Current Genetics (1984, 8:537-542

Current Genetics © Springer-Verlag 1984

Synthesis of a dicyclohexylcarbodiimide-binding proteolipid by cucumber (Cucumis sativus L.) mitochondria Ethan Hack 1 and Christopher J. Leaver

Department of Botany, The King'sBuildings,Universityof Edinburgh, MayfieldRoad, EdinburghEH9 3JH, Scotland

Summary. When isolated cucumber (Cucumis sativus L.) mitochondria were treated with 14C-labelled dicyclohexylcarbodiimide (DCCD), a single polypeptide was predominantly labelled. This polypeptide was soluble in 1-butanol or chloroform :methanol (2 : 1, v/v) and had an apparent molecular mass of approximately 7 kDa; it therefore had the characteristic properties of the DCCDbinding proteolipid subunit of the ATP synthase complexes of mitochondria, chloroplasts, and prokaryotes. When isolated cucumber mitochondria were allowed to synthesize protein in the presence of [as S]methionine and then extracted with 1-butanol or chloroform : methanol (2:1, v/v), a 3ss-labelled proteolipid that migrated more rapidly on SDS-polyacrylamide gels than the pro-teolipid labelled by [14C]DCCD was solubilized. Treatment of mitochondria with unlabelled DCCD after they had been allowed to synthesize protein, specifically converted some of the [as S]methionine-labelled proteolipid to a form that comigrated with the [14C]DCCD-labeUed proteolipid. We therefore conclude that a DCCD-binding proteolipid is synthesized by isolated cucumber mitochondria. Key words: Dicyclohexylcarbodiimide-binding proteolipid - Plant mitochondrial genes - Organelle protein synthesis

Introduction

The biogenesis of mitochondria requires the cooperation of the nuclear and mitochondrial genomes, and the as1 Present address: Department of Botany, Bessey Hall, Iowa

State University,Ames, IA 50011, USA Offprint requests to." C. J. Leaver

sembly of polypeptides encoded by both to give functional enzyme complexes (Tzagoloff 1982). One of the intriguing aspects of this cooperation is that the contribution of the two genomes varies between different organisms. The best-documented example of this variation is found in the ATP synthase (oligomycin-sensitive, protontranslocating ATPase) complex. This complex, which has approximately 10 subunits, can be resolved into two portions, F1 and F0. A functional ATP-synthesizing complex can be reconstituted from the F1 and F0 portions and phospholipids. The F1 portion is hydrophilic and has ATPase activity. Its composition is well defined; it has five subunits. The F0 portion is hydrophobic and functions as a proton channel but does not have catalytic activity by itself. Its composition is not entirely clear (Amzel and Pedersen 1983; Tzagoloff 1982). The bestcharacterized subunit of F0 is the one known as the dicyclohexylcarbodiimide-binding proteolipid subunit because of its two characteristic properties. First, it is soluble in lipid solvents, in particular chloroform: methanol (2 : 1, v/v) and 1-butanol. Second, it covalently binds dicyclohexylcarbodiimide (DCCD) at low DCCD concentrations; this binding causes inhibition of the enzymic activity of the complex and reduces the rate of migration of the proteollpid on SDS-polyacrylamide gels (Cattell et al. 1971; Sebald and Hoppe 1981). In fungi and metazoans, all five subunits of the F1 portion of ATP synthase are encoded by nuclear genes (Sebald 1977). In the yeast Saccharomyces cerevisiae, the DCCD-binding proteolipid subunit (Hensgens et al. 1979; Macino and Tzagoloff 1979) and another subunit of F0, usually referred to as subunit six (Macino and Tzagoloff 1980), as well as an ATPase-associated polypeptide (Macreadie et al. 1983) are encoded by mitochondrial DNA. In other fungi, such as Neurospora crassa, subunit six is encoded in mitochondrial DNA but the DCCD-binding proteolipid is encoded in nuclear

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E. Hack and C. J. Leaver: Synthesis of a proteolipid by cucumber mitochondria

D N A a n d s y n t h e s i z e d in t h e c y t o s o l ( t h e site o f s y n t h e s i s o f t h e A T P a s e - a s s o c i a t e d p o l y p e p t i d e has n o t b e e n defined). I n t e r e s t i n g l y , in Neurospora crassa t h e r e is a s e q u e n c e in m i t o c h o n d r i a l D N A t h a t c o u l d e n c o d e a p r o t e o l i p i d b u t appears n o t to be e x p r e s s e d ( v a n d e n B o o g a a r t et al. 1982). In m a m m a l s , s u c h as h u m a n s ( A n d e r s o n et al. 1 9 8 1 ) , cows ( A n d e r s o n e t al. 1 9 8 2 ) a n d m i c e ( B i b b et al. 1981), a n d p r o b a b l y in at least some l o w e r m e t a z o a n s s u c h as sea u r c h i n s ( R o b e r t s et al. 1983), m i t o c h o n d r i a l D N A includes a gene t h a t c o u l d e n c o d e s u b u n i t six b u t n o n e for t h e p r o t e o l i p i d . I n h i g h e r plants, a n a d d i t i o n a l s u b u n i t , t h e h y d r o p h i l i c a l p h a s u b u n i t o f A T P s y n t h a s e , is s y n t h e s i z e d b y m i t o c h o n d r i a ( H a c k a n d Leaver 1 9 8 3 ; H a c k et al. 1983). T h e site o f synthesis o f t h e s u b u n i t s o f t h e F 0 p o r t i o n is n o t k n o w n . P r e l i m i n a r y e v i d e n c e ( L e a v e r e t al. 1 9 8 2 ) suggests t h a t t h e p r o t e o l i p i d is s y n t h e s i z e d in m i t o c h o n d r i a , as in yeast. I n this p a p e r we describe e x p e r i m e n t s t h a t c o n f i r m this p r e l i m i n a r y evidence.

Materials a n d m e t h o d s

Plant material Seedlings of cucumber (Cucumis sativus L., variety Long Green Ridge) were incubated in water at 4 °C for at least 17 h, then germinated on sterilized cellulose wadding wetted with sterile, distilled water. Trays were kept in continuous darkness with a temperature cycle of 28 °C for 12 h, then 22 °C for 12 h (Becket et al. 1978).

Preparation of mitochondria. Mitochondria were isolated from the cotyledons of five-day-old cucumber seedlings by differential centrifugation and isopycnic sucrose density gradient centrifugation as described previously (Leaver et al. 1983). The yield of mitochondrial protein, measured by a modified Lowry protein assay (Miller 1959), was approximately 0.5 mg/g fresh wt.

In vitro protein synthesis. Mitochondrial translation products were labelled with [3SS]methionine as described, with succinate as energy source (Leaver et al. 1983).

Labelling with [14CIDCCD. Conditions for labelling with [14C]DCCD were based on those described by Cattell et al. (1971) and Sebald et al. (1979), but a short treatment at 25 °C was used instead of a prolonged (four to many hours) treatment at 0 ° - 4 °C. Mitochondria (not labelled with [35S]methionine) were suspended in 0.01 M Tris-HC1 (pH 7.5) and [14C]DCCD (54 Ci/mol), dissolved in absolute ethanol at a concentration of 2 mM or 10 mM, was added to the suspension; ratios of DCCD to protein and final DCCD concentrations are given in the figure legends. The mitochondrial suspensions were then diluted by the addition of 0.01 M Tricine-NaOH (pH. 7.2), 0.4 M mannitol, 0.001 M EGTA and the mitochondria were collected by centrifugation at 12,000 gmax for 3 min. Mitochondria were either used directly for organic solvent extraction or frozen and stored at - 8 0 °C.

Treatment of mitoehondria with unlabelled DCCD. Mitochondria that had been freshly labelled with [3SS]methionine were collected by centrifugation and resuspended in 0.01 M Tris-HC1 (pH 7.5). Dicyclohexylcarbodiimide, dissolved in absolute etha-

no1 at a concentration of 2 mM, 10 mM or 20 mM, was added to mitochondrial suspensions; ratios of DCCD to protein and final DCCD concentrations are given in the figure legends. The treated mitochondria and a control suspension at a protein concentration of 2 mg/ml in 0.01 M Tris-HC1 (pH 7.5), 5% (v/v) ethanol were incubated at 25 °C with shaking for 1 h. The mitochondria were then treated in the same way as mitochondria that were incubated with labelled DCCD.

Organic solvent extraction. Mitochondria were suspended in 0.01 M Tris-HC1 (pH 7.5) to a concentration equivalent to approximately 10 mg/ml of starting protein (the osmotic shock administered in the DCCD treatment causes loss of matrix protein). 50 volumes of 1-butanol or 25 volumes of chloroform : methanol (2 : 1, v/v) were added to the mitochondria, giving a singiephase suspension. After being mixed by vortexing, the suspension was incubated overnight on a roller shaker at 4 °C. The suspension was then centrifuged in a horizontal rotor for 5 min at 12,000 gmax- The pellets were allowed to dry in air. The supernatant was centrifuged again to ensure complete removal of insoluble material. Extracts were dried in vacuo and the residue was resuspended in 1/10 of the original volume of 1-butanol or 1/5 of the original volume of chloroform : methanol (2 : 1, v/v). With both solvents, there was a significant quantity of material that did not dissolve, even after incubation overnight at room temperature. Insoluble material remaining after overnight incubation was removed by centrifugation. Five volumes of diethyl ether, chilled to - 2 0 °C, were added to the butanol extracts and 4 volumes to chloroform:methanol extracts. Protein was allowed to precipitate at - 2 0 °C for at least 24 h. The precipitated protein was collected by centrifugation for 5 rain at 12,000 gmax. The pellet was allowed to dry in air then resuspended in 0.1 ml of l-butanol or chloroform :methanol. It now appeared to be completely soluble and was therefore collected by evaporation to dryness in vacuo.

Electrophoresis. Mitochondria to be analyzed by SDS-polyacrylamide gel electrophoresis were dissolved in sample buffer containing 2% (w/v) sodium dodecyl sulfate, 10% (v/v) glycerol, 0.06 M Tris-HC1 (pH 6.8), 0.01% (w/v) bromophenol blue by heating at 9 5 - 1 0 0 °C for 2 min. Protein extracted with 1-butanol or chloroform : methanol was dispersed in distilled water and dissolved by the addition of an equal volume of 2x sample buffer and incubation at 9 5 - 1 0 0 °C for approximately 2 min. In both cases, disulfide bonds were reduced, after the samples had cooled, by the addition of 1 M dithiothreitol to a final concentration of 0.05 M. The pellets remaining after organic solvent extraction are difficult to dissolve in sodium dodecyl sulfate, and the polypeptides tend to aggregate. In order to minimize this problem, pellets from 0.2 mg mitochondrial protein were suspended in 0.05 ml 9.5 M urea, 0.01 M sodium carbonate (Horst et al. 1980), in which they gradually dissolved. 0.025 ml 9.5 M urea, 4% (w/v) sodium dodecyl sulfate was then added and the suspensions were incubated at 9 5 - 1 0 0 °C for 2 min. After they had cooled, 0.025 ml of 0.24 M Tris-HC1 (pH 6.8), 0.2 M dithio~threitol, 0.04% (w]v) bromophenol blue was added. Remaining insoluble material was removed by centrifugation. Protein derived from approximately 0.1 mg of mitochondrial protein was applied to each track of the gels. Gel electrophoresis was carried out using the buffer system of Laemmli (1970) and 12 --18% (w/v) acrylamide linear gradient gels. Gels were stained with Brilliant Blue R (C.I. 42660), destained, and dried onto Whatman 3MM filter paper. [35S]mcthionine-labelled proteins were detected by autoradiography using Dupont Cronex 4 X-ray film.

E. Hack and C. J. Leaver: Synthesis of a proteolipid by cucumber mitochondria

Fig. 1. Autoradiograph of SDS-polyacrylamide gel of mitochondrial polypeptides, showing effect of DCCD treatment. A-D polypeptides from mitochondria labelled with [35S]methionine by in vitro mitochondrial protein synthesis and treated with: A no DCCD; B 0.02 ~mol DCCD/mg protein (0.1 mM DCCD); C 0.2 ~mol DCCD/mg protein (0.4 mM DCCD); D 0.5 ~mol DCCD]mg protein (1.0 mM DCCD). E, F polypeptides labelled with [14C IDCCD by treatment with: E 0.02 ~mol [14C]DCCD/mgprotein (0.1 mM [14C]DCCD); F 0.2 t~mol [14CIDCCD/mg protein (0.4 mM [14C]DCCD)

Results

Extraction of mitochondria with organic solvents When the proteins of isolated cucumber mitochondria that had been allowed to synthesize protein in the presence of [3SS]methionine were separated by SDS-polyacrylamide gel electrophoresis and analyzed by autoradiography, 1 5 - 2 0 polypeptides were prominently labelled (Fig. 1A). The pattern resembles that observed for other higher plants (Leaver and Gray 1982). Chloroform:methanol (2: 1, v/v), the standard solvent for proteolipids (Folch and Lees 1951, Cattell et al. 1971), efficiently extracted a labelled polypeptide or comigrating polypeptides of molecular mass 6 kDa, together with small quantities of other polypeptides (compare Figs. 1A, 2B, 3B, arrow 1; these tracks were loaded with protein from the same amount of mitochondria and exposed to autoradiography for the same length of time). The 6 kDa polypeptide was detectable by staining gels of extracted

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Fig. 2. Autoradiograph of SDS-polyaerylamide gel of [35S]methionine-labelled mitochondrial polypeptides insoluble in 1-butanol (A, C) or chloroform : methanol (2 : 1, v/v) (B, D). A, B not treated with DCCD. C, D treated with 0.5 ~mol DCCD/mg protein (1.0 mM DCCD)

polypeptides with Brilliant Blue R (not shown). Sigrist et al. (1977) reported that for beef heart mitochondria, extraction with 1-butanol was more selective but somewhat less efficient than extraction with chloroform : methanol. When cucumber mitochondria were treated with 1-butanol, the 6 kDa polypeptide was indeed extracted more selectively than with chloroform:methanol, but extraction was much less efficient (compare Figs. 1A, 2A, B, 3A, B). These results show that cucumber mitochondria synthesize a proteolipid or proteolipids.

Treatment of mitochondria with [14C]DCCD When mitochondria were treated with [14C]DCCD for 1 h at 25 °C and the proteins examined by SDS-polyacrylamide gel electrophoresis and autoradiography, a polypeptide of apparent molecular mass approximately 7 kDa was labelled at both low (0.02 #mol/mg protein) and high (0.2 #mol/mg protein) ratios of DCCD to protein (Fig. 1E, F). Thus cucumber mitochondria contain a DCCD-binding polypeptide.

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E. Hack and C. J. Leaver: Synthesis of a proteolipid by cucumber mitochondria latter was reduced by the bound DCCD. This was tested by treating [3SS]methionine-labelled mitochondria with unlabelled DCCD.

Treatment o f [3SS ]methionine-labelled mitochondria with DCCD

Fig. 3. Autoradiograph of SDS-polyacrylamidegel of polypeptides extracted from mitochondria by 1-butanol (A, E, G) of chloroform : methanol (2 : 1, v/v) (B, C, D, F, H, I). A-F polypeptides from mitochondria labelled with [35Slmethionine by in vitro mitochondrial protein synthesis and treated with: A, B no DCCD; C 0.02 /~mol DCCD/mg protein (0.1 mM DCCD); D 0.2 /~mol DCCD/mg protein (0.4 mM DCCD); E, F 0.5 #mol DCCD/mg protein (1.0 mM DCCD). G-I polypeptides from mitochondria labelled with [14C]DCCD by treatment with: G, H 0.02 #mol DCCD/mg protein (0.1 mM DCCD); 1 0.2/~mol DCCD/mg protein (0.4 mM DCCD)

When mitochondria that had been treated with [14C]DCCD were incubated overnight at 4 °C with chloroform : methanol (2 : 1, v/v), the DCCD-binding polypeptide was extracted (Fig. 3H, !). It could also be extracted, but with low efficiency, by 1-butanol (Fig. 3G). Thus cucumber mitochondria contain a DCCD-binding proteolipid and synthesize a proteolipid, but the [14C]DCCDlabelled proteolipid migrates more slowly on SDS-polyacrylamide gels (apparent molecular mass 7 kDa) than the [35 S]methionine-labelled proteolipid (apparent molecular mass 6 kDa). These results appear at first sight to suggest that these proteolipids are different. However, the binding of DCCD by the ATP synthase proteolipids from a number of sources causes a reduction in thek rates of migration on SDS-polyacrylamide gels (Sebald and Hoppe 1981). It therefore seemed possible that the [3SS]methionine-labelled and 14C DCCD-labeUed proteolipids were the same, and the rate of migration of the

When mitochondria were allowed to incorporate [3s S]methionine into protein, then treated for 1 h with 0.02 pmol DCCD per mg protein, there was a change in the pattern of labelled polypeptides: the 6 kDa band corresponding to the polypeptide(s) soluble in chloroform:methanol was partially replaced by a new band migrating as if it had a molecular mass of 7 kDa (Fig. 1B, arrow 2). Other labelled polypeptides were unaffected by the DCCD treatment. Increasing the ratio of DCCD to protein did not cause a substantial change in the amount of 6 kDa polypeptide converted to the slowlymigrating form but it did have some effect on the migration of other labelled mitochondrial polypeptides (Fig. 1C, D). Even at the highest concentration of DCCD used, there was very little effect on the pattern ofpolypeptides detectable by staining with Brilliant Blue R (not shown). The new [aS S]methionine-labelled band produced by DCCD treatment coincided with the band labelled by [14C]DCCD; this coincidence indicates that the change in migration of the 6 kDa polypeptide is caused by reaction with DCCD. The apparent incompleteness of reaction even at high ratios of DCCD to protein could be due to presence of a different 6 kDa polypeptide, unaffected by DCCD treatment, or to insufficient time for complete reaction (of. Sebald and Hoppe 1981). Chloroform:methanol (2 : 1, v/v) efficiently extracted the form of the 6 kDa polypeptide whose migration was altered by DCCD (Figs. 2D, 3C, D, F, arrow 2). It was also extracted by 1-butanol, but much less efficiently (Figs. 2C, 3E). We therefore conclude that isolated cucumber mitochondria synthesize a DCCD-binding proteolipid.

Discussion

This report brings the number of polypeptides identified as products of plant mitochondrial protein synthesis in at least one higher plant to four: two subunits of cytochrome c oxidase (Leaver and Forde 1980), the alpha subunit of ATP synthase (Boutry et al. 1983; Hack and Leaver 1983), and a DCCD-binding proteolipid. The properties of the proteolipid indicate that it too is a subunit of ATP synthase: like the corresponding polypeptide from other organisms (Sebald and Hoppe 1981), it binds dicyclohexylarbodiimide covalently at low concentrations and the reaction causes a reduction in the

E. Hack and C. J. Leaver: Synthesis of a proteolipid by cucumber mitochondria rate of migration of the polypeptide on SDS-polyacrylamide gels. Preliminary evidence indicates that mitochondrial synthesis of all four polypeptides may be widespread in higher plants. In general, the pattern of major polypeptides synthesized by plant mitochondria and analyzed by SDS-polyacrylamide gel electrophoresis is remarkably constant (Leaver and Gray 1982; Boutry and Briquet 1982; C. J. Leaver and co-workers,unpublished observations). More specifically, the alpha subunit of ATP synthase (Hack et al. 1983) and the two subunits of cytochrome oxidase (E. Hack, unpublished observations), like the DCCD-binding proteolipid, are probably synthesized in cucumber mitochondria, and the DCCDbinding proteolipid, like the other three polypeptides, is probably synthesized in maize mitochondria (Leaver et al. 1982;C. J. Leaver, unpublished observations). That the DCCD.binding proteolipid is a product of mitochondrial protein synthesis does not prove that it is encoded by mitochondrial DNA. So far, however, no proteins synthesized by mitochondria have been found to be encoded by extra-mitochondrial genes, so that synthesis of a protein in an organelle provides strong evidence of an organeUe gene. Indeed, this evidence is arguably stronger than the reverse case, where the presence of DNA capable of encoding a polypeptide has been demonstrated, but the synthesis of the polypeptide has not. Recent evidence indicates extensive duplication of DNA sequences between mitochondria and nuclei in diverse organisms (van den Boogaart et al. 1982; Farrelly and Butow 1983; Gellissen et al. 1983; Kemble et al. 1983) and also between mitochondria and chloroplasts (Stern and Londsdale 1982; Lonsdale et al. 1983; Timmis and Scott 1983). In one case, that ofmitochondrial DNA homologous to the chloroplast gene for the large subunit of ribulose-l,5-bisphosphate carboxylase, the mitochondrial DNA can be expressed in an E. coli transcription-translation system (Lonsdale et al. 1983) but the large subunit does not appear to be synthesized in mitochondria (Leaver and coworkers, unpublished observations). The Neurospora crassa mitochondrial DCCD-binding proteolipid is encoded by nuclear DNA, but N. crassa mitochondria contain a gene that could encode a DCCD-binding proteolipid; this gene is not expressed, except perhaps at an unexamined, specialized stage in the life-cycle of the organism (van den Boogaart et al. 1982). These findings demonstrate the importance of determining whether identified genes are expressed. The site of synthesis of the proteolipid subunit of ATP synthase is of particular interest in relation to the evolution of the mitochondrial genome (for discussion see Fredrick (ed), 1981). It seems probable that ifmitochondria evolved from an endosymbiont in the ancestral eukaryotic cell, the protomitochondrial genome would have encoded all the proteins of the endosymbiont.

541

Through evolution, then, a gradual transfer of genetic information from the mitochondria to the nucleus must have occurred. This transfer has evidently proceeded to different extents in different groups of organisms. Alternatively, if mitochondrial DNA originated from the partitioning of the cell's genetic information, this partitioning must have occurred in different ways or been modified during evolution. Some polypeptides, such as subunits I and II of cytochrome c oxidase and cytochrome b, may be encoded by the mitochondrial genome of all species, but others show variation. The best-documented case of this variation is the proteolipid subunit of ATP synthase. In metazoans and some fungi such as Neurospora crassa, this polypeptide is encoded in the nucleus, synthesized on cytosolic ribosomes as a large precursor and imported into the mitochondria, whereas in at least some yeasts such as Saccharomyces cerevisiae, it is synthesized in the mitochondria. In at least one higher plant, too, the proteolipid is synthesized in mitochondria. So far, therefore in the two cases where diversity in the site of synthesis of mitochondrial polypeptide has been found, the plant polypeptide is synthesized in the mitochondria. In one case, the proteolipid, this coding sites is shared with yeast; in the other, the alpha subunit of ATP synthase, it may be unique to plants. It can be suggested that the relatively large coding function of plant mitochondrial DNA reflects the conservatism of the plant mitochondrial genetic apparatus, although not of plant mitochondrial DNA content. This conservatism may also be shown by ribosomal RNA, whose sequence is more prokaryotic than that of animal or fungal mitochondrial (Chao et al. 1983; Spencer et al. 1984). Acknowledgements. We thank A Liddell for skillfuldeltechnical

assistance and Dr. D. Apps for a gift of [14C]DCCD. This work was supported by a research grant from the Scienceand Engineering Research Council to C. J. L.

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Communicated by R. J. Schweyen Received May 30, 1984