A PEX5 Missense Allele Preferentially Disrupts PTS1 Cargo Import Into Arabidopsis Peroxisomes

Received: 24 July 2018 | Revised: 9 January 2019 | Accepted: 3 March 2019 DOI: 10.1002/pld3.128

ORIGINAL RESEARCH

A PEX5 missense allele preferentially disrupts PTS1 cargo import into Arabidopsis peroxisomes

Khushali J. Patel* | Yun-Ting Kao* | Roxanna J. Llinas | Bonnie Bartel

Department of BioSciences, Rice University, Houston, Texas Abstract The sorting of eukaryotic proteins to various organellar destinations requires recep- Correspondence Bonnie Bartel, Department of BioSciences, tors that recognize cargo protein targeting signals and facilitate transport into the Rice University, Houston, TX. organelle. One such receptor is the peroxin PEX5, which recruits cytosolic cargo Email: [email protected] carrying a peroxisome-targeting signal (PTS) type 1 (PTS1) for delivery into the per- Present addresses oxisomal lumen (matrix). In plants and mammals, PEX5 is also indirectly required for Yun-Ting Kao, Department of Cell Biology and Molecular Genetics, University of peroxisomal import of proteins carrying a PTS2 signal because PEX5 binds the PTS2 Maryland, College Park, Maryland receptor, bringing the associated PTS2 cargo to the peroxisome along with PTS1 Khushali J. Patel, Graduate School of cargo. Despite PEX5 being the PTS1 cargo receptor, previously identified Arabidopsis Biomedical Sciences, Baylor College of Medicine, Houston, Texas pex5 mutants display either impairment of both PTS1 and PTS2 import or defects only in PTS2 import. Here, we report the first Arabidopsis pex5 mutant with an ex- Funding information National Institutes of Health, Grant/Award clusive PTS1 import defect. In addition to markedly diminished GFP-PTS1 import Number: R01GM079177, F31GM125367, and decreased pex5-2 protein accumulation, this pex5-2 mutant shows typical S10RR026399, P30CA91842, and UL1RR024992; National Science peroxisome-related defects, including inefficient β-oxidation and reduced growth. Foundation, Grant/Award Number: MCB- Growth at reduced or elevated temperatures ameliorated or exacerbated pex5-2 1516966; Robert A. Welch Foundation, Grant/Award Number: C-1309 peroxisome-related defects, respectively, without markedly changing pex5-2 protein levels. In contrast to the diminished PTS1 import, PTS2 processing was only slightly impaired and PTS2-GFP import appeared normal in pex5-2. This finding suggests that even minor peroxisomal localization of the PTS1 protein DEG15, the PTS2- processing protease, is sufficient to maintain robust PTS2 processing.

KEYWORDS Arabidopsis thaliana, peroxin, peroxisome import, peroxisome-targeting signal, PEX5

1 | INTRODUCTION carbon to seedlings prior to the establishment of photosynthesis. Moreover, several steps in photorespiration, which allows recy- Peroxisomes are organelles that are essential for many processes cling of metabolites when RuBisCO uses oxygen instead of carbon including metabolism, development, and environmental responses dioxide (reviewed in Dellero, Jossier, Schmitz, Maurino, & Hodges, (reviewed in Reumann & Bartel, 2016; Kao, Gonzalez, & Bartel, 2016), are peroxisomal. Peroxisomes can arise either de novo from 2018). Peroxisomes are crucial to plant growth as they house fatty the endoplasmic reticulum (ER) or via fission of existing peroxisomes acid β-oxidation (reviewed in Graham, 2008), which provides fixed (reviewed in Kao et al., 2018). Pre-peroxisomes derived from the

*These authors contributed equally to this work.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2019 The Authors. Plant Direct published by American Society of Plant Biologists, Society for Experimental Biology and John Wiley & Sons Ltd.

wileyonlinelibrary.com/journal/pld3 | 1 Plant Direct. 2019;1–13. 2 | PATEL et al.

ER (and mitochondria in mammals) can deliver phospholipids and Analysis of mutants defective in peroxisome cargo recep- peroxisome membrane proteins (PMPs) to preexisting peroxisomes tors can provide insight into the import machinery. Only two or fuse to give rise to new peroxisomes (Mullen & Trelease, 2006; Arabidopsis pex5 mutants, pex5-10 and pex5-1, have been de- Sugiura, Mattie, Prudent, & McBride, 2017; van der Zand, Gent, scribed. pex5-10 carries a T-DNA insertion in the fifth exon of Braakman, & Tabak, 2012). PEX5 (Zolman et al., 2005) that results in the skipping of this exon Peroxins (PEX proteins) facilitate peroxisome biogenesis, divi- and production of an internally deleted pex5-10 protein lack- sion, and matrix protein import. Mature peroxisomes can posttrans- ing several predicted PEX14-binding motifs (Figure 1a) (Khan & lationally import fully folded matrix proteins from the cytosol (Lee, Zolman, 2010). The pex5-10 mutant, like pex5 RNAi lines (Hayashi Mullen, & Trelease, 1997; McNew & Goodman, 1994). PEX5 and et al., 2005), has defects in both PTS1 and PTS2 import (Khan & PEX7 are receptors that recognize matrix proteins carrying either a Zolman, 2010; Lingard & Bartel, 2009). pex5-1 is a missense allele C-terminal peroxisome targeting signal (PTS) type 1 (PTS1) or an N- that creates a Ser318Leu substitution (Zolman et al., 2000) in the terminal PTS2, respectively (reviewed in Kao et al., 2018). In yeast, predicted PEX7-binding domain (Figure 1a), and the pex5-1 mutant PEX5 interacts with PEX14 at the peroxisome surface to induce has impaired PTS2 import but wild-type PTS1 import (Woodward transient pores in the membrane that allow cargo import into the & Bartel, 2005a). Similarly, Arabidopsis pex7 mutants and RNAi matrix (Meinecke et al., 2010). In the peroxisome, the PTS2 signal is lines display defects in PTS2 import (Hayashi et al., 2005; Ramón cleaved from the cargo by the peroxisomal protease DEG15 in plants & Bartel, 2010; Woodward & Bartel, 2005a). In addition to PTS2 (Helm et al., 2007; Schuhmann, Huesgen, Gietl, & Adamska, 2008) or import defects, Arabidopsis pex7 mutants show decreased PEX5 Tysnd1 in mammals (Kurochkin et al., 2007). As DEG15 and Tysnd1 levels and defects in PTS1 import (Ramón & Bartel, 2010), indi- are PTS1 proteins, PTS2 processing presumably requires both PTS1 cating that PEX5 and PEX7 are interdependent. As Arabidopsis and PTS2 import. After cargo delivery in yeast, PEX5 in the peroxi- pex5 mutants with exclusively PTS1 import defects have not been somal membrane is ubiquitinated and targeted for recycling or deg- reported, distinguishing the functions of PTS1 and PTS2 import in radation by the peroxisome-tethered ubiquitin-conjugating enzyme plants has been challenging. (Ubc) PEX4 and the RING–finger ubiquitin–protein ligase complex In this study, we describe a novel pex5 missense mutation (pex5- consisting of PEX2, PEX10, and PEX12 (reviewed in Platta, Hagen, 2) recovered from a forward genetic screen for β-oxidation defects. Reidick, & Erdmann, 2014). In yeast, PEX5 that is monoubiquitinated The pex5-2 mutant exhibited reduced growth, low PEX5 levels, and by PEX12 and PEX4 is recycled from the peroxisomal membrane by decreased peroxisomal import of GFP-PTS1 protein. In contrast, the peroxisome-tethered PEX1–PEX6 ATPase complex for additional pex5-2 displayed robust PTS2-GFP import and only slight defects in import rounds (Pedrosa et al., 2018; Platta, Grunau, Rosenkranz, PTS2 protein processing, suggesting that relatively little PTS1 im- Girzalsky, & Erdmann, 2005). In contrast, PEX5 that is polyubiquiti- port may be sufficient to efficiently cleave PTS2 signals. Some pex5- nated by PEX2 and the cytosolic Ubc4 is targeted for proteasomal 2 deficiencies were exacerbated at elevated growth temperature degradation (Platta et al., 2009). and ameliorated at lowered growth temperature, suggesting that Core peroxins that facilitate matrix protein import are con- PEX5 function and/or pex5-2 dysfunction is impacted by tempera- served in the reference plant Arabidopsis thaliana (reviewed in Kao ture. The distinct and overlapping defects of the Arabidopsis pex5-1, et al., 2018; Woodward & Bartel, 2018). With the exception of pex5-2, and pex5-10 mutants will allow continued elucidation of the PEX14 (Hayashi et al., 2000; Monroe-Augustus et al., 2011), known relationships between PTS1 and PTS2 import in plants. null alleles of genes encoding peroxins confer embryonic lethality in Arabidopsis (Boisson-Dernier, Frietsch, Kim, Dizon, & Schroeder, 2 | MATERIALS AND METHODS 2008; Fan et al., 2005; Goto, Mano, Nakamori, & Nishimura, 2011; Hu et al., 2002; McDonnell et al., 2016; Schumann, Wanner, 2.1 | Plant materials and growth conditions Veenhuis, Schmid, & Gietl, 2003; Sparkes et al., 2003). Thus, the roles of most plant peroxins have been elucidated by analyzing Arabidopsis (Arabidopsis thaliana) wild type and mutants were in the partial loss-of-function missense alleles (Burkhart, Kao, & Bartel, Columbia-0 (Col-0) background. pex4-1 (Zolman et al., 2005), pex5- 2014; Burkhart, Lingard, & Bartel, 2013; Gonzalez et al., 2017; 1 (Zolman et al., 2000), pex5-10 (Zolman et al., 2005), and pex6-1 Goto et al., 2011; Kao, Fleming, Ventura, & Bartel, 2016; Mano, (Zolman & Bartel, 2004) were previously described. Wild type trans- Nakamori, Nito, Kondo, & Nishimura, 2006; Ramón & Bartel, formed with 35S:PEX5 (Zolman & Bartel, 2004), 35S:GFP-PTS1 (Zolman 2010; Rinaldi et al., 2017; Woodward et al., 2014; Zolman & Bartel, & Bartel, 2004), or 35S:PTS2-GFP (Woodward & Bartel, 2005a); 2004; Zolman, Monroe-Augustus, Silva, & Bartel, 2005; Zolman, pex4-1 carrying 35S:GFP-PTS1 (Zolman et al., 2005); and pex5-1 car- Yoder, & Bartel, 2000), T-DNA insertions that incompletely abol- rying 35S:GFP-PTS1 (Woodward & Bartel, 2005a) were previously de- ish function (Khan & Zolman, 2010; Ratzel, Lingard, Woodward, & scribed. pex5-1 carrying 35S:PTS2-GFP, pex5-2 carrying 35S:PTS2-GFP, Bartel, 2011; Woodward & Bartel, 2005a; Zolman et al., 2005), or and pex5-2 carrying 35S:PEX5 and 35S:GFP-PTS1 were selected from RNAi approaches (Fan et al., 2005; Hayashi, Yagi, Nito, Kamada, progeny of the corresponding crosses using PCR-based genotyping & Nishimura, 2005; Nito, Kamigaki, Kondo, Hayashi, & Nishimura, with the primers listed in Supporting Information Table S1. All assays 2007; Orth et al., 2007). except the initial characterization (Supporting Information Figure S1) PATEL et al. | 3

FIGURE 1 Arabidopsis pex5 alleles alter different protein domains. (a) Schematic of Arabidopsis (At) PEX5 protein showing Wxxx [F/Y/Q] repeats (green) that may bind PEX14, the region corresponding to the PEX7-binding domain (lavender) in human PEX5 (Dodt, Warren, Becker, Rehling, & Gould, 2001), PTS1-binding tetratricopeptide repeats (TPR; numbered), the C-terminal domain (black), and Arabidopsis pex5 mutations (red). (b) Alignment of the TPR and C-terminal domains of PEX5 orthologs from Arabidopsis thaliana (At; OAO90794.1), Oryza sativa (Os; AAX11255.1), Homo sapiens (Hs; NP_001124495.1), Drosophila melanogaster (Dm; AAF45676.2), Trypanosoma brucei (Tb; AAD54220.1), and Saccharomyces cerevisiae (Sc; KZV12481.1) generated using the Clustal W method of the Megalign program (DNAStar). Identical residues in at least four sequences are highlighted in black, chemically similar residues are in gray, and the pex5-2 mutation is in red. α-helices in the human PEX5 structure (panel c) are indicated by bars under the alignment. (c) Structure of the human (Hs) PEX5 TPR domain (sequence in panel b) in a complex with a PTS1 cargo protein (yellow). The Gly residue analogous to the residue altered in pex5-2 is highlighted in red. The ribbon diagram was generated from the PEX5-sterol carrier protein (SCP2) structure deposited in the Protein Data Bank under ID code 2C0L (Stanley et al., 2006) using UCSF Chimera software (Pettersen et al., 2004) 4 | PATEL et al. used pex5-2 carrying 35S:GFP-PTS1 that had been backcrossed at with rabbit primary antibodies raised against PMDH2 (1:5,000; least once with wild type carrying 35S:GFP-PTS1. Pracharoenwattana, Cornah, & Smith, 2007), the PED1 isoform of Seeds were surface-sterilized with bleach solution (3% NaOCl thiolase (1:10,000; Lingard, Monroe-Augustus, & Bartel, 2009), or and 0.01% Triton X-100), washed twice with sterile water, sus- PEX5 (1:100; Zolman & Bartel, 2004) followed by horseradish per- pended in 0.1% (w/v) agar, and stratified in dark at 4°C for 1–3 oxidase (HRP)-linked goat anti-rabbit secondary antibody (1:5,000 days. Stratified seeds were plated on plant nutrient (PN) media dilution of 0.125 mg/ml, GenScript A00098) or with mouse pri- (Haughn & Somerville, 1986) solidified with 0.6% (w/v) agar and mary antibodies raised against HSC70 (1:100,000, Stressgen HPA- supplemented with 0.5% sucrose (PNS) as indicated. PNS plates 817) followed by HRP-linked goat anti-mouse secondary antibody were supplemented with IBA (from a 100 mM IBA stock in ethanol) (1:5,000, Santa Cruz Biotechnology sc-2031). Antibodies were visu- as indicated. Plates were incubated at 22°C in yellow-filtered light alized using enhanced chemiluminescent HRP substrate (Advansta (Stasinopoulos & Hangarter, 1990) for the indicated number of days, Western Bright K-12045 or Prometheus ProSignal Pico) and ex- and light-grown root lengths were measured and/or seedlings were posed on autoradiography film. Membranes were sequentially used for immunoblotting or confocal microscopy. For temperature probed with various antibodies without stripping. Representative experiments, plates were incubated at 22°C in yellow-filtered light films were scanned with a flatbed scanner, and band intensity was for 1 day and then incubated at 16, 22, or 28°C for seven additional quantified using ImageJ (version 1.42q; Schneider, Rasband, & days for light-grown experiments or wrapped in aluminum foil and Eliceiri, 2012). grown at 16, 22, or 28°C for four additional days for dark-grown experiments. All experiments except the initial characterization 2.5 | Confocal fluorescence microscopy (Supporting Information Figure S1) were repeated at least twice with similar results; representative results are shown. Light-grown seedlings carrying 35S:GFP-PTS1 or 35S:PTS2-GFP were mounted in water and imaged using a Carl Zeiss LSM 710 laser scanning confocal microscope equipped with a meta detec- 2.2 | pex5-2 isolation tor and 40× Plan-Apochromat oil-immersion objectives. GFP was Ethyl methanesulfonate (EMS) mutagenesis of wild-type seeds car- excited with a 488-nm argon laser and fluorescence was collected rying 35S:GFP-PTS1 was previously described (Rinaldi et al., 2016). between 493 and 552 nm. Each image corresponds to a 1.0-μ m

M2 seeds were grown for about 2 weeks in yellow-filtered light on optical section (pinhole = 1 airy unit) and is an average of four PNS supplemented with 100 mM NaCl and 12 μM IBA, and puta- exposures. tive mutants with elongated roots were transferred to soil for seed For quantification of confocal images (collected at a production. M3 lines displaying resistance to 10 μM IBA (with or 1,024 × 1,024 pixel setting; 4.8177 pixels/μm), ImageJ macros without 100 mM NaCl) and wild-type-like sensitivity to 80 nM (listed in Supporting Information) were used to measure the inten- 2,4-dichlorophenoxyacetic acid were retained for further analysis. sity of punctate fluorescence [defined as particles of at least 15 pixels (3.11 μm) in diameter with a circularity of 0.2–1.0] and the total intensity in the image [defined as particles of at least 2 pix- 2.3 | Whole-genome sequencing els (0.415 μm) in diameter with circularity of 0–1.0]. The fraction

Approximately 100 pex5-2 M3 seeds were plated on PNS covered with of [total punctate intensity]/[total intensity] from three images sterile filter paper. Genomic DNA was purified (Thole, Beisner, Liu, (Supporting Information Figures S3 and S4) was calculated as a Venkova, & Strader, 2014) from 18-day-old light-grown seedlings and measure of peroxisomal import. sequenced by the Genome Technology Access Center at Washington University in St. Louis. Sequence reads were aligned with the TAIR 2.6 | Statistical analysis 10 build of the A. thaliana Col-0 genome using Novoalign (Novocraft; http://novocraft.com). SNPs were identified using SAMtools (Li, SPSS Statistics software (Version 24.0.0.0) was used to analyze 2011; Li et al., 2009) and annotated with snpEFF (Cingolani et al., measurements. One-way analysis of variance (ANOVA) followed by 2012). Mutations were filtered using a script prioritizing homozygous the Duncan's post hoc test was used to determine the significance of EMS- d e r i v e d m u t a t i o n s (G- t o - A a n d C- t o - T) c a u s i n g n o n - s y n o n y m o u s differences among genotypes or treatments. amino acid changes or altering splice sites. We disregarded mutations that were present in our laboratory stock of wild-type Col-0. 3 | RESULTS Identifiers of genes with homozygous EMS-consistent mutations are displayed in Supporting Information Figure S2. 3.1 | pex5-2 displays peroxisome-related defects that are rescued by PEX5 overexpression 2.4 | Immunoblot analysis As β-oxidation is exclusively peroxisomal in Arabidopsis (reviewed Seedling extracts were processed for immunoblotting as de- in Graham, 2008), we can use β-oxidation efficiency to assess per- scribed (Kao & Bartel, 2015). Membranes were incubated overnight oxisome function (reviewed in Bartel, Burkhart, & Fleming, 2014). PATEL et al. | 5

The predominant naturally occurring auxin, indole-3-acetic acid identified pex5-2 mutation was causing the observed peroxisome- (IAA), plays critical roles in plant growth and development by mod- related impairments. ulating cell identity, division, and elongation (Woodward & Bartel, We compared pex5-2 with previously reported pex5 alleles: 2005b). One IAA precursor, indole-3-butyric acid (IBA), is con- pex5-10 and pex5-1. The pex5-10 T-DNA insertion results in an in- verted into IAA via β-oxidation in peroxisomes (Strader & Bartel, ternal deletion removing several of the predicted PEX14-binding 2011; Strader, Culler, Cohen, & Bartel, 2010). Thus, in seedlings motifs (Khan & Zolman, 2010; Zolman et al., 2005), and pex5-1 car- with functioning peroxisomes, IBA treatment confers high-auxin ries a Ser318Leu substitution in the putative PEX7-binding domain phenotypes including reduced root and hypocotyl elongation (Woodward & Bartel, 2005a; Zolman et al., 2000). Like pex5-1, the (Strader et al., 2011; Zolman et al., 2000), and IBA-resistance pex5-2 mutant accumulated apparently full-length PEX5 protein screens have uncovered genes needed for peroxisome biogenesis (Figure 2b). Of the three alleles, pex5-2 displayed the most robust and function (reviewed in Bartel et al., 2014). As salt increases IBA resistance (Figure 2a) whereas pex5-1 and pex5-10 displayed Arabidopsis peroxisome abundance (Fahy et al., 2017; Frick & more severe PMDH and thiolase PTS2-processing defects than Strader, 2018; Mitsuya et al., 2010), we reasoned that screening pex5-2 (Figure 2b). for IBA resistance in the presence of salt might uncover factors pex5-2 also displayed growth defects. Unlike pex5-1 shoots, which impacting salinity-induced peroxisome proliferation. We therefore resembled wild type, pex5-2 shoots were smaller than wild type and identified seedlings with elongated roots on normally inhibitory more similar to pex5-10 (Figure 2c). In contrast, pex5-2 seedling roots concentrations of IBA and NaCl. Subsequent analyses of a mutant elongated similar to wild type and pex5-1 on sucrose-supplemented emerging from this screen showed reduced response to IBA in the media (Figure 2a,c). As with IBA responsiveness (Figure 2a), express- presence of NaCl, but this mutant was even less IBA responsive ing wild-type PEX5 in the pex5-2 mutant improved rosette size in in the absence of NaCl (Supporting Information Figure S1a), sug- the mutant (Figure 2c), indicating that these defects were caused by gesting that we had not disrupted a proliferation-related gene. decreased PEX5 function. Moreover, root growth of this mutant was also resistant to inhibi- tion by 2,4-dichlorophenoxybutryic acid (Supporting Information 3.2 | pex5-2 exhibits PTS1 but not PTS2 Figure S1b), which, like IBA, requires peroxisomal chain shortening import defects for activity (Hayashi, Toriyama, Kondo, & Nishimura, 1998). In contrast, the mutant root growth was inhibited similar to wild type by As the pex5-2 mutant presented more complete IBA resistance the synthetic auxin 2,4-dichlorophenoxyacetic acid (Supporting but less severe defects in processing PTS2 proteins than pex5-1, Information Figure S1b), suggesting that general auxin responsive- we directly compared peroxisomal matrix protein import in these ness was intact. We concluded that the mutant defects stemmed alleles by using confocal microscopy to visualize import of PTS1- from decreased peroxisome function and we selected the mutant and PTS2-tagged GFP reporters (Woodward & Bartel, 2005a; for in-depth analysis. Zolman & Bartel, 2004). Wild type showed the expected GFP- Whole-genome sequencing of genomic DNA from this mutant PTS1 and PTS2-GFP puncta in seedling cotyledons and hypoco- revealed a homozygous G-to-A missense mutation in the PEX5 gene tyls (Figure 3a,b), indicating efficient PTS1 and PTS2 import. As (Supporting Information Figure S2), and we named the mutant pex5- previously reported (Woodward & Bartel, 2005a), pex5-1 dis- 2 (Figure 1). The pex5-2 mutation results in a Gly498Arg substitu- played wild-type-like GFP-PTS1 puncta (Figure 3a, Supporting tion in one of the seven tetratricopeptide repeat (TPR) domains Information Figure S3) coupled with largely cytosolic PTS2-GFP (Figure 1) that comprise the PTS1-binding region of PEX5 (Gatto, fluorescence (Figure 3b, Supporting Information Figure S4). In Geisbrecht, Gould, & Berg, 2000; Terlecky, Nuttley, McCollum, Sock, contrast, pex5-2 showed a mixture of cytosolic and punctate & Subramani, 1995). The crystal structure of the human PEX5 TPR GFP-PTS1 fluorescence (Figure 3a, Supporting Information domain (Stanley et al., 2006) reveals that each TPR consists of two α- Figure S3) and punctate PTS2-GFP fluorescence (Figure 3b, helices that pack together to form a PTS1-binding pocket (Figure 1c). Supporting Information Figure S4). Quantification of punctate The Gly498Arg substitution in pex5-2 is in the middle of the first (peroxisomal) versus dispersed (cytosolic) fluorescence re- predicted α-helix of TPR2 (Figure 1b,c) and alters a Gly residue that vealed that pex5-2 imported only a fraction (20%–50% in coty- is conserved in diverse PEX5 orthologs (Figure 1b). ledons; 5%–12% in hypocotyls) of GFP-PTS1, whereas wild type In addition to strong IBA resistance (Figure 2a), the pex5-2 mu- and pex5-1 imported more than 90% of GFP-PTS1 (Supporting tant exhibited a slight defect in processing of the peroxisomal ma- Information Figure S3). Conversely, pex5-2 imported PTS2- late dehydrogenase (PMDH) PTS2 protein (Figure 2b). IBA resistance GFP at least as well as wild type (over 90%) compared to less was closely linked to the pex5-2 mutation; 12 of 12 IBA-resistant F2 than 15% PTS2-GFP import for pex5-1 (Supporting Information seedlings from a backcross to wild type were pex5-2 homozygotes. Figure S4). Although PTS2-GFP fluorescence appeared brighter Moreover, overexpressing wild-type PEX5 using the constitutive in pex5-2 than wild type (Figure 3b), images collected at dif- cauliflower mosaic virus 35S promoter restored both IBA respon- ferent gain settings revealed that peroxisomes were of similar siveness and PTS2 processing to pex5-2 seedlings (Figure 2a,b). sizes in pex5-2 and wild type (Supporting Information Figure These linkage and complementation experiments confirmed that the S4a,b), and immunoblotting revealed more GFP in the pex5-2 6 | PATEL et al.

line compared to wild type or pex5-1 (Supporting Information Figure S4c). As the transgene in pex5-2 was introduced by cross- ing from the wild-type line, this expression difference is likely due to different degrees of silencing in the different lines. We concluded that the pex5-2 lesion in the PTS1-binding TPR domain (Figure 1) specifically impaired PTS1 import while leaving PTS2 import intact.

3.3 | Elevated growth temperature exacerbates pex5-2 physiological and molecular defects

After importing PTS1 cargo, PEX5 is ubiquitinated via the peroxisomal ubiquitination machinery. PEX4 is a ubiquitin-conjugating enzyme implicated in the ubiquitination that facilitates PEX5 retrotranslocation from the peroxisome membrane. The peroxisomal impairments of the pex4-1 mutant (Zolman et al., 2005) are less severe when seedlings are grown at elevated temperature in the dark (Kao & Bartel, 2015). Moreover, PEX5 levels are lower in dark-grown seedlings at elevated growth temperature (Kao & Bartel, 2015). These results suggest the possibility that accumulated PEX5 protein contributes to pex4-1 physiological defects. To examine whether growth temperature also impacted pex5-2 phenotypes, we compared growth at three temperatures. In light-grown seedlings, pex5-2 roots were highly IBA resistant when grown at all tested temperatures (16, 22, or 28°C) (Figure 4a), whereas pex5-2 root growth without sucrose was impaired at the normal growth temperature (22°C), further impaired at 28°C, but wild type-like at 16°C (Figure 4b). Similar to light-grown roots, pex5-2 dark-grown hypocotyls were highly IBA resistant regardless of growth temperature (Figure 4c), whereas pex5-2 hypocotyl growth without sucrose was most impaired at high temperature (Figure 4d). Moreover, growth at 28°C re- sulted in slight accumulation of the precursor form of PMDH in light-grown pex5-2 seedlings (Figure 4e), suggesting worsened PTS2 processing at higher temperature, whereas PMDH processing was nearly complete in pex5-2 seedlings grown at 16°C (Figure 4e), suggesting improved PTS2 processing at low temperature. In contrast to pex5-2, PMDH processing in pex4-1 was FIGURE 2 pex5-2 displays peroxisome-related defects that improved at higher temperature and exacerbated at lower tem- are rescued by PEX5 overexpression. (a) pex5-2 is resistant to the perature (Figure 4e). Temperature seemed to have a less severe inhibitory effects of IBA on root elongation. Bars indicate mean root lengths of 8-day-old seedlings grown in light in the absence or impact on pex5-1 than on pex5-2. For example, PTS2 processing presence of 10 μM IBA on media supplemented with 0.5% sucrose of PMDH was unchanged in pex5-1 grown at various tempera- (n = 12). Error bars indicate standard deviations. Different letters tures (Figure 4e). within bars indicate significant differences between genotypes We also examined PEX5 levels following growth at these three following IBA treatment (one-way ANOVA, p < 0.001). Percentages temperatures. Interestingly, the general decline in PEX5 levels that above bars indicate relative elongation for each line on IBA compared to mock conditions. (b) pex5-2 displays a slight defect in accompanied higher growth temperature in dark-grown wild-type PTS2 processing. Extracts from 8-day-old light-grown seedlings seedlings (Figure 4f; Kao & Bartel, 2015) was not observed in light- were processed for immunoblotting and probed with antibodies grown seedlings (Figure 4e). pex5-2 seedlings generally accumu- to the indicated proteins. PMDH and thiolase are synthesized lated less PEX5 protein than wild type at all temperatures tested as precursor (p) proteins that are processed into mature forms (Figure 4e,f), suggesting that the worsened physiological (Figure 4b) (m) after peroxisome entry. HSC70 was used to monitor loading. and PTS2-processing defects (Figure 4e) of pex5-2 at high tempera- (c) pex5-2 displays slight growth defects. Plants were grown on agar-based medium containing 0.5% sucrose for 8 or 15 days, and ture did not stem from a magnified decrease in overall pex5-2 pro- seedlings were moved to a new plate and photographed tein level at high temperature. PATEL et al. | 7

FIGURE 3 pex5-2 displays defective GFP-PTS1 import (a) and efficient PTS2-GFP import (b). Cotyledon epidermal cells (top rows) and hypocotyl cells (bottom rows) of 4-day-old light-grown seedlings carrying the 35S:GFP-PTS1 (Zolman & Bartel, 2004) or 35S:PTS2-GFP (Woodward & Bartel, 2005a) transgenes were visualized using confocal microscopy (scale bars = 50 μm). The middle rows show digital enlargements of the regions boxed in the top rows (scale bars = 25 μm)

4 | DISCUSSION mutants carried a PEX5 missense mutation (Zolman et al., 2000). This allele, pex5-1, has normal PTS1 import coupled with substantial PTS2 import defects (Figure 3; Woodward & Bartel, 2005a), 4.1 | A novel pex5 allele displaying PTS1-specific presumably because the pex5-1 mutation disrupts a predicted defects PEX7-binding region (Figure 1a). Subsequent analysis of a T-DNA Most Arabidopsis peroxisomal matrix proteins carry a PTS1 insertional mutant, pex5-10 (Zolman et al., 2005), revealed both (Reumann, 2004) and are delivered to peroxisomes via the PTS1 PTS1 and PTS2 import defects (Khan & Zolman, 2010; Lingard & receptor PEX5. One of the first reported Arabidopsis peroxin Bartel, 2009) due to an internal deletion in the PEX5 protein that 8 | PATEL et al.

FIGURE 4 Growth at elevated temperature exacerbates some pex5-2 defects. (a–d) Seedlings were grown in the light (a, b) or the dark (c, d) on medium containing sucrose, containing sucrose and IBA (a, c), or lacking sucrose (b, d). Light-grown root lengths (a, b) or dark-grown hypocotyl lengths (c, d) were normalized to the corresponding mean length on sucrose-supplemented medium. Bars show the means of three (28°C) or four (16°C and 22°C) biological replicates (each with ≥12 seedlings); error bars show standard deviations. Different lowercase letters within bars indicate significant differences within a genotype at different temperatures (one-way ANOVA, p < 0.05). Different uppercase letters above bars indicate significant differences between genotypes at the same temperature (one-way ANOVA, p < 0.05). (e, f) Extracts from seedlings grown at the indicated temperatures in the light (e) or dark (f) were processed for immunoblotting and probed with antibodies to the indicated proteins. PEX5 levels (quantified using ImageJ) were normalized to HSC70 and then to the 22°C Wt level (set at 1.0) to give the numbers below the PEX5 panels (means of three biological replicates, including the one shown). PMDH and thiolase are synthesized as precursor (p) proteins that are processed into mature forms (m) after peroxisome entry. The percentages of unprocessed PMDH (means of three biological replicates, including the one shown) in the mutants are shown below the PMDH panel. HSC70 was used to monitor loading includes several predicted PEX14-binding domains (Figure 1a; et al., 2000), we report the first pex5 mutant specifically impaired Khan & Zolman, 2010). Similarly, expressing a truncated pex5 pro- in PTS1 import. In addition to inefficient PTS1 import (Figure 3, tein (1–657 aa) that lacks TPR7 and the C-terminal region in wild Supporting Information Figure S3), the pex5-2 mutant displayed type or reducing PEX5 levels using RNAi confers both PTS1 and typical peroxisome defects, including poor growth without sucrose PTS2 import defects (Hayashi et al., 2005). In this work, two dec- supplementation (Figure 4b,d), resistance to IBA-mediated inhibi- ades after the first forward genetic screens for Arabidopsis seed- tion of root and hypocotyl elongation (Figures 2a, 4a,c), and slight lings with peroxisome-related defects (Hayashi et al., 1998; Zolman accumulation of an unprocessed PTS2 protein (Figures 2b and 4e). PATEL et al. | 9

The Gly498Arg substitution in pex5-2 is in the second of seven Seedling pex5-2 protein levels were slightly lower than pex5-1 TPR domains in the PEX5 C-terminal region (Figure 1). Although or wild-type PEX5 protein levels (Figure 4e,f). Decreased pex5-2 examination of the crystal structure of the human PEX5 TPR do- protein levels could reflect a TPR-folding defect that increases main (Stanley et al., 2006) suggests that the affected Gly resi- PEX5 susceptibility to degradation. Alternatively or in addition, due does not directly interact with the PTS1 of the cargo protein PTS1 cargo binding, which we expect to be impaired by the pex5-2 (Figure 1c), this residue is conserved in PEX5 orthologs in meta- mutation, might protect PEX5 from degradation. Low growth tem- zoans, trypanosomes, and fungi (Figure 1b). PTS1 import was sub- perature slightly increased pex5-2 protein levels in dark-grown stantially impaired in the pex5-2 mutant (Figure 3a), suggesting seedlings but not light-grown seedlings (Figure 4e,f). However, that the Gly-to-Arg substitution might impede folding of TPR2 or because we observed the most dramatic impacts of temperature the TPR domain in general, thus reducing PTS1 binding. on pex5-2 physiological and molecular defects in light-grown seed- Although the PTS2 pathway is absent in fruit flies (Faust, Verma, lings (Figure 4b,e), it seems likely that pex5-2 defects stem more Peng, & McNew, 2012), nematodes (Gurvitz et al., 2000; Motley, directly from altered pex5-2 function caused by the Gly498Arg Hettema, Ketting, Plasterk, & Tabak, 2000), and diatoms (Gonzalez change and not solely from decreased pex5-2 levels. et al., 2011), plant peroxisomes use both PTS1 and PTS2 pathways. In addition to pex5-2, other Arabidopsis mutants with low Given the reciprocal GFP-PTS1 and PTS2-GFP import defects in PEX5 levels include pex6 and pex26 mutants (Gonzalez et al., pex5-2 and pex5-1 (Figure 3), comparing the phenotypes of these 2017; Zolman & Bartel, 2004), which show defects in retrotrans- mutants can start to illuminate the relative importance of PTS1 locating PEX5 from the peroxisomal membrane (Gonzalez et al., versus PTS2 import. For example, both pex5-1 and pex5-2 exhibit 2017; Ratzel et al., 2011), and pex7 mutants (Ramón & Bartel, IBA resistance (Figures 2a, 4a,c) and growth defects that can be 2010), which are defective in the PEX5-interacting PTS2 receptor ameliorated by sucrose (Figure 4b,d), suggesting that β-oxidation and show import defects of not only PTS2 proteins but also PTS1 requires both PTS1 and PTS2 enzymes. Although all of the en- proteins (Ramón & Bartel, 2010; Woodward & Bartel, 2005a). zymes implicated exclusively in IBA β-oxidation are PTS1 proteins Interestingly, reducing function of PEX2, one of the peroxisomal (Strader et al., 2011; Zolman, Martinez, Millius, Adham, & Bartel, RING ubiquitin-protein ligases, restores PEX5 levels in pex6-1 and 2008; Zolman, Nyberg, & Bartel, 2007), the 3-ketoacyl-CoA thio- pex26-1 but not pex7-1 mutants (Burkhart et al., 2014; Gonzalez lase catalyzing the final step of both IBA and fatty acid β-oxidation et al., 2017), suggesting multiple avenues for PEX5 degradation. (Germain et al., 2001; Hayashi et al., 1998; Strader & Bartel, 2011) It will be interesting to learn whether the apparent pex5-2 insta- is a PTS2 protein. Moreover, the acyl-CoA oxidases acting early bility that we observe can be attributed to the peroxisomal ubiq- in fatty acid β-oxidation include both PTS1- and PTS2-containing uitination machinery. isozymes (Adham, Zolman, Millius, & Bartel, 2005). In contrast to When PEX5 retrotranslocation is inefficient, as in mammalian shared β-oxidation defects, we observe shoot growth defects in pex1 mutants, PEX5 ubiquitination is associated with peroxisome pex5-2 but not pex5-1 (Figure 2c), perhaps because the photore- degradation via specialized autophagy (Law et al., 2017). Similarly, spiration enzymes glycolate oxidase, Ser:glyoxylate aminotrans- genetically preventing autophagy improves peroxisome function in ferase, glyoxylate:Glu aminotransferase, and hydroxypyruvate Arabidopsis pex1 and pex6 mutants (Gonzalez et al., 2018; Rinaldi reductase are all PTS1 proteins (Fukao, Hayashi, & Nishimura, et al., 2017). The allelic series of pex5 mutants may be useful in fu- 2002) that we predict would be efficiently imported into pex5-1 ture dissection of the possible role of PEX5 in promoting autophagy but not pex5-2 peroxisomes. of peroxisomes (pexophagy) in plants.

4.2 | Genetic and environmental controls of PEX5 4.3 | Consequences and causes of PTS1-specific levels and function import defects

Growth temperature can influence peroxisome function in pex One hallmark of pex mutants is reduced PTS2 processing (re- mutants. For example, reduced growth temperature improves im- viewed in Bartel et al., 2014), which can vary in severity in dif- port of catalase, a PTS1 protein, in mammalian cell lines carrying ferent tissues or ages (Kao et al., 2016). The protease DEG15 mutations found in several groups of peroxisome biogenesis disor- cleaves the N-terminal PTS2-containing region (Helm et al., der patients (Fujiwara et al., 2000; Imamura, Tamura, et al., 1998; 2007; Schuhmann et al., 2008). As DEG15 is a PTS1 protein, Imamura, Tsukamoto, et al., 1998). In contrast, growth at elevated PTS2 processing is expected to require robust PTS1 and PTS2 temperature decreases PEX5 levels and improves growth, IBA re- import. Interestingly, however, the strong PTS1 import defect sponsiveness, and PTS2 processing in dark-grown pex4 seedlings observed in pex5-2 (Figure 3a) was accompanied by only mini- (Figure 4f; Kao & Bartel, 2015). The effects of temperature on pex4 mal PTS2-processing defects (Figures 2b, 4e,f), suggesting that defects suggest that the detrimental impact of excess PEX5 in the a small amount of DEG15 import is sufficient for substantial peroxisomal membrane when PEX4 is dysfunctional is partially re- PTS2 processing and that more severe PTS2-processing defects lieved by decreased overall PEX5 levels at elevated temperature probably reflect primarily PTS2 import defects. Of course, the (Kao & Bartel, 2015). ability to detect PTS2-processing defects relies on the stability 10 | PATEL et al. of the precursor protein in the cytosol, which might vary for et al., 2005), providing a valuable asset for future peroxisome different proteins. For example, thiolase precursor levels in- research. crease in pex seedlings treated with the MG132 proteasome inhibitor (Kao & Bartel, 2015), implying that cytosolic thiolase ACKNOWLEDGMENTS is susceptible to ubiquitin-dependent degradation. Differences in cytosolic precursor stability could contribute to the appar- We thank Steven M. Smith for antibodies recognizing PMDH, Lucia ent differences in PMDH and thiolase-processing efficiencies C. Strader for assistance with whole-genome sequencing, Joseph in pex5-2 (Figures 2b and 4e) and other pex mutants (Burkhart Faust for developing the script to facilitate analysis of whole- et al., 2014; Gonzalez et al., 2017, 2018; Kao et al., 2016; genome sequencing data, Mauro A. Rinaldi for mutagenized seeds Monroe-Augustus et al., 2011). of wild type carrying 35S:GFP-PTS1, and Sammira Rouhani for assist- In addition to pex5-2, several other Arabidopsis pex mutants ing with the mutant screen. We are grateful for critical comments display impaired PTS1 import but apparently normal PTS2 import. on the manuscript from Kathryn Smith, Melissa Traver, Zachary The pex2-1, pex4-1, and pex10-2 mutants, which are defective in the Wright, and Pierce Young. This research was supported by grants PEX5-recycling ubiquitination machinery, display punctate PTS2- from the National Institutes of Health (NIH, R01GM079177), the GFP and a mixture of cytosolic and punctate GFP-PTS1 (Burkhart National Science Foundation (MCB-1516966), and the Robert A. et al., 2014). These receptor-recycling mutants have slightly elevated Welch Foundation (C-1309) to BB. Confocal microscopy equip- PEX5 levels (Kao et al., 2016), and pex4-1 exhibits increased PEX5 ment was obtained through a Shared Instrumentation Grant (NIH membrane association (Kao & Bartel, 2015; Ratzel et al., 2011), as S10RR026399). Genomic sequencing at the Genome Technology expected when PEX5 recycling from the peroxisomal membrane is Access Center at Washington University School of Medicine was impaired. Both peroxisomal membrane-associated PEX5 and insuf- supported by the NIH (P30CA91842 and UL1RR024992). KJP was ficient PEX5-PTS1 recognition are detrimental to PTS1 import, in- supported in part by a Spurlino Undergraduate Summer Fellowship, dicating that the localization and quantity of PEX5 are important to YTK was supported in part by the Studying Abroad Scholarship from maintain peroxisome function. the Taiwan Ministry of Education, and RJL was supported in part by Salt (NaCl) increases Arabidopsis peroxisome abundance the NIH (F31GM125367). (Fahy et al., 2017; Frick & Strader, 2018; Mitsuya et al., 2010) and enhances the inhibitory effect of IBA (Supporting Information AUTHOR CONTRIBUTIONS Figure S1a), presumably because increased numbers of peroxisomes host more β-oxidation. Although pex5-2 was selected in a YTK and BB designed the research; YTK and KJP conducted the mu- screen for salinity-related peroxisome proliferation factors, sub- tant screen; KJP performed the physiological and molecular charac- sequent testing revealed that salt still increased IBA responsive- terizations; RJL, YTK, and KJP performed the microscopy; YTK, KJP, ness in pex5-2 (Supporting Information Figure S1a), suggesting and BB wrote the manuscript; all authors revised the manuscript and that the peroxisome proliferation machinery remained functional approved the final version. and that PTS1 import is not necessary for this response. In contrast, salt did not similarly improve IBA responsiveness in pex6-1 REFERENCES roots (Supporting Information Figure S1a), hinting that PEX6 may be involved in salt-induced peroxisome proliferation. Future iden- Adham, A. R., Zolman, B. K., Millius, A., & Bartel, B. (2005). 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