Identi¢Cation and Characterisation of PEX6 Orthologues from Plants

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Short sequence-paper Identi¢cation and characterisation of PEX6 orthologues from plants

Claude P. Kaplan 1;2, Josie E. Thomas 1, Wayne L. Charlton, Alison Baker *

Centre for Plant Sciences, LIBA, University of Leeds, Leeds LS2 9JT, UK

Received 8 February 2001; received in revised form 22 March 2001; accepted 26 March 2001

Abstract

The sunflower (Helianthus annuus) orthologue of PEX6, an AAA ATPase essential for the biogenesis of peroxisomes in yeasts and mammals, was isolated. HaPex6p is immunologically related to Pichia pastoris Pex6p. Like other genes involved in peroxisome biogenesis and function HaPEX6 mRNA and protein levels peak in early post-germinative growth and mRNA levels also increase in senescent tissue. HaPEX6 identifies probable orthologues in Arabidopsis and rice. ß 2001 Elsevier Science B.V. All rights reserved.

Keywords: Peroxin 6; PEX gene; Peroxisome biogenesis; Germination; Glyoxylate cycle

Peroxisome biogenesis is a complex and still genesis [7] but there are con£icting reports concern- poorly understood process that requires the function ing their cellular location and their function. In Yar- of at least 23 protein products (peroxins) encoded by rowia lipolytica a model of peroxisome growth by the PEX genes [1]. Two of these genes, PEX1 and membrane fusion has been proposed in which PEX6, encode members of the AAA family of ATP- Pex6p performs an essential priming function at an ases, a large superfamily of proteins involved in the early stage of biogenesis [8]. In contrast in Pichia ATP-dependent rearrangement of protein complexes pastoris, epistatic analysis of mutants places Pex1p [2]. As is the case for many peroxins the functions of and Pex6p function after the translocation of matrix Pex1p and Pex6p are uncertain. Yeast and mamma- proteins across the peroxisome membrane [9]. lian cells defective in either of these proteins have Although it is presently unclear how these two small peroxisomes with reduced matrix content and very di¡erent models of Pex1p and Pex6p function show reduced stability of the receptor (Pex5p) re- can be reconciled, evidence is emerging that the de- quired for the targeting and import of matrix pro- tails of peroxisome biogenesis and precise functions teins with the C-terminal PTS-1 signal [3,4]. Pex1p of peroxins may not be the same in all species. For and Pex6p interact physically and genetically [5,6] example Pex16p plays an essential role in biogenesis and this interaction is essential for peroxisome bio- of the peroxisome membrane in mammals but not in Yarrowia, and is completely lacking from the Saccha- romyces cerevisiae genome [10]. Likewise the Caeno- * Corresponding author. Fax: +44-113-233-3144; rhabditis elegans genome is lacking the PEX7 gene E-mail: [email protected] which encodes the receptor for a second import path- 1 The ¢rst two authors made equal contributions to this work. 2 Present address: The Technology Partnership plc, Melbourn way for matrix proteins [11]. Compared to yeasts and Science Park, Cambridge Road, Melbourn, Royston, Hertford- mammals, only PEX1 [12], PEX5 [13,14] and PEX14 shire SG8 6EE, UK. [15] from plants have been characterised. Mutation

BBAMCR 10414 22-5-01 174 C.P. Kaplan et al. / Biochimica et Biophysica Acta 1539 (2001) 173^180 of a putative Arabidopsis PEX16 orthologue SSE1 Total RNA from 2 day seedling cotyledons was resulted in an abnormality of seed storage reserve used for RT-PCR with primers S1 and AS2. The ¢rst deposition but no peroxisomal phenotype was re- ¢ve cycles were performed with an annealing temper- ported [16]. Plant peroxisomes are involved in a ature of 36³C followed by 35 cycles with an anneal- number of roles that are essential and in some cases ing temperature of 61³C. PCR products of the ex- unique, such as photorespiration [17] and formation pected size were eluted from a polyacrylamide gel of the plant growth regulator indole acetic acid from and re-ampli¢ed with S1 and AS3 using the same indole butyric acid [18]. Given that the complement cycling conditions. The products of the second and function of PEX genes may di¡er in di¡erent PCR reaction were puri¢ed, cloned and sequenced. species, and the important physiological roles of The PCR product corresponding to HaPEX6 was plant peroxisomes, the isolation and characterisation used to screen the sun£ower cDNA library. of plant orthologues of PEX genes will provide im- A 20-mer peptide CAELKKYEMLRDQFE- portant insights into these processes and provide GASSK-COOH corresponding to the carboxy-termi- tools for their biotechnological manipulation. nus of HaPex6p was synthesised coupled to bovine Sun£ower seeds (Tall single, Saunders seed mer- serum albumin with m-maleimidobenzoyl-N-hydrox- chants, Cambridge, UK) were sterilised 10 min in ysuccinimide ester and used to immunise a sheep. 0.5% hypochlorite solution and grown in vermiculite For a¤nity puri¢cation of anti-HaPex6p antibodies at 22^24³C in a 16/8 h light/dark cycle. RNA was the peptide was coupled to a Sulpholink column isolated according to [19] except that after the ¢rst (Pierce) and antibodies isolated according to the phenol/chloroform extraction DNA was selectively manufacturer's protocol. An antibody recognising degraded by the addition of 3 M sodium acetate the second AAA domain was raised in rabbit by pH 3.4 followed by ethanol precipitation and wash- immunisation of a fusion protein comprising a func- ing of the pellet with sodium acetate and 70% etha- tional IgG binding domain of Staphylococcus aureus nol. Poly(A) RNA was isolated by oligo(dT) chro- protein A (162 amino acids) and residues 624^669 of matography. A 2 day sun£ower seedling cDNA HaPex6p. For immunoblotting 10 sun£ower seed- library was prepared using the ZAP-cDNA Synthesis lings from each time point were ground in liquid kit (Stratagene) following the manufacturer's instruc- nitrogen, resuspended in SDS sample bu¡er, heated tions. Sun£ower RNA was separated by denaturing for 5 min at 95³C and centrifuged at 2292Ug for 15 formaldehyde gel electrophoresis. Nucleic acid trans- min. 50 Wl of the soluble fraction was analysed by fer and hybridisation were according to the Hybond- SDS^PAGE, blotted with a¤nity-puri¢ed anti-Ha- N protocol book (Amersham). Pex6p (1:100 dilution relative to the original serum) Degenerate primers S1 5P-GGGGAATTCTGG- and detected by enhanced chemiluminescence. For GANGA(T/C)(A/G)TNGG-3P, AS2 5P-GGGGGAT- pre-incubation with peptide a 1:10 dilution of anti- CC(G/C)CNA(T/C)(G/A)TACAT(G/A)TT-3P and body was mixed with 500 Wg/ml peptide, incubated AS3 5P-GGGAAGCTTGTICCNGGNGGNCC-3P for 30 min at 37³C then diluted 10-fold and applied were designed based on the sequences of S. cerevisiae to the membrane. In vitro transcription^translation (L20789) and P. pastoris Pex6p (Z22566) and a se- and immunoprecipitation were carried out according quence reported from the 5P-UTR of the maize gene to [21]. Sun£ower cotyledon homogenates were pre- B-peru (X70791) [20]. This was identi¢ed in a DNA pared and protein and enzymes assayed as described database search as having weak homology to several [22]. members of the AAA family, but especially PEX6. A 135 bp fragment of HaPEX6 was ampli¢ed by Close examination of the B-peru sequence reveals semi-nested RT-PCR and used as a probe to screen a that there is a piece of DNA comprising four exons sun£ower cDNA library. Two clones were isolated: bounded by well conserved plant splice junctions that Hap8.2 which contained a ca. 3.5 kb insert and has the potential to encode 93 amino acids with high Hap8.3 which contained an insert of 2.7 kb. Restric- similarity to Pex6 proteins. Most likely this is a part tion mapping and sequencing revealed that both of Zea mays PEX6 that has been isolated due to a clones contained double inserts but that Hap8.2 con- cloning artefact. tained a 2.5 kb EcoRI fragment corresponding to the

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Fig. 1. Deduced amino acid sequence of HaPex6p. P-loop motifs are boxed, Walker B motifs underlined, AAA family signature italic. The two regions used to raise antibodies are highlighted in bold. Internal methionines that are probably used for initiation of translation in vitro are shown in bold italic.

5P part of the HaPEX6 cDNA while Hap8.3 con- gram. The most similar entry is a hypothetical Ara- tained a 1.5 kb XhoI fragment corresponding to the bidopsis protein F10O3.18 on chromosome I (Swall 3P part of HaPEX6. The two clones were identical in accession number Q9SA70, P = 3.7e3227) followed 1.1 kb of sequence overlap so were ligated together by a hypothetical rice protein ZWH0005.1 (Swall at the unique internal XhoI site to produce Hap8.4. accession number Q9SMA4 P = 2.7e3197). The Re-screening the library by nested PCR failed to iso- next nine entries are peroxin-6 from mammals and late any longer clones. Hap8.4 (2875 bp) was fully yeasts with rat Pex6p being the most similar sequenced (EMBL accession number AJ305171). A (P = 1.2e3119) and Hansenula polymorpha the least genomic Southern blot indicates that HaPEX6 is a similar (P = 4.7e393). The CDC48 proteins form the single copy gene in sun£ower (data not shown). next most closely related members of the AAA fam- The HaPex6p sequence (Fig. 1) was used to search ily (P s 7.8e379). the Swall protein database with the BLASTp pro- The cDNA clone encodes a polypeptide of 909 ami-

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Fig. 2. Alignment of part of the second AAA domain of HaPEX6 with orthologues from plants, mammals and yeasts and compared with two other plant AAA proteins. Sequences were aligned with Clustal W. C- and G-boxes are regions in which sequence conservation is higher within AAA subfamily members than between them [23]. no acids (Fig. 1) with a calculated molecular mass for Pex6ps, suggesting that the clone contains most of 99 574 and a pI of 5.75. The calculated mass is but probably not quite all of the open reading frame. about 5% less than the experimentally determined The protein has 34^37% identity and 56^58% simi- value (Fig. 4C) and at the lower end of the size range larity to yeast and mammalian Pex6ps and like them

BBAMCR 10414 22-5-01 C.P. Kaplan et al. / Biochimica et Biophysica Acta 1539 (2001) 173^180 177 contains two AAA domains, the ¢rst of which is weakly conserved and has non-canonical Walker motifs. The P-loop has the consensus G/AXXXXGKT/S (boxed in Fig. 1). In the ¢rst AAA cassette only the GK are conserved in all Pex6p orthologues (not shown), while the P-loop of the second AAA cassette is perfectly conserved in all Pex6ps (Fig. 2). In contrast other AAA proteins with two AAA domains including CDC48 and Pex1p from Arabidopsis have two perfectly conserved P-loops. The AAA family contains hundreds of members involved in many bio- Fig. 3. HaPex6p is immunologically related to P. pastoris logical functions. A detailed sequence comparison Pex6p. The Hap8.4 cDNA clone was transcribed and translated [23] identi¢ed various sub-domains within the AAA in vitro and the resulting 35S-labelled polypeptides immunopre- cassette, some of which are highly conserved across cipitated with the antibodies indicated. Precipitated proteins all AAA protein sub-families while others show high- were separated by SDS^PAGE and radioactivity was detected by phosphoimaging. er conservation within sub-families than between them. A Clustal W alignment of part of the second AAA domain of HaPex6p, the putative Arabidopsis in the conserved second AAA cassette just N-termi- Pex6p and Pex6p from Homo sapiens, Rattus norve- nal to the P-loop and including the C-box. (Antigens gicus, P. pastoris and S. cerevisiae is shown in Fig. 2. are shown in bold in Fig. 1). In neither case was any For comparison an alignment between HaPex6p and product immunoprecipitated with the corresponding two other plant AAA proteins, Arabidopsis CDC48 preimmune serum (lanes 3 and 5). Antibodies raised and Arabidopsis Pex1p is also shown. Highlighted are against P. pastoris (Pp) Pex6p immunoprecipitated two of the domains, the C- and G-boxes [23], which HaPex6p (lane 6) but anti-ScCDC48 did not (lane show more conservation within than between fami- 7). A weak precipitation was seen with anti PpPex1 lies. It is clear that there is much greater conservation (lane 8). Thus the polypeptide encoded by Hap8.4 is between Pex6 proteins from all three kingdoms (7/22 immunologically related to PpPex6p and more and 17/32 identities in the C- and G-box respectively) weakly to PpPex1p. than in the three plant AAA proteins HaPex6p, At- Sequence similarity and immunological evidence Pex1p and AtCDC48 (2/23 and 11/31 identities). strongly suggest that Hap8.4 encodes the sun£ower To provide experimental evidence for the identity orthologue of Pex6p. If this gene is involved in per- of HaPEX6 clone, immunoprecipitation was used to oxisome biogenesis it should show an appropriate assess whether the protein encoded by the Hap8.4 expression pattern. In oilseeds like sun£ower and cDNA is immunologically related to known Pex6ps. Arabidopsis, peroxisomal L-oxidation and the glyoxy- When Hap8.4 was transcribed and translated in late cycle play a crucial role in the mobilisation of wheat germ lysate in the presence of [35S]methionine, reserves and the production of energy and carbon two products of 44 kDa and 46 kDa were synthesised skeletons to fuel post-germinative growth and seed- (Fig. 3, lane 1). Although the 7th residue from the N- ling establishment. During the ¢rst few days follow- terminus is methionine, the Kozak consensus is poor ing germination peroxisomes increase in size and/or and the product sizes suggest the utilisation of two number [24,25]. Homogenates were prepared from a internal methionines at positions 479 and 514/516. time course of sun£ower seedling development and This would result in the translation of a polypeptide assayed for the two marker enzymes of the glyoxy- consisting of the second (conserved) AAA cassette late cycle, isocitrate lyase (ICL) and malate synthase and the carboxy-terminus of the protein. Consistent (MS), and catalase as a general marker for glyoxy- with this both polypeptides were immunoprecipitated somes. Hydroxypyruvate reductase (HPR), a marker by antibodies raised against the C-terminal peptide for peroxisomal photorespiration, and chlorophyll of HaPex6p (lane 2), and by an antibody raised were also measured as indicators of the development against amino acids 624^669 (lane 4) which lies with- of photosynthetic competence (Fig. 4A). As de-

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Fig. 4. HaPEX6 shows a similar expression pro¢le to peroxisomal enzymes during post-germinative growth. (A) Extracts from sun- £ower cotyledons grown for the time indicated were assayed for the enzymes ICL, MS, catalase, HPR and chlorophyll. (B) Total RNA was extracted from a similar developmental time course and from green (G) and senescent (S) cotyledons and probed with 32P- radiolabelled Hap8.4. Radioactivity was detected by phosphoimaging. Equal amounts of RNA were loaded on each lane, based on spectrophotometric quantitation and ethidium bromide staining of a duplicate gel. (C) Protein extracts were obtained from whole seedlings at the time points indicated. Protein extract equivalent to 0.5 seedling was loaded on each lane, separated by SDS^PAGE, and immunoblotted with antibody raised against the C-terminal 20 amino acids of HaPex6p. (D) Extracts from the 72 h time point were blotted as in C except that the blot in the right-hand lane (+) was probed with antibody that had been pre-incubated with the C-terminal peptide. 6 scribed previously for sun£ower [26] and cucumber map location of two mutants, B11 and ibr4, isolated [27] ICL, MS and catalase increase in speci¢c activity due to their resistance to the inhibitory e¡ect of the over the ¢rst few days of post-germinative growth auxin indole-3-butyric acid on root elongation. B11 peaking, in this instance, at 96 h before declining. falls into the same phenotypic class as another mu- In contrast chlorophyll and HPR begin to accumu- tant, B44, which is a missense mutation in Pex5p, the late after 48 h, coincident with the onset of greening import receptor for PTS1 targeted peroxisomal pro- as the cotyledons begin to emerge. teins [18]. F10O3.18 is therefore a strong candidate Total RNA was prepared from a similar develop- for the defective gene in the B11 mutant. Primers mental time course and probed with a HaPEX6 designed against F10O3.18 ampli¢ed a partial probe. The size of the transcript was estimated to cDNA from a 2 day Arabidopsis library (data not be between 2900 and 3200 nucleotides, in reasonable shown) showing that it is expressed at this develop- agreement with the size of the Hap8.4 cDNA allow- mental stage along with other Arabidopsis PEX genes ing for polyadenylation. The HaPEX6 transcript is ([12], W.L. Charlton, unpublished data). These genes detectable at 48 h but increases in abundance up to and antibodies will prove useful tools to investigate 96 h before decreasing (Fig. 4B). This corresponds to the role of Pex6p in peroxisome biogenesis in plants. the period when the radical elongates and the coty- We thank Dr Andreas Beyer (Ruhr Universita«t, ledons just begin to emerge, and coincides with the Bochum, Germany) for drawing our attention to rapid increase in glyoxylate cycle marker enzymes the sequence similarity between PEX6 and the se- (Fig. 4A). The transcript was also detectable at a quence in the 5P-UTR of B-peru and Prof. Suresh low level in mature green leaves, and was more abun- Subramani (Univerity of California, San Diego, dant in senescent leaves. During senescence, glyoxy- CA, USA) for antibodies against Pichia pastoris late cycle enzymes are synthesised once again and Pex1p and Pex6p. taken up into peroxisomes [28]. AtPex1p shows a similar expression pattern [12]. In good agreement with the Northern data, Western blots on total pro- References tein extracts of sun£ower seeds with the anti-C-terminal peptide antibody detected a protein of 105 [1] L.J. Olsen, The surprising complexity of peroxisome biogen- kDa that peaks at 96 h then declines (Fig. 4C). esis, Plant Mol. Biol. 38 (1998) 163^189. [2] R.D. Vale, AAA proteins: lords of the ring, J. Cell Biol. 150 The detection of this polypeptide can be completely (2000) F13^F19. abolished by the pre-incubation of the antiserum [3] A. Spong, S. Subramani, Cloning and characterisation of with the peptide antigen (Fig. 4D). PAS5: A gene required for peroxisome biogenesis in the In summary, we have cloned a gene encoding an methylotropic yeast Pichia pastoris, J. Cell Biol. 123 (1993) AAA family member from sun£ower, which, on the 535^548. basis of sequence identity, immunological cross-reac- [4] T. Yahraus, N. Braverman, G. Dodt, J.E. Kalish, J.C. Mor- rell, H.W. Moser, D. Valle, S.J. Gould, The peroxisome bio- tivity and expression pro¢le can be identi¢ed as sun- genesis disorder group 4 gene PXAAA1 encodes a cytoplas- £ower Pex6p. Highly related ORFs were found in the mic ATPase required for stability of the PTS1 receptor, genomes of Arabidopsis and rice. Interestingly the EMBO J. 15 (1996) 2914^2923. Arabidopsis ORF F10O3.18 is very close to the [5] K.N. Faber, J.A. Heyman, S. Subramani, Two AAA family

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