see online supplemental information Table S1). The OPC8:0/6:0-CoA produced from step two is then shortened by several rounds of b-oxidation, and JA is produced after the release of CoA. The last reaction is expected to be activated through acetyl-CoA thioesterases, such as ACH, which is also phosphorylated (Figure 2; also see online supplemental information Table S1). In Figure 2, we also highlight the role of the plastid in JA biosynthesis and show known phospho-proteins that are involved in the pathway. Peroxisome function in defence responses is not limited to JA biosynthesis, but also has an emerging role in salicylic acid (2-hydroxybenzoic acid) and indole-acetic acid biosynthesis. Several key enzymes involved in these biosynthetic pathways are known phospho-proteins (Figure 2; also see online supplemental information Table S1). Additional studies are required to unravel the biological significance of these PTMs and their impact on the plant innate immunity. Other Functions The photorespiration pathway involves chloroplasts, mitochondria, and peroxisomes [84], and recent data indicates the phosphorylation of many photorespiratory enzymes (Figure 2; also see online supplemental information Table S1). The effect of protein phosphorylation on photorespiration was proposed and reviewed previously [85]. The vital pentose phosphate shunt is an NADPH regeneration source [86] in peroxisomes and involves 6-phosphogluconate dehydrogenase, 6-phosphogluconolactonase-5, and NADP-isocitrate dehydrogenase. Interestingly, all three enzymes are phosphorylated (see online supplemental information Tables S1 and S2), although there is no position assignment for the phosphate group on NADP-isocitrate dehydrogenase (see online supplemental information Table S2). Multiple proteins involved in: the metabolism of glutamine, aspartate, and L-methionine; allantoin and anthocyanin biosynthesis; oxalate and sulfur compound metabolism; and the mevalonate pathway, are also phospho-proteins (see online supplemental information Table S1). The role of peroxisomes in response to environmental factors is evolving, and in online supplemental information Table S1, we show several phosphorylated peroxisomal proteins implicated in response to hormones and biotic and abiotic stresses.

Concluding Remarks This review highlights protein phosphorylation as a recognized event in peroxisomes and links this process to essentially every aspect of peroxisome biology, from biogenesis and remodeling, to metabolism and pexophagy. Although many of the phospho-regulatory events take place outside the peroxisome to regulate peroxisome biology, many occur inside this organelle. This is supported by the localization of several protein phosphatases and kinases in peroxisomes. Our compilation of peroxisomal phosphorylation sites also creates a launchpad for additional functional studies (see Outstanding Questions) on the role of covalent modifications on the biochemistry of each protein and its implication for peroxisome biology. Author Contributions

A.K., D.M., and G.M. all participated in the writing of this review. 378

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Outstanding Questions What are the cytosolic protein kinases and phosphatases that regulate cytosolic peroxisome related events (e.g., cargo recognition and docking, and receptor recycling)? What is the effect of phosphorylation on newly identified phospho-proteins or pathways? What is the mechanism of regulation for CDPK1 and its targets? What are the specific cellular/peroxisomal events that control the activity of the identified protein kinases and phosphatases that regulate peroxisome function?

Acknowledgments The authors wish to thank past and current lab members for ongoing interest in protein phosphorylation as a protein regulatory mechanism. A.K. is funded by the Research Council of Norway grant number 251310/F20 and D.M. and G.M. are supported by the Natural Sciences and Engineering Research Council of Canada.

Disclaimer Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Supplemental Information Supplemental information associated with this article can be found, in the online version, at https://doi.org/10.1016/j. tplants.2018.12.002.

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