Four putative, glyoxysome membrane proteins are instead immunologically-related protein body membrane proteins

Plant Science

106

(1995) 215-226

Four putative, glyoxysome membrane proteins are instead immunologically-related protein body membrane proteins Jeff Bunkelmann, Francisco J. Corpas, Richard N. Trelease* Department of Botany, Arizona State University, Tempe, AZ 85287-1601. USA

Received 30 November 1994; revision received 27 January 1995; accepted 30 January 1995

Abstract Previous research revealed that peroxisomes/glyoxysomes in plants, mammals, and yeasts possessed a prominent, integral peroxisome membrane protein (PMP) with a molecular mass in the range 21-26 kDa. We also found a major,

low molecular mass polypeptide in the glyoxysome membrane fractions from four oilseed species: putative PMP26 in cotton (Gossypium hirsutum L.), PMP22 in cucumber (Cucumis sativus L.), PMP24.5 in sunflower (Heliauthus annuus L.), and PMP24 in castor bean (Ricks communis L.). Rabbit antiserum, produced against all the proteins which were solubilized from membranes recovered from isolated cotton glyoxysome fractions, recognized these four putative PMPs on Western blots as well as similar molecular mass polypeptides in mitochondria and protein bodies isolated from cotton and cucumber cotyledons. Postembedding, immunogold analyses of cotton and cucumber cotyledons, however, revealed that antibodies which were affinity-purified to the putative cotton glyoxysome 26-kDa polypeptide specifically bound to membranes of protein bodies, but not to membranes of glyoxysomes nor mitochondria. Antibodies to the bean tonoplast intrinsic protein ((r-TIP), a known protein body membrane protein (PBMP), also recognized the four putative PMPs on Western blots and co-localized with anti-cotton PMP26 IgGs on thin sections to membranes of protein bodies. Intact protein bodies were not the source of the PBMPs in the oilseed organelle fractions; unmunogold labeling revealed immunoreactive, small indiscrete vesicles interspersed among glyoxysomes and mitochondria in gradient fractions. Thus, putative prominent PMPs believed previously by us and others to be important in biogenesis and/or function of plant peroxisomes were discovered to be antigenically-related PBMPs of similar low molecular mass whose function in oilseeds has yet to be elucidated. Keywords: Glyoxysome; Peroxisome membrane protein; Protein body; Cotyledon; Endosperm

1. Introduction Peroxisomes are organelles that contain

single membrane-bound at least one H,O?-forming

* Corresponding author, Tel.: (+I-602) (+I-602) 965 6899.

965 2669;

Fax:

oxidase and catalase [l] and are found in almost all eukaryotic cells. These organelles participate in a variety of organism/tissue specific functions including P-oxidation of fatty acids and lipid synthesis [2,3]. Although the biogenesis and specific metabolic functions of several matrix enzymes are well defined, research on the peroxisome mem-

0168-9452/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDi 016%9452(95)04083-7

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brane has resulted in identification and partial characterization of only a few peroxisome integral membrane proteins (PMPs) in mammals, yeasts, and plants [4]. The presence of at least one prominent, low molecular mass polypeptide of 21-26 kDa is a conserved feature of this organelle in almost every species examined. Hart1 and Just [5] identified several prominent PMPs from rat liver with molecular masses of 69/70,36, and 22 kDa. PMP69 levels were doubled by oral administration of clotibrate, a peroxisome proliferator, while PMP22 levels were relatively unchanged. Bodnar and Rachubinski [6] also found similar PMPs in rat liver: PMP69/70, 50, 36, 22, and 15. Three GTP-binding proteins, PMP25, 27, 29 were located in the livers of clotibratetreated rats [7]. The importance of a PMP(s) in peroxisome biogenesis was demonstrated when a transfected rat gene encoding a 35-kDa PMP (peroxisome assembly factor 1 or PAF-1) restored peroxisome biogenesis in peroxisome-deficient CHO cells [8]. Low molecular mass PMPs were identified in several yeast species. Three PMPs were found in Candida tropicalis (PMP34, 29, 24); PMP24 was induced by growth on an oleic acid substrate [9]. Three major PMPs were identified in Saccharomyces cerevisiae including a PMP24 [lo]. In Hansenula polymorpha cells, a 22-kDa polypeptide was one of the four constitutive PMPs identified by Sulter et al. [ 111. Growth on ethanol induced the appearance of PMP24 in H. polymorpha. Most research on plant PMPs has been restricted to the glyoxysomes of oilseed seedlings. A prominent 24.5 kDa polypeptide was one of the four PMPs (57, 49, 31 kDa) identified in darkgrown sunflower seedlings [ 121. Chapman and Trelease [13] reported six PMPs in cotton cotyledons including a major membrane polypeptide of 24 kDa (renamed PMP26). Kruse and Kind1 [14] reported four PMPs (PMP63, 32, 24, and 2 1.5) in glyoxysomes of cucumber seedlings, whereas Cot-pas et al. [15] recently identified six PMPs in cucumber glyoxysomes (PMPs73, 61, 52, 36, 30, 22) to which they raised polyclonal antibodies. Preisig-Muller and Kind1 [16] identified a 53-kDa protein in cucumber glyoxysome fractions as a membrane-bound Dna-j protein. Donaldson and Gonzalez [ 171 identified nine putative PMPs

(93, 88, 75, 72, 57,46,

32, 30, and 24) in the membranes of endosperm glyoxysomes from germinated castor beans (3 days dark, post-imbibition). Only PMP24 and 30 were unique to glyoxysomes because similar molecular mass peptides were present in the endoplasmic reticulum fractions. Halpin et al. [18] only observed five PMPs in castor bean endosperm including a 33-kDa and a major 24kDa polypeptide. Struglics et al. 1191 reported that potato tuber peroxisomes possessed six PMPs, one of which was a prominent 22-kDa polypeptide. As documented above, a prominent 21- to 26kDa protein apparently exists in membranes of peroxisomes from a diversity of species. The PMP generally is regarded as important to peroxisome biogenesis/function, e.g. the PMP22 in rat liver likely is a porin [20]. It is important to note, however, that of the putative PMPs in this low molecular mass range, only the rat liver PMP22 was authenticated as a PMP via immunocytochemical localization to the peroxisome membrane [21,22]. Because of the common occurrence and possible common function among eukaryotic peroxisomes, we focused our study on these low molecular masses PMPs which were previously identified by us in four different oilseeds [12,13,15]. The approach here was to raise polyclonal antibodies to these putative PMPs by immunizing rabbits with the proteins solubilized in dodecylmaltoside/ aminocaproate from membranes recovered from cottonseed glyoxysome fractions. The intent was to use this antiserum and/or selected affmitypurified IgGs to test for immuno-relatedness among eukaryotic PMPs after the oilseed proteins were immunocytochemically authenticated as PMPs. Our results indicate that the prominent membrane proteins consistently identified by us and others in glyoxysome membrane fractions are not PMPs, hence do not share a common function with low molecular mass proteins ostensibly localized in membranes of other eukaryotic peroxisomes.

2. Material and methods 2.1. Plant material and growth conditions All seeds were germinated and grown in the dark at 30°C except as indicated. Cotton seeds,

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Gossypium hirsutum L. cv. Coker 1OOAglandless (kindly supplied by Dr Donald Hendrix, USDA Western Cotton Research Laboratory, Phoenix, AZ), were grown in darkness for 24 or 48 h according to Chapman and Trelease [23]. Cucumber seeds, Cucumis sativus L. (J.W. Jung Seed Company, Madison, WI) were aerated in distilled water for 30 min at 3O”C, transferred to water-saturated vermiculite, and dark-grown for 48 h. Sunflower seeds, Helianthus annuus L. (Tempe Feed & Tack, Tempe, AZ) were aerated in distilled water for 12 h, scrolled in paper strips for 24 h, transferred to vermiculite under a dim green light, and then grown for an additional 24 h in darkness according to Jiang et al. [ 121. Dehulled castor bean seeds, Ricinus communis L. (kindly supplied by Dr Thomas S. Moore, Jr.) were briefly rinsed in 0.5% (v/v) sodium hypochlorite from commercial bleach solution and grown in darkness for 72 h in sterilized, water-saturated vermiculite.

2.2. Isolation of organelles Cotton glyoxysomes and mitochondria were isolated from 48-h-old cotyledons according to the method described by Chapman and Trelease [24] with the following modifications. The homogenizing buffer contained in addition 1 mM phenylmethylsulfonyl fluoride (PMSF) and 2 mM dithiothreitol (DTT), and the organelles were fractionated dropwise from the linear sucrose gradient. Cucumber glyoxysomes and mitochondria were isolated from 48-h-old cotyledons according to the protocol for isolating cotton glyoxysomes, except for the modifications described by Corpas et al. [ 151. Glyoxysomes in endosperm of 72-h-old castor bean seedlings were isolated according the protocol described for cucumber glyoxysomes. Sunflower glyoxysomes were purified from cotyledons of 48-h-old seedlings as described by Jiang et al. [12]. Protein bodies were isolated from cotyledons of 24-h-old cotton seedlings in glycerol medium according to the method described by Yatsu and Jacks (251. 2.3. Preparation of glyoxysome fractions

membrane

proteins from

Membrane proteins were detergent-solubilized from the glyoxysome membrane fractions as described by Chapman and Trelease [ 131 with the

211

following modifications. After centrifugation of carbonate-washed membranes at 100 000 x g (45 min, 4”C), the integral membrane proteins were solubilized by resuspending the carbonate-washed pellet in a solution containing 56 ~1 of 10% (w/v) laurylmaltoside (dodecyl-&D-maltoside, Calbiothem) and 300 ~1 of 50 mM Tris-HCl, 0.75 M aminocaproic acid, pH 7.2 [26]. The resuspension was incubated on ice for 30 min with brief vortexing every 10 min, centrifuged at 100 000 x g (30 min, 4”C), and the supernatant (highly enriched with membrane proteins) was collected and stored at -80°C. Similar procedures were carried out by Jiang et al. [12] and Corpas et al. [15].

2.4. Electrophoresis of proteins Proteins were separated by SDS-PAGE (BioRad Protean II, 18 x 20 cm plates) according to Laemmli [27] using a 4% stacking gel and a linear 10-l 5% T running gel. Membrane and whole organelle proteins were mixed with an equal volume of 2x SDS sample buffer and heated for 15 min at 45°C. Samples and low molecular mass standards (Bio-Rad) were loaded onto a 1.5~mm gel and electrophoresed at 15 mA (constant current), 14”C, until the bromophenol blue front was 1 cm from the bottom of the gel. Protein bands were visualized by silver staining according to a modification [12] of the method described by Heukeshoven and Demick [28]. For immunoblotting, proteins were transferred from the SDS gel to a PVDF membrane (Immobilon P, Millipore) using a semi-dry blotter (Bio-Rad Trans-Blot SD) and a buffer system modified from Shiigger and von Jagow [26]. Four sheets of 3MM Whatman chromatography paper, the PVDF membrane, and the gel were soaked in anode solution (0.3 M Tris, 0.1 M glycine) for 20 min and then layered onto the anode plate. Four sheets of Whatman paper soaked in cathode solution (0.3 M aminocaproic acid, 0.03 M Tris) were layered over the gel. The proteins were electrophoresed onto the membrane at 1.2 to 1.5 rnA.cm-* for 2 h at room temperature. Protein blots were probed with antiserum or affinitypurified antibodies followed by anti-rabbit IgGsalkaline phosphatase conjugate (Sigma) and stained as described by Kunce and Trelease [29].

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2.5. Electron microscopy

Cucumber and cotton cotyledons (48 h, darkgrown) were sliced with a razor blade into
Science IO6 (1995) 215-226

through a 0.2-pm cellulose acetate filter. The sections were incubated overnight at room temperature on a drop of affinity-purified IgGs or antiserum diluted with blocking buffer containing 2% (v/v) Tween-20 (Sigma). After rinsing in buffer without Tween-20, the sections were incubated for 1 h on a drop of Protein A-gold (10 nm, EY Laboratories Inc.) or NanogoldfM (1.4 nm, Nanoprobe Inc.) diluted with blocking buffer at 1:lOO or 1:75 (v:v), respectively. Sections were rinsed with 0.85% (w/v) NaCl, 50 mM Na-phosphate (pH 7.4) and deionized water. Sections probed with Nanogold were silver-enhanced (LIS Silver, Nanoprobe Inc.) for 10 min according to manufacturer’s directions. After poststaining in 2% aqueous uranyl acetate for 3 min, the sections were examined at 60 or 80 kV in a Philips EM 201 transmission electron microscope.

kD

66*

+-70

45=D ~36 +-31 ~26 +24

Fig. 1. Silver-stained SDS polyacrylamide gel (lo-15% linear gradient) showing polypeptides in whole glyoxysomes (lane a), carbonate-wash supematant (lane b), cotton and laurylmaltoside-solubilized membranes (lane c). Putative PMPs (lane c) are indicated by the arrows to the right of the gel. Positions of low molecular mass standards are identified to the left of the gel. Amounts of protein applied to each lane are: a, 30 pg; b, 20 pg; and c, 24 pg.

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2.4. Antisera production and affinity pur@ation

of

antibodies

For preparation of cotton PMP antiserum, all of the proteins (120 c(g) solubilized in aminocaproateflaurylmaltoside from membranes recovered from density-gradient isolated glyoxysomes were diluted to 1.5 ml with PBS (4.3 mM Na2HP04, 1.4 mM KH2P04, 137 mM NaCl, 2.7 mM KCl, pH 7.4), mixed with 3 ml Freund’s complete adjuvant (Sigma), and injected subcutaneously into the four haunches of a New Zealand white rabbit. Two booster injections with membrane proteins and incomplete adjuvant (1:2) were done at 15 days (80 pg protein) and 30 days (70 hg protein) after the first injection. Eight days after the last injection, the rabbit was exsanguinated and the serum collected by centrifugation. Antibodies to cotton PMP26 were affinity-purified to the cotton PMPs blotted onto PVDF membranes according to the method described by McCammon et al. [lo] except that microfuge tubes containing the excised pro-

tein bands were used during the puritication procedure. Antisera to PMPs from several eukaryotic species were kindly provided by the following colleagues: antiserum to the seed-specific, bean (Phaseohs vulgaris L.) tonoplast intrinsic protein (o-TIP) from Dr Russel L. Jones; antiserum to rat liver PMPs and affinity-purified antibodies to the PMP24 in C. tropicalis from Dr Richard A. Rachubinski; monospecific antiserum to rat liver PMP22 from Dr Wilhelm Just; castor bean PMP antiserum from Dr J. Michael Lord; and monospecific antiserum to castor bean PMP24 from Dr Robert P. Donaldson. 2.7. Protein and enzyme assays Protein concentrations in organelle or PMP fractions were determined with Bio-Rad (Coomassie-stain method) and Pierce BCA protein assay reagents, respectively, according to the manufacturer’s directions. Bovine plasma gamma

B kD

“& 24.5+

t24.5

Fig. 2. SDS gel (A) and Western blot (B) of membrane polypeptides recovered from glyoxysome fractions of sunflower (Sun), cotton (Cot), and cucumber (Cut) cotyledons and castor bean (Cas) endosperm. A, a prominent, low molecular mass polypeptide (24.5,26, 22, and 24 kDa) was present in sunflower, cotton, cucumber, and castor bean fractions, respectively. B, rabbit antiserum (dilution of 1:lOO)against the polypeptides recovered from membranes in the cotton glyoxysome fractions recognized these four putative PMPs. Amounts of protein applied to each lane are: A, 5 Pg; B, 15 pg.

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220

globulin (Bio-Rad) was the standard. Catalase (EC 1.11.1.6) was assayed according to Ni et al. [31]. Cytochrome c oxidase (EC 1.9.3.1) was assayed according to Tolbert et al. [32]. 3. Results Fig. 1 illustrates that glyoxysomes in darkgrown cotton cotyledons contain at least five putative, prominent peroxisome membrane proteins (PMPs): PMP70, -36, -31, -24, and a most prominent PMP26 (lane c). The protein profile of whole glyoxysomes (lane a) separated in an SDS gel showed numerous polypeptides with a wide range of molecular masses. An almost identical polypeptide pattern was observed in the carbonate-wash supernatant (lane b); this pattern indicated that the initial lysing of the glyoxysomes in 100 mM K-phosphate did not sufftciently solubilize many of the matrix proteins from the pelleted membranes and that an alkaline Nacarbonate wash was necessary to extract these re-

A

sidual polypeptides. A second carbonate wash of the pelleted membranes solubilized an insignificant amount of protein (data not shown); therefore, this additional step was routinely omitted from our protocol. Two criteria set by us for an authentic, integral PMP (lane c) was for the polypeptide to be a minor band among all glyoxysome polypeptides separated in a SDS gel (lane a), and for the polypeptide to be essentially absent from the carbonate-wash supematant (lane b). The five polypeptides listed above fit these criteria. Therefore, rabbits were immunized with the laurylmaltoside-aminocaproate polypeptides (lane c) solubilized from membranes in density-gradient fractions possessing highly-purified glyoxysomes. Fig. 2A shows that membranes recovered from glyoxysome fractions isolated from sunflower, cotton, and cucumber cotyledons, and from castor bean endosperm, each contain a prominent, low molecular mass polypeptide: putative PMP24.5, -26, -22, and -24, respectively. The Western blot in Fig. 2B illustrates that IgGs in the cotton anti-

c

kD

97*

t26

Fig. 3. Silver-stained SDS gels and Western blots of polypeptides present in fractions of isolated gtyoxysomes and protein bodies (Pb) from cotton (AC) and cucumber (B) cotyledons. Each type of organelle possessed tides on SDS gels; however, the anti-cotton antiserum at a dilution of 1:1000 recognized a species-specific, peptide in all three organelles. Amounts of protein applied to each lane were: A, 13 pg; B, 15 fig; C, 40

(Px), mitochondria (Mi), a distinct set of polypeplow molecular mass polypg (gel) and 60 pg (blot).

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:

Fig. 4. Electron micrographs of thin sections through cotton (A,C) and cucumber (B,D) cotyledons probed with either affinitypurified, cotton anti-26 kDa IgGs diluted 1:10 (A,B) or with bean a-TIP antiserum diluted I:100 (C,D). As indicated by the arrows, PMP26 and o-TIP antibodies primarily localized to the membranes of intact (PB) and vacuolating protein bodies (PBV) but not to glyoxysome (G) membranes. (AC) Protein A-gold (10 nm), x 46 000 and x 39 000, respectively. (B,D) Nanogold (1.4 nm) followed by silver-enhancement, x 29 000 and x 41 000, respectively. LB, lipid body; P, plastid. Bar = 0.5 pm.

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f

Fig. 5. Western blot analysis with anti-TIP antiserum (1:lOO). These antibodies recognized the same prominent polypeptides in each of the four oilseed species as those shown in Fig. 2. Protein, 12 rg per gel lane.

serum recognized each of the four prominent, lowmolecular mass polypeptides as well as several higher molecular-mass polypeptides in each species. The sunflower PMP24.5 and cotton PMP26 were the most antigenic on PVDF membranes. Fig. 3 illustrates the results of experiments designed to determine whether these polypeptides were unique to membranes recovered from glyoxysome fractions. Comparisons of SDS-PAGE polypeptide profiles between cotton (Fig. 3A) and cucumber (Fig. 3B) glyoxysome and mitochondria fractions revealed patterns which were unique for each organelle. A unique pattern was also found when profiles of cotton glyoxysomes were compared to cotton protein bodies (Fig. 3C). These results indicated that cross-contamination of whole organelles among the organelle fractions did not occur in both species. Western blot analyses, how-

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ever, revealed that antigenically-related polypeptides, namely a 26-kDa polypeptide occurred not only in purified cotton glyoxysome fractions, but also in cottonseed mitochondria and protein body fractions (Fig. 3A,C). Similar results were found for cucumber in that a 22-kDa polypeptide occurred in purified cucumber glyoxysome and mitochondria fractions (Fig. 3B; cucumber protein body fractions were not examined). Immunocytochemical localizations (Fig. 4) were employed to determine whether the 26-kDa polypeptide was indeed localized in the membranes of these three organelles. IgGs affinity-purified to cotton 26-kDa polypeptides recovered from the purified glyoxysome fractions were used as probe. Gold-labeled, putative PMP26 IgGs bound primarily to the membranes of intact and vacuolating protein bodies in cotton (Fig. 4A) and cucumber (Fig. 4B) cotyledons rather than to membranes of glyoxysomes or mitochondria (the latter organelle not appearing in the micrographs). These completely unexpected results indicated that the consistent appearance of the prominent 26- and 22-kDa polypeptides in SDS gels of cotton and cucumber glyoxysome (and mitochondria) fractions, respectively, were due to a persistent contamination with protein bodies and/or their membranes. Support for this latter contention was provided by Western blot (Fig. 5) and immunocytochemical (Fig. 4 C,D) analyses employing antiserum to a known PBMB, namely the seed-specific, bean tonoplast intrinsic protein (a-TIP). Fig. 5 illustrates that antibodies in this antiserum crossreacted with the four putative PMPs: 24.5, 26, 22, and 24 kDa in sunflower, cotton, cucumber, and castor bean fractions, respectively. Immunocytochemical localizations with this antiserum revealed specific recognition of antigens in the membranes (future tonoplasts) of cotton (Fig. 4C) and cucumber (Fig. 4D) protein bodies, exhibiting virtually identical patterns observed with antibodies to the cotton 26-kDa polypeptide (Fig. 4A,B). Electron microscopic examinations of the mitochondria and glyoxysome fractions from which cucumber or cotton membrane proteins were recovered did not reveal contamination by intact protein bodies. However, when IgGs to the cotton

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Fig. 6. Electron micrographs of sections through purified cotton glyoxysome (A) and mitochondria (B) isolated in sucrose gradients. Sections were probed with aftinity-purified cotton anti-26 IgGs diluted 1:10 and Nanogold (I .4 nm) followed by silver-enhancement. Antibodies localized primarily to membranes of vesicles (arrows) interspersed among both types of organelles. G, glyoxysome; M, mitochondria. Magnifications: A, x20000; B, x 31000. Bar = 0.5 pm.

26-kDa polypeptide were used as a probe on thin sections through these fractions, the likely source of the contamination was discovered. Membrane fragments (vesicles), interspersed among the cotton glyoxysomes (Fig. 6A) and mitochondria (Fig. 6B), consistently were labeled with numerous gold particles. These vesicles often contained material of the same electron density as the matrix of protein bodies in situ. When anti-TIP was used as probe, membranes of small vesicles were labeled virtually the same as shown in Fig. 6 (data not shown). 4. Discussion The glyoxysomes of cotton cotyledons possessed at least five putative PMPs (24, 26, 3 1, 36, and 70 kDa) according to our SDS PAGE criteria for authentic, integral PMPs (Fig. 1). Identification of these polypeptides as integral membrane proteins required washing the membranes recovered from

the glyoxysome fraction with an alkaline sodium carbonate solution (Fig. 1, lane b), a treatment known to effectively solubilize residual matrix and peripheral membrane proteins [33,34]. Solubilization in laurylmaltoside, a nonionic detergent, was equally important in identifying these as integral membrane proteins because comparisons of polypeptide profiles obtained after solubilization in buffered SDS or laurylmaltoside/aminocaproic acid revealed that the latter was more selective in solubilizing only the membrane proteins (data not shown). Preferential solubilization of mitochondrial membrane versus matrix proteins in this laurylmaltoside solution also was observed by Schlgger and von Jagow [26]. The general occurrence of a prominent, lowmolecular mass polypeptide in the glyoxysome fractions of all four oilseed species examined (Fig. 2A) gave credence to the developing concept that a similar polypeptide existed in the membrane of most eukaryotic peroxisomes [4,11,24]. The

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immunocross-reactivity with the cotton antiserum (Fig. 2B) suggested a possible structural/functional relationship among these polypeptides. In addition, the immunological kinship between the 26-kDa (cotton) and 22-kDa (cucumber) polypeptides in purified glyoxysome and mitochondria fractions (Fig. 3) seemed to indicate a common function in the two oilseed organelles. This was supported by published studies. For example, Bodnar and Rachubinski [6] reported an immunologically-related 15kDa polypeptide in the membranes of rat liver peroxisomes, mitochondria, lysosomes, and ER. Hart1 and Just [5] showed that antiserum to the rat liver PMP36 cross-reacted with a 36-kDa mitochondrial membrane protein. Sulter et al. [35] showed that antibodies against the Hansen&a polymorpha PMP3 1 recognized antigens by immunogold labeling in the boundary membranes of this yeast’s mitochondria and peroxisomes. These collective data provided strong circumstantial evidence for a common polypeptide in glyoxysomes and mitochondria. Specific, immunogold labeling at the ultrastructural level, however, was needed for definitive evidence. Our immunocytochemical results (Figs. 4, 6) convincingly revealed that the prominent, lowmolecular mass polypeptide was not endogenous to oilseed glyoxysomes or mitochondria, but was a PBMP. Whether or not another membrane protein exists in common with mitochondria and glyoxysomes remains to be elucidated. Ultrastructural observations of the mitochondrial and glyoxysomal fractions from cotton (Fig. 6 and [23]), cucumber [ 151 and sunflower (not shown) did not reveal significant contamination with intact protein bodies. Hence, a plausible explanation for the persistent occurrence of PBMPs in highly purified glyoxysome and mitochondria fractions was that protein body membrane fragments (vesicles) co-migrated or equilibrated with these organelles in the sucrose gradient fractions. Mettler and Beevers [36] reported that protein body membranes isolated from dry castor bean seeds equilibrated at 1.21 g .cms3 in a sucrose gradient. Likewise, most of the protein body membranes from 3-day-old cucumber cotyledons equilibrated at 1.20 g.cme3 except for a minor, impure band at 1.23 g.cmm3 1371.Mader and Chrispeels [38] reported that protein body

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membranes from bean seeds sedimented at 1.18 g. cmm3. These data indicate that protein body membranes (vesicles) exhibit a broad range in densities which overlap with the equilibrium densities of mitochondria (1.18 g.cmm3) and glyoxysomes (1.24 g . cmm3) in sucrose gradients. The specific immunogold labeling of vesicle membranes with the anti-26 kDa (Fig. 6) and antiTIP (not shown) antibodies strongly indicated that the source of PBMPs in the glyoxysome and mitochondrial fractions was from vesicles as postulated above. Johnson et al. [39] reported that TIP was a very abundant membrane protein and likely was highly concentrated in protein body membranes. Our dense immunogold labeling of the membrane vesicles support this contention. Hence, a minor contamination of protein body membranes (vesicles) in highly-purified glyoxysome and mitochondria fractions could indeed account for the persistent occurrence of a prominent, low molecular mass polypeptide in glyoxysome fractions as observed by us and others [13,14,17,18]. An important consequence of discovering that the prominent, putative PMPs were instead PBMPs is that we have data to add to those accumulating for these proteins. Their molecular mass being in the 25-kDa range and their crossreactivity with anti-o-TIP antibodies (Fig. 5) strongly indicate that these PBMPs belong to a family of homolgous membrane intrinsic proteins (MIPS) which function as channel-type transport proteins [40,41]. They have been shown to be widely distributed among seeds of monocots and dicots [39]. Our data show that four oilseed crop plants also possess these proteins (this was shown previously only for castor beans), and that although immunologically related, they each are of distinctly different molecular mass. In addition, we have shown the definitive and specific localization of these proteins to membranes of protein bodies in cotton and cucumber cotyledons which was shown previously by immunocytochemistry in several legumes and Arabidopsis [39,42]. Acknowledgements

This research was supported by USDA NRICGP grant 92-37304-7755 to R.N.T. and by Scientific Program of NATO to F.J.C. The

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authors sincerely thank Lilian W. Jiang and Stefan Kleff for their assistance in isolating peroxisomes and Dr Kevin C. Vaughn for his helpful discussions on immunocytochemistry. References 111C. de Duve and P. Baudhin, Peroxisomes (microbodies and related particles). Physiol. Rev., 46 (1966) 323-357.

PI A.H.C. Huang, R.N. Trelease and T.S. Moore, Jr., Plant Peroxisomes. American Society of Plant Physiologists Monograph Series. Academic Press, New York, 1983, pp. 67-155. 131 H. van den Bosch, R.B.H. Schutgen, R.J.A. Wanders and J.M. Tager, Biochemistry of peroxisomes. Annu. Rev. Biochem., 61 (1992) 157-197. [41 C. Causeret, M. Bentejac and M. Bugaut, Proteins and enzymes of the peroxisomal membrane in mammals. Biol. Cell, 77 (1993) 89-104. [51 F.-U. Hart1 and W.W. Just, Integral membrane polypcptides of rat liver peroxisomes: topology and response to different metabolic states. Arch. Biochem. Biophys., 255 (1987) 109-119. 161 A.G. Bodnar and R.A. Rachubinski, Characterization of the integral membrane polypeptides of rat liver peroxisomes isolated from untreated and clotibrate-treated rats. B&hem. Cell Biol., 69 (1991) 499-508. [71 K. Verheyden, M. Fransen, P.P. van Veldhoven and G.P. Mannaerts, Presence of small GTP-binding proteins in the peroxisomal membrane. Biochim. Biophys. Acta., 1109 (1992) 48-54. PI T. Tsukamoto, S. Miura and Y. Fujiki, Restoration by a 35 K membrane protein of peroxisome assembly in a peroxisome-deficient mammalian cell mutant. Nature, 350 (1991) 77-81. [91 W.M. Nuttley, A.G. Bodnar, D. Mangroo and A. Rachubinski, Isolation and characterization of membranes from oleic acid-induced peroxisomes of Candida tropicalis. J. Cell Sci., 95 (1990) 463-70. UOI M.T. McCammon, M. Veenhuis, S.B. Trapp and J.M. Goodman, Association of glyoxylate and beta-oxidation enzymes with peroxisomes of Saccharomyces cerevisae. J. Bacterial., 172 (1990) 5816-5827. 1111 G.J. Sulter, W. Harder and M. Veenhuis, Structural and functional aspects of peroxisomal membranes in yeast. FEMS Micro. Rev., 11 (1993) 285-296. WI L.W. Jiang, I. Bunkelmann, L. Towill, S. Kleff and R.N. Trelease, Identification of peroxisome membrane proteins (PMPs) in sunflower (Helianthus annuus, L.) cotyledons and influence of light on the PMP developmental pattern. Plant Physiol., 106 (1994) 293-302. 1131 K.D. Chapman and R.N. Trelease, Characterization of membrane proteins in enlarging cottonseed glyoxysomes. Plant Physiol. B&hem., 30 (1992) l-10. [I41 C. Kruse and H. Kindl, Integral proteins of the glyoxysoma1 membranes. Ann. N.Y. Acad. Sci., 386 (1982) 499-501.

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