Matrix proteins are inefficiently imported into Arabidopsis peroxisomes lacking the receptor-docking peroxin PEX14

Abstract

Mutations in peroxisome biogenesis proteins (peroxins) can lead to developmental deficiencies in various eukaryotes. PEX14 and PEX13 are peroxins involved in docking cargo-receptor complexes at the peroxisomal membrane, thus aiding in the transport of the cargo into the peroxisomal matrix. Genetic screens have revealed numerous Arabidopsis thaliana peroxins acting in peroxisomal matrix protein import; the viable alleles isolated through these screens are generally partial loss-of-function alleles, whereas null mutations that disrupt delivery of matrix proteins to peroxisomes can confer embryonic lethality. In this study, we used forward and reverse genetics in Arabidopsis to isolate four pex14 alleles. We found that all four alleles conferred reduced PEX14 mRNA levels and displayed physiological and molecular defects suggesting reduced but not abolished peroxisomal matrix protein import. The least severe pex14 allele, pex14-3, accumulated low levels of a C-terminally truncated PEX14 product that retained partial function. Surprisingly, even the severe pex14-2 allele, which lacked detectable PEX14 mRNA and PEX14 protein, was viable, fertile, and displayed residual peroxisome matrix protein import. As pex14 plants matured, import improved. Together, our data indicate that PEX14 facilitates, but is not essential for peroxisomal matrix protein import in plants.

Fig. 1

A series of pex14 alleles. a Recombination mapping of pex14-1 and pex14-4. Mapping with the PCR-based markers ILL2 (Davies et al. 1999), LFY (Konieczny and Ausubel 1993), MBK5 (Zolman et al. 2001), and MNA5 (Zolman et al. 2001) localized the defects to a region on the bottom of chromosome 5 that contains the PEX14/PED2 gene. b PEX14 has 12 exons (thick boxes) separated by 11 introns (lines). The positions of the pex14-2 and pex14-3 T-DNA insertions are indicated with triangles. The positions of the pex14-1 and pex14-4 lesions are marked with asterisks. pex14-1 has a 19-bp deletion beginning at position 1722 (where 1 is the A of the initiator ATG) resulting in one out-of-frame amino acid followed by an early stop codon. pex14-4 has a G-to-A mutation at position 2220 which alters the 5′ splice site of the eighth intron, resulting in 19 out-of-frame amino acids followed by an early stop codon. c Schematic showing putative domains in PEX14 and the locations of the defects in the four pex14 alleles. The bracket indicates the protein fragment used to generate the α-PEX14 antibody. d The four pex14 alleles display reduced (pex14-1, pex14-4) or undetectable (pex14-2, pex14-3) levels of full-length PEX14 mRNA. RNA extracted from 10-day-old light-grown seedlings was subjected to RNA gel-blot analysis probed with PEX14 (top panel). The bottom panel shows the ethidium bromide-stained gel prior to transfer. e The four pex14 alleles lack detectable full-length PEX14 protein, but pex14-1 and pex14-3 accumulate pex14 truncation products. Immunoblot of proteins from 8-day-old light-grown seedlings from the indicated lines probed with α-PEX14, α-PEX5, α-PEX6, α-PEX7, α-PEX13, and α-HSC70 (loading control) antibodies. The first and second panels are different exposures of the α-PEX14 panel to show the presence of the lower molecular mass pex14-1 and pex14-3 products in the longer (second) exposure. Positions of molecular mass markers (in kDa) are indicated on the left

Fig. 2

pex14 mutant seedlings are IBA resistant. a Root elongation on IBA. After 8 days of growth under yellow-filtered light on medium supplemented with the indicated concentration of IBA, seedlings were removed from the agar, and the length of the primary root was measured. Error bars represent standard deviations of the means (n ≥ 12). b Lateral root initiation. Lateral roots emerged from the primary root were counted (upper panel) and the lengths of the primary roots were measured (lower panel) 4 days after transfer of 4-day-old seedlings to unsupplemented medium or medium supplemented with the indicated concentration of IBA or NAA. Error bars represent standard deviations of the means (n ≥ 8)

Fig. 3

pex14 mutant seedlings are sucrose dependent. a Hypocotyl elongation in the dark. Seedlings were grown with or without 0.5% sucrose for 1 day under white light and 5 days in darkness, after which hypocotyl lengths were measured. Error bars represent standard deviations of the means (n ≥ 14). b Light-grown root elongation. Primary roots were measured after 8 days of growth with or without 0.5% sucrose under white light. Error bars represent standard deviations of the means (n ≥ 12)

Fig. 4

pex14 mutant growth defects. a, b Light-grown 14-day-old wild-type Col-0 (Wt), pex14, and pex5 seedlings on unsupplemented medium (a) or medium supplemented with 0.5% sucrose (b). c 21-day-old plants transferred to soil after 14 days on medium supplemented with 0.5% sucrose. d 28-day-old plants transferred to soil after 14 days on medium supplemented with 0.5% sucrose. e 41-day-old plants transferred to soil after 14 days on medium supplemented with 0.5% sucrose

Fig. 5

pex14 mutants display transient defects in PTS2 protein processing. Immunoblots of proteins prepared from 3- and 5-day-old seedlings (a), 6- and 9-day-old seedlings (b), or 35-day-old rosette leaves (c) probed with α-thiolase and α-PMDH2 antibodies, which recognize precursor (p) and mature (m) polypeptides, and α-HSC70, a loading control. Positions of molecular mass markers (in kDa) are indicated on the left. Upon processing, the N-terminal 4-kDa peptide containing the PTS2 is removed from the 48.5 kDa thiolase precursor and the 37.5 kDa PMDH precursor

Fig. 6

pex14 mutants display defects in peroxisome import of both PTS1- and PTS2-tagged matrix proteins. a Confocal microscopic images of cotyledon epidermal cells from 3-day- and 9-day-old light-grown seedlings of Col-0 (Wt) and pex14-1 or pex14-2 expressing GFP-ICL from the ICL promoter (Lingard et al. 2009). b, c Confocal microscopic images of cotyledon epidermal cells from 3-day- and 9-day-old light-grown seedlings of Col-0 (Wt) and pex14-1 expressing 35S-GFP-PTS1 (b) (Zolman and Bartel 2004) or 35S-PTS2-GFP (c) (Woodward and Bartel 2005). In a–c, the corresponding 3-day-old wild-type and mutant images were acquired with identical microscope settings and the 9-day-old wild-type and mutant images were acquired with identical microscope settings. Scale bar 20 μm. d–f Immunoblot of proteins prepared from 3- and 10-day-old seedlings probed sequentially with α-GFP, α-HSC70, a loading control (d), α-ICL (e), and α-thiolase (f). Precursor (p) and mature (m) PTS2 proteins (PTS2-GFP and thiolase) are marked on the right, and the position of GFP-PTS1 is marked on the left. An asterisk marks the position of a protein that cross-reacts with the ICL antibody. Positions of molecular mass markers (in kDa) are indicated on the left

Fig. 7

pex14-2 displays reduced organellar association of PTS1- and PTS2-cargo proteins and delayed PEX13 accumulation. Extracts from 3- and 10-day-old light-grown wild-type and pex14-2 seedlings were separated by centrifugation into soluble and organellar pellet fractions. For each sample, 1% of the total homogenate (H), 1% of the soluble fraction (S), and 25% of the pellet fraction (P) were separated using SDS–PAGE and processed for sequential immuno-blotting using the indicated antibodies. HSC70 and the mitochondrial membrane complex V subunit α (mito ATPase) were used as cytosolic and organellar controls, respectively. Precursor (p) and mature (m) proteins contain or lack the N-terminal PTS2 peptide, respectively. Positions of molecular mass markers (in kDa) are indicated on the left