Immunological identification of yeast SCO1 protein as ... - Springer Link

Mol Gen Genet (1991) 229:413~420 002689259100325T © Springer-Verlag 1991

Immunological identification of yeast SCO1 protein as a component of the inner mitochondrial membrane Paul Buchwald, Gaby Krummeck, and Gerhard R~del Labor ffir Molekutare Biologic und Allgemeine Pathologic, Institut ffir Pathologic der Universitfit Ulm, Neuherbergstrasse 11, 8000 Mfinchen45, FRG Received March 20, 1991

Summary. The S C O ! gene of Saccharomyces cerevisiae encodes a 30 kDa protein which is specifically required for a post-translational step in the accumulation of subunits 1 and 2 of cytochrome e oxidase (COXI and COXII). Antibodies directed against a //-Gal::SCO1 fusion protein detect SCOI in the mitochondrial fraction of yeast cells. The SCO1 protein is an integral membrane protein as shown by its resistance to alkaline extraction and by its solubilization properties upon treatment with detergents. Based on the results obtained by isopycnic sucrose gradient centrifugation and by digitonin treatment of mitochondria, SCO1 is a component of the inner mitochondrial membrane. Membrane localization is mediated by a stretch of 17 hydrophobic amino acids in the amino-terminal region of the protein. A truncated SCO1 derivative lacking this segment, is no longer bound to the membrane and simultaneously loses its biological function. The observation that membrane localization of SCO1 is affected in mitochondria of a rho ° strain, hints at the possible involvement of mitochondrially coded components in ensuring proper membrane insertion. Key words: SCO1 Cytochrome oxidase - Membrane protein - Mitochondria - Yeast

Introduction In the yeast Saccharomyces cerevisiae cytochrome c oxidase (COX) is composed of 9 subunits (Power et al. 1984). Formation of the three largest subunits (COXI, COXII and COXIII), which are encoded by mitochondrial DNA, requires the products of several nuclear genes (Michaelis etal. 1982; McEwen etal. 1986; Kloeckener-Gruissem et al. 1987; Tzagoloff and Dieckmann 1990). Most of these trans-acting factors seem to affect post-transcriptional steps in the expression of indiOffprint requests to: P. Buchwald

vidual COX subunits, at the level of RNA maturation, translation, post-translational modification or assembly (for reviews see: Fox 1986; Attardi and Schatz 1988; Grivell 1989; Tzagoloff and Dieckmann ]990). Some nuclear-coded factors involved in expression of a COX subunit have additional functions in the biogenesis of other proteins. For example, the product of the nuclear PET54 gene seems to be required for translation of COX3 mRNA and for excision of intron aI5/~ from C O X I pre-mRNA (Costanzo and Fox 1986; Valencik et al. 1989). Similarly, the MRS1 protein has a dual function in removal of intron bI3 from the mitochondrial cytochrome b pre-mRNA and of intron aI6 from CO J(1 pre-mRNA (Kreike et al. 1986; Bousqet et al. 1990). A further example for a bifunctional protein essential for COX biogenesis is provided by the product of the PET2858 gene, which is involved in the aminoterminal processing of COXII (the only mitochondrially synthesized protein with an amino-terminal extension) and also of cytochrome b2 (Pratje and Guiard 1986). Assembly of COX subunits is governed by at least three different proteins encoded in the nucleus. COX10 and COX11, homologues of the ORF1 and ORF2 products of the cytochrome oxidase operon of P. denitrificans, apparently provide an essential function at a posttranslational stage of enzyme assembly. COX11 - and probably also COX10, - is tightly associated with the mitochondrial membrane (Nobrega et al. 1990; Tzagoloft et al. 1990). The third component is the product of the nuclear gene SCO1, which is required for accumulation of COXI and COXII (Schulze and R6del 1988). Pulse-chase experiments revealed that both subunits and to a lesser extent COXII! - are rapidly degraded in the absence of SCOI protein, suggesting that SCOI might be necessary for proper assembly of COXI and COXII into the inner mitochondrial membrane (Krummeck and R6del 1990). In line with the assumption that COX assembly is disturbed we observe in a scol null mutant a decrease in the content of the nuclear-coded COX subunits. In vitro translation of SCO1 mRNA results in the

414 formation of a 33 kDa protein, which can be imported into isolated mitochondria (Schulze and R6del 1989). During import the protein is cleaved to a 30 kDa form, presumably by processing of the amino terminal end, which shows the characteristics of mitochondrial targeting sequences (for a review see: Hartl et al, 1989). From the observation that the imported protein resists alkaline extraction, we concluded that SCO1 is an integral membrane protein. This conclusion, however, assumes, that the protein attains its authentic localization under our conditions of in vitro import. In this paper we show by use of antibodies, raised against a/~-Gal:: SCO1 fusion protein, that SCO1 is indeed an integral component of the mitochondrial inner membrane. From our analysis of rho ° mutants (which are completely devoid of mitochondrial DNA) we conclude that proper membrane localization of the SCOI protein requires the presence of mitochondrially encoded factors. To define the region responsible for membrane anchoring, we constructed a SCOI derivative from which the single hydrophobic region with the potential to span a membrane was deleted. We show that this truncated protein is no longer bound to the membrane and simultaneously has lost its biological function.

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Strains. The E. coli strains used in this work were JM83 (Vieira and Messing i982), C600 (Appleyard 1954) and D H 5 e (BRL). Yeast strains were DL1 ( M A T e , leu2-3, leu2-112, his3-11, his3-15, ura3-228, ura3-251, ura3-372). (Van Loon et al. 1983); KL14-4A ( M A T e , hisl, trp2, [rho°]) (Wolf et al. 1973), NP3 ( M A T e , leu2, ura3, scol1) and GR20 ( M A T e , leu2-3, leu2-112, his3-11, his3-15, ura3-228, ura3-251, ura3-372, sco l :: URA3) (Schulze and R6del 1988).

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Media. Complete media containing glucose (YPD) or galactose (YPGal) and minimal media were as described (Sherman et al. 1986). Plasmids. pBSB10E is a derivative of pBluescript K S ( + ) (Stratagene) carrying the 1.7 kb E c o R I fragment of pB10E containing the SCO1 gene (Schulze and R6del 1989). pUBYI was obtained by ligation of a blunt-ended 1057 bp D d e I - B s t Y I fragment of pBSB10E into plasmid p U C I 9 (Yanisch-Perron et al. 1985) that had been cleaved with SmaI and BamHI (Fig. 1/II). A 1062 bp B s t Y I fiagment was cloned into plasmids p T R B O (Biirglin and De Robertis 1987) and p A T H 1 0 (Dieckmann and Tzagoloff 1985) to create p T R B Y I and pATHBYI, respectively (Fig. 1/III, IV). In order to express SCOI protein at high levels in yeast, plasmid p A H D E H 1 2 (Fig. I/V) was constructed by cloning the SCO1 gene into plasmid pAAH5 (Ammerer 1983). To this end, the 1.6 kb D r a I - - E c o R I fragment of plasmid pBSB10E (Fig. l/I) was filled in with D N A polymerase I, ligated to a HindIII linker (Boehringer, Mannheim) and cloned as a HindIII fragment into pAAH5. YIp-dell8 and Y E p - d e l l 8 were obtained by

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Fig. 1. A Cloning strategy for the plasmids pUBYI, pTRBYI, pATHBYI and pAHDEH12, Plasmids pTRBYI and pATHBYI code for the fl-Gal::SCO1 and TrpE::SCOI fusion proteins, respectively, pAHDEH12 is a derivative of pAAH5 with the ADCI promoter (PADel) and ADC1 terminator (ter) together with the authentic regulatory elements of SCOI. For details of the constructions see Materials and methods. The initiation (ATG) and termination codons (TAA) of the authentic SCO1 gene and of the fusion genes are indicated. Open reading frames are shown as bars (open bar, lacZ from plasmid pTRB0; open bar with arrow, SCOI ;filled bar, lacZ portions of plasmid pUC19 ; hatched bar, trpE from plasmid pATH10). Thin lines indicate vector sequences or 3' flanking sequences of the SCO1 gene. Only relevant restriction sites are shown: RI, EcoRI; DI, DraI; D, DdeI; BI, BstYI; S, SmaI; H, HindIII; HL, HindIII linker. The figure is not drawn to scale. B Fusion sites of/~-Gal::SCO1 and TrpE::SCO1 proteins. Part of the amino acid sequence and the coding sequence of the SCOt reading frame is shown in the upper part (I). In the lower part, fusion sites of the plasmids pUBYI (II), pTRBYI (III) and pATHBYI (IV) are indicated. Sequences derived from SCO1 are given in bold type, normal type indicates lacZ sequences from pUC19, lower-case letters lacZ sequences of pTRB0, and lower case italics trpE sequences of pATH10. Numbers refer to the position of the respective amino acid in the original sequence context. Restriction sites used to obtain the fusion constructs are indicated by arrows (D, DdeI; BI, BstYI).

415 cloning the 1.6 kb B a m H I - SalI SCOI fragment of plasmid pBSB10E, obtained after in vitro mutagenesis (see below), into plasmids YIp351 and YEp351, respectively (Hill et al. 1986).

In vitro mutagenesis. To delete the hydrophobic stretch of 17 consecutive hydrophobic amino acids (amino acids 76-92) of the SCOI protein, PCR was performed with Taq polymerase (Amersham). Plasmid pBSBI0 (10 ng) (Schulze and R6del 1989) was used as template and the following oligonucleotides as primers: dell : 5' ataagatcTCCCGTGGAAAACTCGATCGAGCC 3' del2: 5' ataagatctcAGGGAGAAACGCAGATTGGAAACA 3' (Capital letters indicate authentic sequences, lower case letters represent non-homologous sequences which include the recognition sequence for BglII.) After 30 cycles of amplification (30 sec at 93 ° C; 30 sec at 42 ° C; 5 rain at 72 ° C), the PCR product was analysed on a 1% agarose gel, electrophoretically eluted, digested with BglII, and re-ligated. Inflame ligation was confirmed by D N A sequence analysis of plasmids, isolated from E. coli transformants.

Preparation of inclusion bodies. Inclusion bodies formed by /%Gal::SCO1 fusion protein were prepared from JM83 transformed with plasmid pTRBYI, as described (Buchner and Rudolf 1991) with several modifications. E. coli transformants were grown overnight at 37° C, harvested by centrifugation at 2000xg for 5 min, washed once with H 2 0 and resuspended in buffer A (100 mM TRIS-HC1 pH 7.5, 20 mM EDTA). After incubation with lysozyme (final concentration 0.25 mg/ml) at 4 ° C for 1 h cells were disrupted by repeated freezing in liquid nitrogen followed by thawing on ice. After removal of DNA by treatment with DNase I (final concentration: 2 ~tg/ml) for 1 h at 37 ° C, the suspension was centrifuged at 13 000 x g for 45 min at 4 ° C. The pellet was washed once with buffer B (100 mM TRIS-HC1 pH 7.5, 20 mM EDTA, 0.5 M NaC1, 2% Triton X-100) and three times with buffer A. The resulting inclusion bodies were screened for the presence of the fusion protein by analytical SDS-polyacrylamide gel electrophoresis and subsequently used for immunization of a rabbit without further purification (see below). For the preparation of TrpE:: SCO1 fusion protein, E. coli C600 cells, transformed with plasmid pATHBYI, were treated as described (Dieckmann and Tzagoloff 1985). Insoluble proteins were separated on SDS-polyacrylamide gels, blotted onto Immobilon PVDF transfer membrane (Millipore) and stained with Ponceau S (Serva). Strips containing the TrpE:: SCO1 fusion protein were cut out and used for affinity purification of the antibodies raised against the/%Gal::SCOI fusion protein. Immunological procedures. A female rabbit (Chinchilla Hybrid, Thomae) was immunized subcutanously with 300 gg of/~-Gal::SCO1 fusion protein at a concentration of 0.8 mg/ml in PBS buffer (Harlow and Lane

1988), mixed with an equal volume of Freund's complete adjuvant (Sigma). Booster injections with Freund's incomplete adjuvant (Sigma) were carried out 2, 6 and 8 weeks after the first injection. After 12 weeks serum I/4 was obtained by final bleeding. SCOJ-specific antibodies were detected in Western analyses with anti-rabbit IgG and peroxidase-anti-peroxidase (PAP, Dianova) according to the manufacturer's instructions, in the presence of TBS buffer (Harlow and Lane 1988) and 0.5% Blotto (non-fat dried milk).

Isolation of mitochondria. Mitochondria were prepared from yeast cells grown in either glucose or galactose media to 0 D 6 o o =1.5-2.0, according to Daum et al. 0982). Preparation of mitochondrial membranes. Freshly prepared mitochondria were suspended in 0.6 M mannitol, 10 mM TRIS-HC1 pH 7.5 at a concentration of 5 mg/ml. After addition of 5 volumes 10 mM TRIS-HC1 pH 7.5 and incubation for 45 rain on ice, submitochondrial particles were formed by sonication with a Branson 250 sonifier at 80% duty cycle for 20 s on ice. Membranes were centrifuged at 47 000 x g for 1 h at 4 ° C and resuspended in 10 mM Tris-HC1 pH 7.5 (0.5 ml/mg protein of the initial mitochondrial preparation). Analysis of proteins. Proteins were separated by electrophoresis on 15% acrylamide gels in the presence of SDS (Laemmli 1970). Gels were either stained with Coomassie Brillant Blue R or blotted onto Immobilon PVDF transfer membrane for subsequent immunological detection of SCOI. Miscellaneous techniques. DNA manipulations were done as described (Maniatis et al. 1982). Alkaline treatment of mitochondria was performed as described by Fujiki et al. (1982). Oligonucleotides were synthesized on a DNA synthesizer from Applied Biosystems (Modell 321A). Results

Generation of antibodies directed against SCO1 protein Plasmids pTRBYI and pATHBYI, bearing lacZ." : SCO1 and trpE::SCO1 fusion constructs, respectively (for details of construction see Materials and methods) were used to transform E. coli. Inclusion bodies prepared from the transformants were analyzed on an SDS-polyacrylamide gel and revealed novel proteins of molecular weights 149 kDa and 70 kDa, respectively (Fig. 2), as expected from the D N A sequence of the fusion constructs. In the case of transformants bearing the lacZ::SCO1 fusion construct, a single contaminating protein band of about 115 kDa is detectable. This polypeptide is not seen in control preparations from untransformed E. coli and most likely represents a degradation product of the fusion protein. In the case of inclusion body preparations obtained from transformants with the

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[ Fig. 2. Expression of SCOI fusion proteins in E. coll. Inclusion bodies were prepared from E. coli strains transformed with lacZ: : SCOI (lane 1) and trpE: : SCO1 fusion constructs (lane 2), separated by SDS gel electrophoresis and stained with Coomassie Brilliant Blue. Lane 3 shows the protein pattern of the untransformed E. eoli strain C600

trpE: :SCO1 fusion construct, several additional protein bands besides the fusion protein are detectable. This observation can be explained on the basis of the less efficient expression of the TrpE fusion protein as compared to that of the/?-Gal fusion protein. Because of the high yield and purity of the/%Gal:: SCOI fusion protein, the corresponding inclusion body preparations were used to immunize rabbits, without further purification. Serum I/4, obtained 12 weeks after the initial immunization, detects /%Gal::SCOI and TrpE:: SCO1 fusion proteins in lysates of E. coli transformants (data not shown). Antibodies raised against other E. coli proteins could be efficiently removed by affinity purification of the serum using the isolated TrpE :: SCO1 fusion product (data not shown). This purification, however, was unnecessary, as the antibodies showed no cross-reactivity with yeast proteins (see be-

low). Detection o f SCO1 in yeast mitochondria

SCO1 antiserum was used to detect SCO1 protein in yeast extracts. Crude mitochondria and post-mitochondrial supernatants were prepared from wild type DL1, the scol null mutant GR20 and from DL1 cells carrying the SCO1 gene on plasmid pAHDEH12, under the control of the ADC1 promoter (see Materials and methods). Proteins were separated by SDS-PAGE and analyzed by Western blotting with SCO1 antiserum. A protein with an estimated molecular weight of 29 30 kDa is detected in mitochondria of wild type, but not of GR20 (Fig. 3, lanes 5 and 6). The identity of this protein with SCO1 is further demonstrated by its elevated level in the wild-type transformant (Fig. 3, lane 4). The additional band with a higher electrophoretic mobility, seen in mitochondria of the transformant (Fig. 3, lane 4), presumably represents a degradation product of SCO1. Due to the lower concentration of SCO1 protein, this polypeptide is not detected in mitochondria of untransformed wild-type cells (Fig. 3, lane 6). The SCO1 protein is undetectable in post-mitochondrial supernatant

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(Fig. 3, lanes 1-3), showing that it is located exclusively within the mitochondrial compartment. SCO1 & an intrinsic component o f the mitochondrial membrane

After import of SCO1 protein synthesized in vitro into isolated mitochondria, it is found in tight association with the mitochondrial membrane, as shown by its resistance to alkaline extraction (Schulze and R6del 1989). The same method was used to localize the SCOI protein in vivo. Wild-type mitochondria were extracted with sodium carbonate as described in Materials and methods. Soluble and insoluble fractions were subjected to Western analysis. Figure. 4A shows that the SCO1 protein is found predominantly in the insoluble fraction. The same result is obtained with mitochondrial extracts obtained from the wild-type transformant bearing a highcopy-number plasmid carrying the SCO1 gene (data not shown). These results show that the SCO1 protein is an intrinsic membrane protein and that moderate overexpression has no effect on the intramitochondrial localization. We next analyzed the solubility properties of the SCO1 protein. Sub-mitochondrial particles were prepared by sonic disruption of wild type mitochondria as described in Materials and methods, treated with increasing concentrations of detergents and separated into soluble and insoluble fractions by centrifugation. Figure 5 shows the results obtained with the non-ionic detergent n-octyl-fl-D-glucopyranoside (Baron and Thompson 1975). As with other membrane proteins, a concentration of 1.4 mg detergent per mg mitochondrial protein is required to solubilize the SCOI protein. At lower detergent concentrations, which are sufficient to release soluble proteins from mitochondria, SCOI protein is still retained in the membrane fraction. Similar results were obtained with other detergents including

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one of these (with 17 consecutive hydrophobic amino acid residues) is long enough to span a membrane. To test whether this region in the amino terminal one-third of the protein is responsible for the observed membrane localization, we constructed a SCO 1 derivative that completely lacks this portion by in vitro mutagenesis. In vitro mutagenesis was performed as described in Materials and methods, and the resulting scol allele, scol-dell8, was introduced into the scol null mutant GR20. Transformants bearing the scol-dell8 allele either in a single copy integrated into chromosomal D N A or in multiple copies on an autonomously replicating vector, remained respiration-deficient, showing that the truncated SCO1 version is non-functional. After alkaline extraction of mitochondria isolated from the transformants, the SCOI derivative protein is almost completely recovered from the soluble fraction (Fig. 4 B). The fact that membrane anchoring is not detected in the absence of the hydrophobic stretch of 17 amino acids suggests that this sequence is the main determinant responsible for the membrane localization of SCO1.

SCO1 is a protein of the inner mitochondrial membrane

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Fig. 5. Release of SCOI protein from mitochondrial membranes by treatment with n-Octyl-/~-D-glucopyranoside. Mitochondrial membranes were isolated as described in Materials and methods, suspended in 10 m M TRIS HC1, pH 7.5 to a concentration of i rag/ ml in the presence of the indicated concentrations of n-Octyl-fl-Dglucopyranoside (.) and centrifuged for 1 h at 23 000 x g (2 ° C). Supernatants were separated by SDS-PAGE and transfered to P V D F membranes for Western analysis with either anti-porin, antiCytcl or anti-SCO1 sera. The resulting bands were evaluated by densitometry with an LKB Ultroscan X L

Triton X-100 and sodium deoxycholate (data not shown). Taken together, these data clearly show that the SCOI protein is an intrinsic component of the mitochondrial membrane. Membrane association of SCO1 is mediated by a stretch of17 hydrophobie amino acids

The hydropathy profile of the SCO1 protein shows some hydrophobic stretches (Schulze and R6del 1989). Only

Based on our current knowledge of the protein determinants responsible for intramitochondrial sorting (Pfanner and Neupert 1990), the presence of a cleavable presequence hints at a localization of the SCOI protein in the inner mitochondrial membrane. Proteins of the outer mitochondrial membrane usually lack such a cleavable presequence. To test whether the SCO1 protein is a component of the outer or the inner membrane, wild-type mitochondria were treated with increasing concentrations of digitonin, as described in Materials and methods. Controlled lysis of mitochondria by this detergent has successfully been used to follow the intramitochondrial fate of the Rieske FeS protein during import in both N. crassa and yeast mitochondria (Hartl et al. 1986; Fu et al. 1990). Release of marker proteins from outer membrane (porin), inner membrane (cytochrome cl) and matrix (malate dehydrogenase) was assayed immunologically and enzymatically and compared to the behaviour of the SCOt protein. Figure 6 summarizes the results of this experiment. While porin is the first marker protein to be released from digitonin-treated mitochondria, solubilization of cytochrome cl and of SCO1 requires a higher concentration of the detergent. At this digitonin concentration, the inner membrane is already partially solubilized, as shown by the release of a significant portion of the malate dehydrogenase from the matrix compartment. These data suggest that the SCO1 protein is a component of the inner mitochondrial membrane. Independent confirmation of this conclusion comes from assays of inner and outer membranes separated from submitochondrial particles in a linear sucrose gradient. Most of the SCO1 protein co-fractionates with cytochrome ca in the inner membrane fractions, while porin is found predominantly in the outer membrane fractions (Fig. 7).

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3.85 20 30 Fractions Fig. 7. Separation of submitochondrial particles by isopycnic sucrose gradient centrifugation. Membrane particles from disrupted wild-type mitochondria (see Materials and methods) were separated on a linear sucrose gradient (0.85 M-1.6 M) as described (Pain et al. 1990). Subfractions were analysed via Western blotting as described in the legend of Fig. 5. The peak value for each protein was set to 100 arbitrary units 10

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with digitonin. Freshly prepared wild-type mitochondria were suspended in a buffer containing 0.6 M sorbitol and 20 mM HEPESKOH (pH 7.4) plus the indicated concentrations of digitonin and incubated for 5 min on ice. Each fraction was separated into supernatant and pellet by centrifugation for 30 rain at 13000 x g (2° C). Supernatants were assayed for malate dehydrogenase activity (the highest value obtained was set at 100 U), pellet fractions were screened for porin, Cyt cl and SCO1 by Western analysis as described in the legend of Fig. 5. The values obtained with the pellet fraction of untreated mitochondria were set as equivalent to 100 arbitrary units

Membrane association of SCO1 requires mitochondrially encoded components Based on our studies on the phenotype of scol mutants we proposed a function of the SCOt protein in a posttranslational step in the accumulation of COX subunits I and II, possibly at the level of assembly (Schulze and R6del 1989; Krummeck and R6del 1990). To test whether the association of SCO1 with the inner mitochondrial membrane requires the presence of a mitochondrially encoded factor, mitochondria were isolated from strain KL14-4A [rho°], which completely lacks mitochondrial DNA, and were subjected to alkaline treatment with sodium carbonate. Soluble and insoluble material was collected and probed with SCO1 antiserum. In contrast to the results obtained with wild-type mitochondria, most of the SCOI protein from KL14-4A is detected in the soluble fraction (Fig. 4C). This clearly does not result from mutational alterations of this protein, since KL14-4A [rho°] is able to complement the respiration deficiency of scol mutants. Discussion In this paper we determined the intramitochondrial localization of the SCO1 protein, which is required for

formation of a functional cytochrome oxidase complex. Based on the observation that the SCO1 protein is resistant to extraction at p H 11.5, it must be regarded as an intrinsic membrane protein. In line with this view, the detergent concentrations required for solubilization of SCOI are of the same order as those required for extraction of other membrane proteins. These data are in complete agreement with the previous finding that SCO1 protein synthesized in vitro behaves like a membrane protein after import into isolated mitochondria (Schulze and R6del 1989). Obviously the SCO1 protein reaches its normal intramitochondrial location under the conditions of in vitro import. When controlled lysis of mitochondria with digitonin is performed, the SCO1 protein shows the characteristics of a component of the inner mitochondrial membrane: it is released from mitochondria at the same digitonin concentration as cytochrome cl, which is anchored in the inner mitochondrial membrane with most of the protein exposed to the intermembrane space (Hase et al. 1987). At this digitonin concentration, the bulk of the outer membrane protein porin has already been released from the membrane fraction. Membrane localization of the SCO1 protein is mediated by a stretch of hydrophobic amino acids in the amino-terminal one-third of the protein. Deletion of this segment by in vitro mutagenesis completely abolishes membrane association and renders the truncated protein extractable with sodium carbonate. Concomitantly, we observe a loss in the biological function of the protein, indicating that membrane association is an essential requirement for function. The presence of a single membrane-anchoring stretch predicts that part of the protein is exposed to the intermembrane space, while another part is oriented towards the mitochondrial matrix. Current studies address the question of how the SCO1 pro-

419 rein is oriented in the m e m b r a n e and whether the hydrophobic segment plays a role in the i m p o r t pathway, e.g. as a stop-transfer sequence. Our observation that SCO1 is alkali-extractable in mitochondria isolated f r o m rho ° cells indicates that stable localization of SCO1 in the inner mitochondrial m e m b r a n e depends on the presence of (a) mitochondrially encoded component(s). We favour the idea that one or more of the mitochondrially synthesized C O X subunits could be required to correctly anchor the S C O I protein in the membrane, e.g. by protein-protein contacts. Such protein interactions could explain the unusually short length of the SCO1 t r a n s m e m b r a n e segment 17 hydrophobic amino acids. In this context, it will be interesting to see whether a functional m e m brane anchor requires specific amino acids or just depends on the presence of hydrophobic amino acids. Other explanations for the different localization of SCO1 in rho ° mitochondria, however, are also possible. For example, alterations in the mitochondrial m e m b r a n e potential (which is required for mitochondrial import) due to the loss of cytochrome oxidase activity could impair the i m p o r t and/or assembly of proteins. However, we can exclude the possibility that the altered localization of the SCO1 protein in the rho ° strain KL14-4A results f r o m mutational alteration of the SCO1 gene, because in crosses of this strain with scol mutants we observe complementation of the S C O I deficiency phenotype. SCO1 has been shown to act at a post-translational stage in the accumulation of C O X subunits I and II, possibly in p r o m o t i n g subunit assembly ( K r u m m e c k and R6del 1990). In addition to SCO1, two other proteins, encoded by the nuclear genes COXIO and C O X l l , have been proposed to act at the level of C O X subunit assembly. These gene products are homologous to the ORF1 and ORF3 products, respectively, of the Paracoccus denitrificans cytochrome oxidase operon (Raitio et al. 1987)~ The possibility that the third unidentified reading frame of the Paracoccus operon, ORF2, might encode a product homologous to the SCOI protein was excluded by comparing the respective protein sequences. Interestingly, like the S C O I protein, COX11 has been shown to be an intrinsic c o m p o n e n t of the mitochondrial m e m brane (Tzagoloff et al. 1990). Similarly, COX10 seems to be membrane-associated as predicted from its hydropathy profile (Nobrega et al. 1990). The observation that of the components required for post-translational steps in C O X formation, all those so far identified are m e m brane proteins, raises the possibility that all three proteins act in concert. However, the phenotypes of the respective mutants clearly show that the function of SCO1 is not identical to that of COX10/COX11. In both coxlO and c o x l l mutants, the a m o u n t of immunologically detectable C O X I is strongly reduced, while the steady-state concentration of C O X I I is not altered as c o m p a r e d to wild type cells. In contrast, C O X I I is immunologically almost undetectable in scol null mutants (unpublished data). A m a j o r function of S C O I might therefore be to confer stability on COXII. In addition, however, SCO1 is also involved in conferring stability

on COXI. This is evident f r o m the observation that strains deleted for the SCO1 gene are affected in accumulation of C O X I and C O X I I , whereas strains carrying the scol-1 allele are preferentially affected in C O X I I accumulation. Thus, the effect on C O X I in scol null mutants cannot be only secondary to the lack of COXII. Currently, a number of possible mechanisms can be envisaged as to how the SCO1 protein can confer stability on COX subunits. A m o n g these are chaperone-like functions in the (un)folding of proteins to allow attachment of prosthetic groups, correct m e m b r a n e insertion or ordered interaction with other subunits of heterooligomeric complexes. Acknowledgements. We thank S. Hennig for expert technical assistance, Prof. O. Haferkamp, Ulm, for support, Dr. K. Czerny, Munich, for help in the preparation of antiserum, Prof. G. Schatz, Basel, and Dr. A. Haid, Munich, for antisera against cytochrome cl and porin, and U. Michaelis, P. Skowronek and J. Buchner for critical reading of the manuscript. Part of this work was supported by the Deutsche Forschungsgemeinschaft (SFB 184, project A7).

References Ammerer G (1983) Expression of genes in yeast using the ADC1 promoter. Methods Enzymol 101 : 192-201 Appleyard RK (1954) Segregation of new lysogenic types during growth of a doubly lysogenic strain derived from E. coli K12. Genetics 39 :440-452 Attardi G, Schatz G (1988) Biogenesis of mitochondria. Annu Rev Cell Biol 4:289-333 Baron C, Thompson TE (1975) Solubilization of bacterial membrane proteins using alkyl glucosides and dioctanoyl phosphatidylcholine. Biochim Biophys Acta 382:276285 Bousquet I, Dujardin G, Poyton RO, Slonimski PP (1990) Two group I mitochondrial introns in the cob-box and coxI genes require the same MRSI/PETI57 nuclear gene product for splicing. Curr Genet 18:117-124 Buchner J, Rudolf R (1991) Renaturation, purification and characterization of recombinant Fab fragments produced in E. coli. Bio/Technology 9 : 157-162 Biirglin TR, DeRobertis EM (1987) The nuclear migration signal of Xenopus laevis nucleoplasmin. EMBO J 6:2617-2625 Costanzo MC, Seaver EC, Fox TD (1986) At least two nuclear gene products are specifically required for translation of a single yeast mitochondrial mRNA. EMBO J 5:3637-3641 Daum G, B6hni PC, Schatz G (1982) Import of proteins into mitochondria: cytochrome bz and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria. J Biol Chem 257:13028-13033 Dieckmann CL, Tzagoloff A (1985) Assembly of the mitochondrial membrane system: CBP6, a yeast nuclear gene necessary for synthesis of cytochrome b. J Biol Chem 260:1513-1520 Fox TD (1986) Nuclear gene products required for translation of specific mitochondrially coded mRNAs in yeast. Trends Genet 2: 97-100 Fu W, Japa S, Beattie DS (1990) Import of the iron-sulfur protein of the cytochrome bcl complex into yeast mitochondria. J Biol Chem 265 : 16541-16547 Fujiki Y, Hubbard AL, Fowlern S, Lazarow PB (1982) Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J Cell Biol 93:97-102 Grivell LA (1989) Nucleo-mitochondriai interactions in yeast mitochondrial biogenesis. Eur J Biochem 182:477-493 Harlow E, Lane D (1988) Antibodies: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

420 Hartl F-U, Schmidt B, Wachter E, Weiss H, Neupert W (1986) Transport into mitochondria and intramitochondrial sorting of the Fe/S protein of ubiquinol-cytochrome c reductase. Cell 47:939-951 Hartl F-U, Pfanner N, Nicholson DW, Neupert W (1989) Mitochondrial protein import. Biochim Biophys Acta 988:1-45 Hase T, Harabayashi M, Kawai K, Matsubara H (1987) A carboxyterminal hydrophobic region of yeast cytochrome cl is necessary for functional assembly into complex III of the respiratory chain. J Biochem 102:411-419 Hill JE, Myers AM, Koerner TJ, Tzagoloff A (1986) Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast 2:163-167 Kloeckener-Gruissem B, McEwen JE, Poyton RO (1987) Nuclear functions required for cytochrome c oxidase biogenesis in Saccharomyces cerevisiae: multiple trans-acting nuclear genes exert specific effects on expression of each of the cytochrome c oxidase subunits encoded on mitochondrial DNA. Curr Genet 12:311-322 Kreike J, Schulze M, Pillar T, K6rte A, R6del G (1986) Cloning of a nuclear gene MRSI involved in the excision of a single group I intron (bI3) from the mitochondrial COB transcript in S. cerevisiae. Curr Genet 11:185-191 Krummeck G, R6del G (1990) Yeast SCO1 protein is required for a post-translational step in the accumulation of mitochondrial cytochrome c oxidase subunits I and II. Curr Genet 18:13-15 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York McEwen JE, Ko C, Kloeckner-Gruissem B, Poyton RO (1986) Nuclear functions required for cytochrome c oxidase biogenesis in Saccharomyces cerevisae. J Biol Chem 261:11872-11879 Michaelis G, Mannhaupt G, Pratje E, Fischer E, Naggert J, Schweizer E (1982) Mitochondrial translation products in nuclear respiration-deficientpet mutants of Saccharornyces cerevisiae. In: Slonimski P, Borst P, Attardi G (eds) Mitochondrial genes. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp 311-321 Nobrega MP, Nobrega FG, Tzagoloff A (1990) COXIO codes for a protein homologous to the ORF1 product of Paracoccus denitrificans and is required for synthesis of yeast cytochrome oxidase. J Biol Chem 265 : 14220-14226 Pain D, Murakami H, Blobel G (1990) Identification of a receptor for protein import into mitochondria. Nature 347:444449 Pfanner N, Neupert W (1990) The mitochondrial protein import apparatus. Annu Rev Biochem 59:331-353 Power SD, Lochrie MA, Sevario KA, Patterson TE, Poyton RO

(1984) The nuclear-coded subunits of yeast cytochrome c ocidase : I. Fractionation of the holoenzyme into chemically pure polypeptides and the identification of two new subunits using solvent extraction and reverse phase high performance liquid chromatography. J Biol Chem 259 : 6564-6570 Pratje E, Guiard B (1986) One nuclear gene controls the removal of transient pre-sequences from two yeast proteins: one encoded by the nuclear the other by the mitochondrial genome. EMBO J 5:1313-1317 Raitio M, Jalli T, Saraste M (1987) Isolation and analysis of the genes for cytochrome c oxidase in Paracoceus denitrificans. EMBO J 6:2825 2833 Schulze M, R6del G (1988) SCO1, a yeast nuclear gene essential for accumulation of mitochondrial cytochrome c oxidase subunit II. Mol Gen Genet 211:492-498 Schulze M, R6del G (1989) Accumulation of the cytochrome c oxidase subunits I and II in yeast requires a mitochondrial membrane-associated protein, encoded by the nuclear SCO1 gene. Mol Gen Genet 216:37-43 Sherman F, Fink GR, Lawrence CW (1986) Methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York Tzagoloff A, Dieckmann CL (1990) PET genes of Saccharomyces cerevisiae. Microbiol Rev 54:211-225 Tzagoloff A, Capitanio N, Nobrega MP, Gatti D (1990) Cytochrome oxidase assembly in yeast requires the product of COXll, a homolog of the P. denitrificans protein encoded by ORF3. EMBO J 9:2759-2764 Valencik ML, Kloeckener-Gruissem B, Poyton RO, McEwen JE (1989) Disruption of the yeast nuclear PET54 gene blocks excision of mitochondrial intron aI5fl from pre-mRNA for cytochrome c oxidase subunit. 1. EMBO J 8 : 3899-3904 Van Loon APGM, Van Eijk G, Grivell LA (1983) Biosynthesis of the ubiquinol-cytochrome c reductase complex in yeast. Discoordinate synthesis of the 11 kD subunit in response to increased gene copy number. EMBO J 2:1765-1770 Vieira J, Messing J (1982) The pUC plasmid, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259-268 Wolf K, Dujon B, Slonimski PP (1973) Mitochondrial genetics V. Multifactorial mitochondrial crosses involving a mutation conferring paramomycin resistance in Saccharomyces cerevisiae. Mol Gen Genet 125:53-90 Yanish-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of M13mp8 and pUCI9 vectors. Gene 33:103-119

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