Arabidopsis 22-kilodalton peroxisomal membrane protein. Nucleotide sequence analysis and biochemical characterization

Abstract

We sequenced and characterized PMP22 (22-kD peroxisomal membrane protein) from Arabidopsis, which shares 28% to 30% amino acid identity and 55% to 57% similarity to two related mammalian peroxisomal membrane proteins, PMP22 and Mpv17. Subcellular fractionation studies confirmed that the Arabidopsis PMP22 is a genuine peroxisomal membrane protein. Biochemical analyses established that the Arabidopsis PMP22 is an integral membrane protein that is completely embedded in the lipid bilayer. In vitro import assays demonstrated that the protein is inserted into the membrane posttranslationally in the absence of ATP, but that ATP stimulates the assembly into the native state. Arabidopsis PMP22 is expressed in all organs of the mature plant and in tissue-cultured cells. Expression of PMP22 is not associated with a specific peroxisome type, as it is detected in seeds and throughout postgerminative growth as cotyledon peroxisomes undergo conversion from glyoxysomes to leaf-type peroxisomes. Although PMP22 shows increased accumulation during the growth of young seedlings, its expression is not stimulated by light.

Figure 1

A, Clustal W alignment of the deduced amino acid sequences of Arabidopsis PMP22, mouse Mpv17 (P19258), human Mpv17 (P39210), mouse PMP22 (P42925), and rat PMP22 (Q07066). The thick underlining indicates the conserved regions mentioned in the text. B, Arabidopsis PMP22, mouse Mpv17, and rat PMP22 according to the algorithm of Kyte and Doolittle (1982) with an amino acid window of 19. Hydrophobic residues are depicted as positive and hydrophilic residues as negative.

Figure 2

Southern blot showing 10 μg of Arabidopsis genomic DNA digested with HindIII (lane 1), EcoRI (lane 2), PstI (lane 3), XbaI (lane 4), and PvuII (lane 5) probed with the [³²P]PMP22 open reading frame, washed under stringent conditions, and detected by autoradiography.

Figure 3

A, Immunoblot showing 150 μg of total protein extract from dark-grown Arabidopsis cell-suspension culture (lane 1) and 0.25 μg of recombinant purified hexahistidine-PMP22 (lane 2) probed with affinity-purified anti-PMP22 antibodies and detected by enhanced chemiluminescence. B, Equal amounts of protein (150 μg) extracted from various organs of Arabidopsis plants and from dark-grown tissue-cultured cells were separated by SDS-PAGE and probed with affinity-purified anti-PMP22 antibodies. Lane 1, Dark-grown tissue-culture cells; lane 2, flowers; lane 3, siliques; lane 4, stems; lane 5, upper leaves; lane 6, basal leaves; and lane 7, roots.

Figure 4

Subcellular localization of Arabidopsis PMP22 in leaf tissue by differential centrifugation. Arabidopsis leaves were fractionated as described in Methods. Equal amounts of protein (120 μg) were separated by SDS-PAGE, transferred to nitrocellulose, and probed with affinity-purified antibodies against PMP22 (A) or the 23-kD protein of the PSII oxygen-evolving complex (B). C, Catalase activity, a peroxisomal matrix marker (shaded bars) and chlorophyll (white bars) were measured in each fraction. Catalase recovery was 102% and chlorophyll recovery was 61%. Lane 1, Homogenate; lane 2, 1,000g supernatant; lane 3, 1,000g pellet; lane 4, 25,000g supernatant; lane 5, 25,000g pellet.

Figure 5

Subcellular localization of PMP22 in tissue culture cells by Suc-density-gradient centrifugation. A postnuclear supernatant prepared from dark-grown Arabidopsis suspension-cultured cells was separated on a 0.7 to 2.1 m continuous Suc-density gradient. Fractions were assayed for catalase (a peroxisomal marker), fumarase (a mitochondrial marker), NADH:Cyt c reductase (an ER marker), and protein. The recoveries of the enzyme activities relative to the postnuclear supernatant were: catalase, 76%; fumarase, 96%; and NADH Cyt c reductase, 91%. Equal volumes of the Suc-gradient fractions were separated by SDS-PAGE and probed with affinity-purified anti-PMP22 antibodies and anti-castor bean isocitrate lyase antibodies.

Figure 6

A, PMP22 is resistant to extraction by Na₂CO₃ and partitions in the detergent phase of Triton X-114. A 20,000g organelle pellet (P) and the corresponding supernatant (S) were prepared from dark-grown tissue-cultured cells and analyzed for PMP22 and isocitrate lyase (ICL) by SDS-PAGE and immunoblotting (20,000g). The 20,000g pellet fraction enriched in PMP22 was treated with 0.1 m Na₂CO₃ (pH 11.0) and centrifuged at 100,000g to produce a membrane pellet and supernatant fraction. A postnuclear supernatant prepared from dark-grown tissue-cultured cells was fractionated using 2% (v/v) Triton X-114 phase separation, and the various fractions were analyzed by SDS-PAGE and immunoblotting with antibodies against isocitrate lyase and affinity-purified anti-PMP22 antibodies. Lane 1, Postnuclear supernatant; lane 2, Triton X-114-insoluble pellet; lane 3, detergent phase; lane 4, aqueous phase; lane 5, “glycoprotein”-rich pellet; and lane 6, postaqueous supernatant. In all lanes 150 μg of protein was analyzed. B, Accessibility of Arabidopsis PMP22 to protease. The peroxisome-enriched 20,000g pellet fraction was subjected to hypotonic lysis followed by a wash with 250 mm NaCl. Salt-washed membranes (250 μg) were incubated with the protease thermolysin at the indicated concentrations (see Methods). After the incubation the protease was inhibited and the membrane-bound and protease-solubilized peptides were separated by ultracentrifugation. Triton X-100 was included in a duplicate incubation containing 100 μg mL⁻¹ thermolysin, which was not subjected to ultracentrifugation before analysis by SDS-PAGE and immunoblotting. All samples were analyzed by SDS-PAGE and immunoblotting with affinity-purified anti-PMP22 antibodies.

Figure 7

Insertion of Arabidopsis PMP22 into isolated peroxisomes. Radiolabeled PMP22 was prepared by in vitro transcription and translation in wheat germ lysate and incubated with peroxisomes isolated from 3-d postimbibition sunflower cotyledons (see Methods). Lane 1, 40% of the translation product added to the other reactions; lane 2, import reaction carried out in the presence of the ATP-regeneration system; lane 3, import in the presence of the ATP-regeneration system followed by protease treatment; lane 4, same as lane 3 but in buffer containing 1% Triton X-100 and 250 mm NaCl before the addition of protease; lane 5, import in the absence of ATP and the ATP-regenerating system; lane 6, import in the absence of ATP and the ATP-regenerating system followed by treatment with protease; lane 7, same as lane 3 but no peroxisomes were added to the import assay; lane 8, same as lane 2 but glyoxysomes were replaced with 50 μg of washed red blood cells from calf ascites; lane 9, same as lane 8 but treated with protease; lanes 10 to 15, Na₂CO₃ (pH 11.0) treated pellets and supernatants derived from import reactions (lane 10 is the pellet and lane 11 the supernatant of the import reaction in lane 2, lane 12 is the pellet and lane 13 the supernatant of the import reaction in lane 3; and lane 14 is the pellet and lane 15 the supernatant of the import reaction in lane 5).

Figure 8

A, Expression of PMP22 during seedling development. Total protein was extracted from an equal number (based on dry weight at sowing) of Arabidopsis seedlings at the indicated number of hours postimbibition. Protein samples were separated by SDS-PAGE and probed with affinity-purified anti-PMP22 antibodies, anti-castor bean isocitrate lyase antibodies (ICL), or affinity-purified anti-spinach glycolate oxidase antibodies (GO). B, Effects of light on expression of PMP22 in tissue cultures. Arabidopsis suspension-cultured cells were grown in either continuous light (lane 1) or total dark (lane 2), subcultured, and cultured for a further 5 d under the same conditions. Total protein was extracted from these samples and subjected to SDS-PAGE and immunoblotting with affinity-purified anti-PMP22 antibodies.