The Early-Acting Peroxin PEX19 Is Redundantly Encoded, Farnesylated, and Essential for Viability in Arabidopsis thaliana

Peroxisomes are single-membrane bound organelles that are essential for normal development in plants and animals. In mammals and yeast, the peroxin (PEX) proteins PEX3 and PEX19 facilitate the early steps of peroxisome membrane protein (PMP) insertion and pre-peroxisome budding from the endoplasmic reticulum. The PEX3 membrane protein acts as a docking site for PEX19, a cytosolic chaperone for PMPs that delivers PMPs to the endoplasmic reticulum or peroxisomal membrane. PEX19 is farnesylated in yeast and mammals, and we used immunoblotting with prenylation mutants to show that PEX19 also is fully farnesylated in wild-type Arabidopsis thaliana plants. We examined insertional alleles disrupting either of the two Arabidopsis PEX19 isoforms, PEX19A or PEX19B, and detected similar levels of PEX19 protein in the pex19a-1 mutant and wild type; however, PEX19 protein was nearly undetectable in the pex19b-1 mutant. Despite the reduction in PEX19 levels in pex19b-1, both pex19a-1 and pex19b-1 single mutants lacked notable peroxisomal β-oxidation defects and displayed normal levels and localization of peroxisomal matrix and membrane proteins. The pex19a-1 pex19b-1 double mutant was embryo lethal, indicating a redundantly encoded critical role for PEX19 during embryogenesis. Expressing YFP-tagged versions of either PEX19 isoform rescued this lethality, confirming that PEX19A and PEX19B act redundantly in Arabidopsis. We observed that pex19b-1 enhanced peroxisome-related defects of a subset of peroxin-defective mutants, supporting a role for PEX19 in peroxisome function. Together, our data indicate that Arabidopsis PEX19 promotes peroxisome function and is essential for viability.

Peroxin (PEX) proteins function in de novo peroxisome biogenesis, division, and matrix protein import.Peroxisome matrix proteins are synthesized in the cytosol and usually are targeted to the peroxisome by a carboxyl-terminal three-amino acid peroxisome-targeting signal 1 (PTS1), which is recognized by the cytosolic PTS1 receptor, PEX5 [12].A less common targeting mechanism uses an N-terminal nine-amino acid PTS2, which is recognized by the cytosolic PTS2 receptor, PEX7 [13,14].Cargo-bound PEX5 and PEX7 associate with the docking proteins, PEX13 and PEX14, residing in the peroxisome membrane (reviewed in [15]).PEX5 forms part of a transient pore that facilitates cargo entry into the peroxisome [16], after which PEX5 is recycled with the assistance of PEX4, a ubiquitin-conjugating enzyme, and the ubiquitin-protein ligases PEX2, PEX10, and PEX12 (reviewed in [15]).Ubiquitinated PEX5 is retrotranslocated out of the peroxisome by the PEX1 and PEX6 ATPases [15], deubiquitinated, and released into the cytosol to be used for additional import.Inefficiently retrotranslocated PEX5 can be poly-ubiquitinated and degraded by the proteasome [15].
Peroxisomes can multiply by fission of pre-existing peroxisomes and by budding from the endoplasmic reticulum (ER; reviewed in [1]).Three peroxins, PEX19, PEX3, and PEX16 are implicated in de novo biogenesis of peroxisomes (reviewed in [17]).In mammals and plants, PEX16 resides in the ER membrane and recruits PEX3 [18,19], which in turn docks PEX19 [20].Some organisms, such as Saccharomyces cerevisiae, lack PEX16 and PEX3 appears to target to the ER directly (reviewed in [21]).Budding of pre-peroxisomes from the ER and PMP insertion require PEX3 and PEX19 in yeast [22]; pex3 and pex19 mutants appear to lack peroxisomes [23,24].PEX19 acts as a chaperone for peroxisome membrane proteins (PMPs), binding PMPs near their transmembrane domains [25] and allowing transfer to PEX3 and insertion in the membrane [26,27].PEX19 promotes PMP targeting to peroxisomes in human fibroblasts [28]; nuclear localization of PEX19 results in mislocalization of PMPs to the nucleus [29].
Although plants have homologs of the three early-acting peroxins [36], neither pex3 nor pex19 mutants has emerged from forward-genetic screens for mutants with defective peroxisome (reviewed in [10]), perhaps because PEX3 and PEX19 each have two isoforms in Arabidopsis.However, Arabidopsis RNAi lines targeting PEX3, PEX16, or PEX19 have enlarged peroxisomes that display reduced matrix protein import [37], confirming a role in plant peroxisome biology for the early-acting peroxin homologs.Moreover, Arabidopsis PEX19 binds to PEX10 [35] and PXA1 [38] in vitro, consistent with a PMP chaperone function.In this work, we explore the roles of PEX19 in Arabidopsis and reveal that Arabidopsis PEX19 is redundantly encoded, farnesylated, and essential for embryogenesis.
Seeds were surface-sterilized with 30% (v/v) commercial bleach, 0.01% (v/v) Triton X-100 and stratified for 1-3 days at 4°C in 0.1% agar.For experiments that included era1-2, 10 μM gibberellin (GA 3 ) was included in the stratification medium for all lines.Stratified seeds were plated on plant nutrient (PN) medium [44] or on PN supplemented with 0.5% sucrose (PNS), with or without IBA.IBA was dissolved in ethanol at 100 mM and control media were normalized to the same ethanol content.Seedlings transferred from plates to soil were grown at 22°C under continuous illumination.
For assays of light-grown seedlings, seeds were stratified for 1-3 days and plated on the indicated media.After 8 days of growth at 22°C under continuous light filtered through yellow long-pass filters, which slow the breakdown of indolic compounds [45], seedling roots were measured.For assays of dark-grown seedlings, seeds were stratified for 1-2 days, plated on the indicated media, placed under yellow light for one day, and then placed in darkness for 4 days.Hypocotyls of germinated seedlings were measured following the 4-day dark period.

Statistical analysis
One-way ANOVA analysis followed by Duncan's test was performed using the SPSS Statistics software program (version 22.0.0.1).For each treatment condition (e.g., no sucrose, IBA), mean root or hypocotyl lengths that were not significantly (P < 0.001) different from each other are designated with the same letter above the bar.

Confocal microscopy
The import of PTS2 proteins was observed using plants expressing 35S:PTS2-GFP, which encodes a GFP extended with the N-terminal 49 amino acid residues from the PED1 isoform of thiolase [54].Fluorescence in plants expressing 35S:YFP-PEX19A and 35S:YFP-PEX19B were compared to plants expressing 35S:YFP-ECH2, which marks the peroxisome matrix [5], 35S:ER-YFP-HDEL (ER-yk), which marks the ER lumen [55], and 35S:YFP [46], which marks the cytoplasm.For confocal imaging, cotyledons from 5-day-old light-grown seedlings were mounted in water under a cover slip.Images of epidermal cells were collected using a Carl Zeiss LSM 710 laser scanning confocal microscope equipped with a Meta detector.GFP samples imaged through a 40x oil immersion objective were excited with a 488-nm argon laser; emission was collected between 494 and 560 nm.Each image averaged 8 exposures using a 23μm pinhole corresponding to a 0.7 μm optical slice.YFP samples imaged through a 63x oil immersion objective were excited with a 488-nm argon laser; emission was collected between 493 and 555 nm.Each image averaged 4 exposures using a 44.8-μm pinhole corresponding to a 0.8 μm optical slice.

Fractionation
Fractionation of seedling extracts into organellar and cytosolic fractions was modified from Burkhart et al., 2014.Seeds (1 mg) were plated on PNS medium and incubated under white light for 5 days.Seedlings were minced with scissors in 1 mL ice-cold fractionation buffer [150 mM Tris pH 7.6, 10 mM KCl, 1 mM EDTA, 1 mM DTT, 100 mM sucrose, 1 mM PMSF, 1 mM NEM, 1x plant protease inhibitor cocktail (Sigma P9599)].Minced samples were transferred to a 1 mL Dounce homogenizer, homogenized for 20 strokes, and filtered through Miracloth (Millipore).Samples were centrifuged for 10 minutes at 640 rpm at 4°C to pellet unlysed cells, giving the homogenate fraction.Homogenate (200 μL) was centrifuged at 12,000 rpm for 20 minutes at 4°C, giving the supernatant fraction.The pellet was washed once with 200 μL fractionation buffer and centrifuged at 12,000 rpm for 20 minutes at 4°C, giving the wash fraction.The pellet was resuspended in fractionation buffer equal to the homogenate volume, giving the pellet fraction.Following fractionation, an aliquot of each fraction was added to equal volume of NuPAGE 2x sample buffer (Invitrogen), and 15 μL of each sample was processed for immunoblotting.

PEX19 is encoded by two genes in Arabidopsis
There are two isoforms of PEX19 in Arabidopsis thaliana [35] and closely related plants (Fig 1A).Although PEX19 duplications appear to have occurred more than once in the plant lineage, some plants carry only a single PEX19 gene (Fig 1B).The two Arabidopsis PEX19 genes were initially named AtPEX19-1 (At3g03490) and AtPEX19-2 (At5g17550) [35].For clarity in describing mutant alleles, in this work we refer to AtPEX19-1 as PEX19A and AtPEX19-2 as PEX19B.PEX19A and PEX19B are 84% identical at the amino acid level (Fig 1A) and are encoded by mRNAs that are 79% identical at the nucleotide level.To characterize the in vivo roles of the two PEX19 isoforms, we obtained two T-DNA alleles, one containing an insert in the third exon of PEX19A (SALK_020100), which we named pex19a-1 (Fig 1C ), and one containing an insert in the first intron of PEX19B (SAIL_76_C06), which we named pex19b-1 (Fig 1C).

Arabidopsis PEX19 is farnesylated
PEX19 is farnesylated in yeast and mammals [30,32].Farnesylation is a post-translational modification in which a 15-carbon hydrophobic moiety is attached to the Cys (C) residue in the carboxyl-terminal CaaX motif, where "a" is an aliphatic residue and "X" is Ser, Met, Ala, Gln, or Cys (reviewed in [33,59]; Fig 2A).Similarly, geranylgeranylation attaches a 20-carbon hydrophobic moiety to the Cys residue of a carboxyl-terminal CaaL (Fig 2B).Both prenylation variants can facilitate protein-membrane or protein-protein interactions [33].The conservation of a PEX19 farnesylation motif (CaaM) across several distantly related organisms (Fig 1A) suggests that this sequence is important for PEX19 function.
To determine if PEX19 is farnesylated in Arabidopsis, we generated an antibody to PEX19B and used immunoblotting to examine protein extracts prepared from three prenylation-defective mutants.We assayed a mutant (era1-2) with a fast-neutron induced deletion of the ENHANCED RESPONSE TO ABSCISIC ACID gene, which encodes the β-subunit of protein farnesyltransferase (Fig 2A) [39], a mutant (ggb-3) carrying a T-DNA insertion in the GERA-NYLGERANYLTRANSFERASE BETA (GGB) gene, which encodes the β-subunit of protein geranylgeranyltransferase (Fig 2B) [60], and a mutant (plp-4) disrupted in both types of prenylation because of a T-DNA insertion in the PLURIPETALA gene, which encodes the common α-subunit of both prenylation enzymes (Fig 2A and 2B) [61].The anti-PEX19 antibody detected an approximately 30-kDa protein in wild-type seedling extracts that migrated more slowly in the plp-4 mutant (Fig 2C ), implying that PEX19 is fully prenylated in wild type and that prenylation increases the electrophoretic mobility of the PEX19 protein.We also observed the more slowly migrating form of PEX19 in the era1-2 mutant, implying that PEX19 is largely farnesylated in wild-type Arabidopsis (Fig 2C).In contrast, PEX19 migration resembled wild type in ggb-3 (Fig 2C), indicating that Arabidopsis PEX19 is not appreciably .This analysis suggested that PEX19B is more abundant in seedlings than PEX19A.
To determine where PEX19A and PEX19B proteins accumulated, we compared PEX19 levels in various tissues from pex19a-1 to detect PEX19B and tissues from pex19b-1 to detect PEX19A.We examined PEX19 levels in roots and shoots from 8-day-old seedlings and from rosette leaves, cauline leaves, flowers, and green siliques from 31-day-old plants.In wild-type plants, PEX19 protein was detected in all examined tissues except mature rosette leaves, with highest accumulation in seedling shoots (Fig 3).PEX19 protein accumulation resembled wild type in the pex19a-1 mutant (Fig 3).In pex19b-1, we only detected PEX19 protein in seedlings aerial tissues, suggesting that PEX19B is the predominant isoform in many tissues but that PEX19A may function along with PEX19B in seedling aerial tissues.However, because our antibody detects PEX19A less effectively than PEX19B (Fig 2D ), we cannot rule out the possibility that PEX19A is present in tissues in addition to seedling shoots.We compared PEX19 protein accumulation patterns to peroxisomal malate dehydrogenase (PMDH) and found that PMDH accumulated more uniformly than PEX19 in various aerial tissues (Fig 3).The relatively higher PEX19 to PMDH ratio in seedlings versus mature leaves (Fig 3) suggests that PEX19, and perhaps peroxisome biogenesis, might be more important in rapidly growing leaves than in mature leaves.
We indirectly examined peroxisomal matrix protein import using the PTS2 protein PMDH to determine if the pex19a-1 or pex19b-1 displayed compromised PTS2-protein processing.The N-terminal PTS2-containing region is cleaved inside the peroxisome matrix following import, resulting in a molecular mass shift that can be detected by immunoblotting.The pex7-2 mutant displays a clear PTS2-processing defect [41]  PEX19 is implicated in inserting PMPs into membranes, and yeast pex19 mutants display reduced PMP levels [34].Therefore, we examined levels of three PMPs, peroxisomal ascorbate peroxidase (APX3) and the membrane peroxins PEX10 and PEX14 in pex19 mutants.We found that the levels of these PMPs were similar to wild type in pex19a-1, pex19b-1, the prenylation mutants, and the 35:HA-PEX19 lines (Fig 4C ), suggesting that altering PEX19 levels or prenylation did not dramatically alter PMP stability in Arabidopsis.
Because PMP levels were not altered in pex19 mutants, we examined whether membrane associations of PMPs were altered by using centrifugation to fractionate wild-type and mutant seedling extracts.We found PEX14 and APX3 in the pellet fraction in wild type, pex19a-1, and pex19b-1 (Fig 5), suggesting that these PMPs remain membrane-associated despite the low PEX19 protein levels in the pex19b-1 mutant.In addition, we detected PEX19 in the soluble fraction in wild-type and pex19a-1 seedlings (Fig 5), confirming previous reports that PEX19 is cytosolic in Arabidopsis [35] as it is in yeast [34] and mammals [29].Moreover, the peroxisomal matrix protein receptor, PEX5, was distributed similarly between the soluble and pellet fraction of wild type, pex19a-1, and pex19b-1 (Fig 5), suggesting that PEX5 localized normally in pex19a-1 and pex19b-1.
To enable rescue of the pex19a-1 pex19b-1 lethality, we transformed wild-type plants with constructs expressing N-terminally YFP-tagged PEX19A or PEX19B from the CaMV 35S promoter (35S:YFP-PEX19A and 35S:YFP-PEX19B).We found that both YFP-PEX19 proteins were localized similarly in a pattern that was neither punctate as exhibited by the YFP-ECH2 peroxisomal matrix protein [5]  To determine if the embryo lethality observed in the pex19a-1 pex19b-1 double mutant was due to the loss of PEX19 function rather than unrelated mutations in these lines, we crossed wild type carrying the 35S:YFP-PEX19A or 35S:YFP-PEX19B construct to plants heterozygous for pex19a-1 and homozygous for pex19b-1 and sought plants homozygous for pex19a-1, pex19b-1, and the reporter transgene in the progeny from the cross.We obtained such lines, wild-type seedlings expressing HA-PEX19 fully process the PTS2 region of PMDH.Protein from 8-day-old light-grown seedlings was separated using 10% PAGE and processed for immunoblotting with antibodies recognizing PMDH or HSC70 (loading control).The positions of the molecular mass markers (in kDa) are indicted at the left.PMDH is synthesized as a precursor (p) with a cleavable PTS2 signal that is processed into the mature (m) protein in the peroxisome; this processing is impaired in the pex7-2 mutant.(E) The pex19b-1 mutant displays normal import of peroxisomally-targeted GFP.GFP fluorescence of cotyledon epidermal cells from 5-day-old light-grown seedlings carrying the 35S:PTS2-GFP construct was imaged using confocal microscopy.doi:10.1371/journal.pone.0148335.g004Homogenates (H) from 5-day-old light-grown seedlings were fractionated using centrifugation to give a supernatant (S) containing soluble proteins, a wash (W) fraction, and a pellet (P) containing membraneassociated proteins.Fractions were separated using 10% PAGE and processed for immunoblotting with antibodies recognizing the indicated proteins.The positions of the molecular mass markers (in kDa) are indicted at the right.Antibodies recognizing HSC70 (a cytosolic protein) and mitochondrial ATP synthase (a membrane protein) were used to monitor fractionation.indicating that the lethality of pex19a-1 pex19b-1 double mutant was restored by either YFP--PEX19A or YFP-PEX19B.Because these lines were viable, we assessed peroxisome function in the rescue lines.Both wild type and the pex19a-1 pex19b-1 double mutant expressing YFP--PEX19A or YFP-PEX19B responded to IBA similarly to wild type (Fig 6F).However, we did observe a slight impairment in processing of PMDH in lines expressing YFP-PEX19B (Fig 6G ), suggesting that expressing YFP-tagged PEX19 (unlike HA-tagged PEX19; Fig 4D) was slightly detrimental to peroxisome function.We concluded that the lethality of the pex19a-1 pex19b-1 double mutant (Table 1) was indeed caused by loss of PEX19 function and that both PEX19 isoforms encode functional PEX19.

Discussion
PEX19 is an early-acting peroxin that binds and delivers a variety of PMPs to PEX3 for membrane insertion (reviewed in [17]).We found that Arabidopsis PEX19 accumulated most abundantly in aerial seedling tissues and was present at much reduced levels in mature rosette leaves (Fig 3), implying that PEX19 could be particularly important in early stages of plant development when peroxisomes are necessary to metabolize fatty acids and convert IBA to IAA.Despite a marked reduction in PEX19 levels in the pex19b-1 mutant (Fig 3), we found that Arabidopsis pex19a-1 and pex19b-1 single mutants displayed wild-type β-oxidation phenotypes (Fig 4A and 4B).Similarly, reducing levels of PEX19A or PEX19B via RNAi does not impart notable β-oxidation defects [37].Moreover, we found that both pex19a-1 and pex19b-1 processed PTS2 proteins normally and displayed wild-type levels and membrane association of tested PMPs (Figs 3-5).Therefore, we attempted to isolate a pex19a-1 pex19b-1 double mutant,  but double mutant seedlings were not recovered (Table 1).This lethality could be rescued by either YFP-PEX19A or YFP-PEX19B, indicating that the two PEX19 isoforms function redundantly and that the nearly undetectable amount of PEX19A remaining in the pex19b-1 mutant relying on YFP-PEX19A or YFP-PEX19B as the sole source of PEX19 respond to IBA similarly to wild-type seedlings.Error bars show standard deviations of mean 8-day-old light-grown root lengths (n 9).(G) Expression of YFP-PEX19B confers slight PTS2-processing defects.Protein extracted from 8-day-old lightgrown seedlings was separated in triplicate using 10% PAGE and processed for immunoblotting with antibodies recognizing PEX19 or GFP (to detect YFP-PEX19; top and middle panels) and PMDH (bottom panels).Membranes were subsequently probed with α-HSC70 (loading control).The positions of the molecular mass markers (in kDa) are indicted at the left.PMDH is synthesized as a precursor (p) with a cleavable PTS2 signal that is processed into the mature (m) protein in the peroxisome. is sufficient to provide PEX19 function in our growth conditions.Like the pex19a-1 pex19b-1 double mutant, embryo lethality has been reported for null alleles of most membrane peroxins in Arabidopsis (reviewed in [1]), and PEX19 mutations in the human peroxisome deficiency disease, Zellweger Syndrome, can result in death in infancy [66,67].PEX19 is farnesylated in yeast [23] and mammals [30].Although farnesylation increases the strength of PEX19-PMP interactions [31,32], farnesylation is not strictly required for PEX19 function in yeast or mammalian cells as overexpression of PEX19 derivatives that cannot be farnesylated rescues pex19 mutant defects [68].Using prenylation mutants, we observed that PEX19 is largely farnesylated in Arabidopsis (Fig 2).Like era1-1, the Arabidopsis farnesyltransferase β-subunit mutant, the yeast ram1 farnesyltransferase β-subunit mutant accumulates unprenylated PEX19 [32].Interestingly, yeast ram1 mutants display reduced levels of several PMPs [32].In contrast, the robust IBA responses of era1-2 and the plp-4 prenylation mutant (Fig 4A and 4B) and the normal levels of PMPs (Fig 4C) and PTS2 processing (Fig 4D ) that we observed in these mutants suggests that farnesylation of Arabidopsis PEX19 is not essential for peroxisome biogenesis or function in our growth conditions, even when unfarnesylated PEX19 is present at wild-type levels (Fig 2C).Thus a functional role for PEX19 prenylation, which is implied by the evolutionary conservation of this modification (Fig 1A ), is not revealed in our general physiological and molecular assays for peroxisome function.
Because reducing PEX19B levels did not seem to impact peroxisome function in an otherwise wild-type background, we constructed various double mutants with pex19b-1 to assess peroxisome function in sensitized backgrounds.The pex2-1 and pex10-2 partial loss-of-function mutants display relatively minor peroxisome-related defects that are dramatically enhanced in a pex2-1 pex10-2 double mutant [40].In contrast to this enhancement, the pex19b-1 pex2-1 and pex19b-1 pex10-2 double mutant resembled the respective pex2-1 and pex10-2 single mutants in IBA resistance and sucrose independence (Fig 7), despite the documented interaction between PEX19 and PEX10 in Arabidopsis [35], yeast [69], and mammalian cells [31].In contrast, pex19b-1 worsened the PTS2-processing defect of the pex13-4 partial loss-of-function allele and exacerbated the sucrose dependence of the pex14-2 null allele (Fig 7).This double mutant analysis is consistent with the possibility that the early steps in matrix protein import carried out by the receptor-docking peroxins PEX13 and PEX14 are more sensitive to PEX19 levels than are the PEX5-recycling steps carried out by the PEX2 and PEX10 RING-finger peroxins.Alternatively, it is possible that the more severe initial defects of the pex13 and pex14 alleles used in these experiments rendered these mutants more sensitive to reduced PEX19B levels.Future experiments with weaker pex13 [70] and pex14 [71] alleles might resolve this question.
In summary, we found that Arabidopsis PEX19 is farnesylated and essential for embryonic viability and that PEX19B encodes the predominant PEX19 isoform in Arabidopsis.The reduced PEX19 levels in the pex19b-1 mutant do not markedly impair peroxisome function in isolation but negatively impact peroxisome function in two mutants with existing defects.The mutants and reporters developed in this work will enable future examination of PEX19 function in plants.

Fig 1 .Fig 2 .
Fig 1. PEX19 is encoded by two genes in Arabidopsis.(A) Alignment of PEX19A and PEX19B from various plants (green) with the zebra fish (Danio rerio) and human (Homo sapiens) homologs (red), highlighting the carboxyl-terminal CaaM farnesylation motif (purple) and the domains implicated in PEX3 (brown) and PMP (blue) binding in human PEX19 [56-58].Sequences were aligned using MegAlign program (DNAStar) and the Clustal W method.Residues identical in at least seven sequences are boxed in black, chemically similar residues are boxed in gray.The sites of the T-DNA insertions in the pex19a-1 and pex19b-1 are indicated by triangles above the sequences.(B) Phylogenetic tree showing relationships of proteins in panel A generated by the

Fig 3 .
Fig 3.The pex19b-1 mutant lacks detectable PEX19 protein in various tissues.8-day-old wild-type, pex19a-1, and pex19b-1 seedlings were separated into roots and aerial tissues (shoots); other tissues were collected from 31-day-old plants: rosette leaf (beginning to senesce), oldest cauline leaf, open flowers, and green siliques (third elongated silique from the apex).Extracts were separated using 10% PAGE and processed for immunoblotting with antibodies recognizing PEX19, PMDH, and HSC70.The positions of the molecular mass markers (in kDa) are indicted at the right.An asterisk marks a protein that cross-reacts with the PEX19 antibody.doi:10.1371/journal.pone.0148335.g003 (Fig 4D) whereas pex19a-1, pex19b-1, the prenylation mutants, and the 35:HA-PEX19 lines all appeared to process PMDH similarly to wild type in seedlings (Fig 4D), where PEX19 is particularly abundant (Fig 3).Moreover, PMDH processing resembled wild type in various tissues and growth stages of the pex19 mutants (Fig 3), suggesting efficient import of matrix proteins in the pex19 mutants at additional developmental time points.We used confocal microscopy to directly examine PTS2 protein import in pex19b-1.Wildtype seedlings expressing 35S:PTS2-GFP [54] displayed the expected punctate pattern, indicating efficient matrix protein import, whereas pex7-2 displayed extensive cytosolic fluorescence [41] (Fig 4E).In pex19b-1, PTS2-GFP fluorescence resembled wild type (Fig 4E), again indicating successful import of PTS2-targeted proteins in this mutant.

Fig 5 .
Fig 5. PEX19 is a soluble protein, and PMPs remain membrane-associated in pex19 mutants.Homogenates (H) from 5-day-old light-grown seedlings were fractionated using centrifugation to give a supernatant (S) containing soluble proteins, a wash (W) fraction, and a pellet (P) containing membraneassociated proteins.Fractions were separated using 10% PAGE and processed for immunoblotting with antibodies recognizing the indicated proteins.The positions of the molecular mass markers (in kDa) are indicted at the right.Antibodies recognizing HSC70 (a cytosolic protein) and mitochondrial ATP synthase (a membrane protein) were used to monitor fractionation. doi:10.1371/journal.pone.0148335.g005

Fig 6 .
Fig 6.YFP-PEX19 is cytosolic and rescues the embryo lethality of the pex19a-1 pex19b-1 double mutant.(A-E) YFP-PEX19 is mostly cytosolic.Cotyledon epidermal cells from 5-day-old light-grown seedlings carrying various YFP-tagged constructs were imaged using confocal microscopy.YFP directed to the peroxisome (YFP-ECH2) displays punctate fluorescence (A) and ER-directed YFP (ER-YFP-HDEL) displays reticulated fluorescence (B).YFP-PEX19A (D) and YFP-PEX19B (E) fluorescence patterns are neither punctate nor reticulated but resemble untagged YFP fluorescence (C), suggesting cytosolic localization.Each pair of images captures the same cells imaged through the middle (left column) or subcortical region (right column) of the cells.See S1 Fig for corresponding bright-field images.(F) Seedlings

S1
Fig. YFP-PEX19 is mostly cytosolic.Cotyledon epidermal cells from 5-day-old lightgrown seedlings carrying various YFP-tagged constructs were imaged using confocal microscopy.YFP directed to the peroxisome (YFP-ECH2) displays punctate fluorescence (A) and ERdirected YFP (ER-YFP-HDEL) displays reticulated fluorescence (B).YFP-PEX19A (D) and YFP-PEX19B (E) show fluorescence patterns that are neither punctate nor reticulated but that resemble untagged YFP fluorescence (C), suggesting cytosolic For each construct, each row of images captures the same cells imaged through the middle (top row) or subcortical region (bottom row) of the cells.Columns show YFP fluorescence (left), bright field (middle), and merged images (right).(PDF) S1 Table.PCR-based markers used for genotyping mutant alleles.(PDF)