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

Abstract

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.

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 [–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 MegAlign program. The Arabidopsis PEX19A and PEX19B duplication is found in closely related plants, such as Arabidopsis lyrata and Capsella rubella, but not in more distantly related plants, such as Medicago or Brachypodium. (C) PEX19A and PEX19B gene diagrams showing T-DNA insertion sites with triangles, introns as lines, and exons as boxes.

Fig 2. Arabidopsis PEX19 is farnesylated.

(A) Proteins with a C-terminal CaaX motif (Cys-aliphatic-aliphatic-X; where X can be Ser, Met, Ala, Asn, or Cys) can be farnesylated by a protein farnesyl-transferase complex composed of PLP and ERA1, cleaved of the three carboxyl-terminal residues, and methylated (me) on the carboxyl group of the prenylated Cys residue [59]. (B) Proteins with a C-terminal a CaaL motif (Cys-aliphatic-aliphatic-Leu) can be geranylgeranylated by protein geranylgeranyl-transferase complex composed of PLP and GGB [59]. (C) PEX19 is farnesylated in vivo. Protein from 8-day-old light-grown seedlings was separated using 12% PAGE and processed for immunoblotting with antibodies recognizing PEX19 and HSC70 (loading control). The positions of the molecular mass markers (in kDa) are indicted at the right. The positions of unfarnesylated (u) and farnesylated (f) PEX19 are indicated at the left. An asterisk marks a protein that cross-reacts with the PEX19 antibody. (D) HA-PEX19 expression decreases farnesylation of endogenous PEX19. Protein extracted from 4-day-old light-grown seedlings was separated using 12% PAGE and processed for immunoblotting with antibodies recognizing PEX19 (top panel), the HA epitope (middle panel), and HSC70 (bottom panel; loading control). The positions of the molecular mass markers (in kDa) are indicted at the left. The positions of unfarnesylated (u), farnesylated (f), and HA-tagged PEX19 are indicated at the right. An asterisk marks a protein that cross-reacts with the PEX19 antibody.

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.

Fig 4. PEX19 farnesylation is not required to maintain peroxisome function.

(A) Light-grown pex19a-1, pex19b-1, and seedlings expressing HA-PEX19 display wild-type IBA sensitivity. The era1-2, ggb-3, and plp-4 prenylation mutants also are IBA sensitive. pex7-2 is an IBA-resistant control. Error bars show standard deviations of mean 8-day-old root lengths (n ≥ 7). Different letters above bars indicate significantly different means (one-way ANOVA, P < 0.001). (B) Dark-grown pex19a-1, pex19b-1, and seedlings expressing HA-PEX19 display wild-type IBA sensitivity and sucrose independence. The prenylation mutants are also IBA sensitive and sucrose independent. pex7-2 is an IBA-resistant control. Error bars show standard deviations of mean 5-day-old hypocotyl lengths (n ≥ 8). Different letters above bars indicate significantly different means (one-way ANOVA, P < 0.001). (C) pex19a-1, pex19b-1, prenylation mutants, and seedlings expressing HA-PEX19 display wild-type levels of several PMPs. Protein extracted from 4-day-old light-grown seedlings was separated using 10% PAGE and processed for immunoblotting. The membrane was serially probed with antibodies recognizing the indicated proteins. (D) pex19a-1, pex19b-1, prenylation mutants, and 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.

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 membrane-associated 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.

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 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 light-grown 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.

Fig 7. Reduced PEX19 function modulates defects of other peroxin mutants.

(A) pex19b-1 does not markedly alter the IBA responsiveness of pex13-4, pex14-2, pex2-1, or pex10-2. Error bars show standard deviations of mean 8-day-old light-grown root lengths of germinated seedlings (n ≥ 7). Different letters above bars indicate significantly different means (one-way ANOVA, P < 0.001). (B) pex19b-1 exacerbates the sucrose dependence of dark-grown pex14-2 seedlings. Error bars show standard deviations of mean 5-day-old hypocotyl lengths of germinated seedlings (n ≥ 5). No pex13-4 or pex19b-1 pex13-4 seeds germinated on medium lacking sucrose (n = 15). Different letters above bars indicate significantly different means (one-way ANOVA, P < 0.001). (C) pex19b-1 worsens the PTS2-processing defect of light-grown pex13-4 seedlings. Protein extracted from 8-day-old light-grown seedlings was separated using 10% PAGE and processed for immunoblotting with antibodies recognizing the indicated proteins. PMDH and thiolase are synthesized as precursors (p) with a cleavable PTS2 signals that are processed into the mature (m) proteins in the peroxisome.