Domain structure of mitochondrial and chloroplast targeting peptides

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Eur. J. Biochem. 180, 535-545 (1989) @) FEBS 1989 Domain structure of mitochondrial and chloroplast targeting peptides Gunnar von HEIJNE, Johannes STEPPUHN and Reinhold G. HERRMANN Department of Molecular Biology, Karolinska Institutet, Center for Biotechnology, Huddinge Hospital Botanisches Institut der Ludwig-Maximilian-Universitat, Munchen (Reccived October 18/November 28, 1988) - EJB 88 1227 Representative samples of mitochondrial and chloroplast targeting peptides have been analyzed in terms of amino acid composition, positional amino acid preferences and amphiphilic character. No highly conserved homology blocks are found in either class of topogenic sequence. Mitochondrial-matrix-targeting peptides are composed of two domains with different amphiphilic properties. Arginine is frequently found either at position - 10 or - 2 relative to the cleavage site, suggesting that some targeting peptides may be cleaved twice in succession by two different matrix proteases. In stroma-targeting chloroplast transit peptides three distinct regions are evident: an uncharged amino-terminal domain, a central domain lacking acidic residues and a carboxy-terminal domain with the potential to form an amphiphilic P-strand. Targeting peptides that route proteins to the mitochondrial intermembrane space or the lumen of chloroplast thylakoids have a mosaic design with an aminoterminal matrix- or stroma-targeting part attached to a carboxy-terminal extension that shares many characteristics with secretory signal peptides. Both mitochondria and chloroplasts import most of their proteins from the cytoplasm in the form of preproteins with amino-terminal extensions (targeting or transit peptides). In most cases, the targeting peptides are removed by intraorganellar proteases during of shortly after import. Since both mitochondrial and chloroplast targeting peptides can direct the import of normally cytoplasmic proteins they should carry most of the information required for correct sorting; yet, the only common themes suggested so far are the possible existence of an amphiphilic a-helix in mitochondrial targeting peptides [l - 31 and three homology blocks of conserved amino acids in chloroplast transit peptides [4-51. In this paper, we present the results of an extensive analysis of collections of nonhomologous mitochondrial targeting (mtp) and chloroplast targeting (ctp) peptides. We find no signs of conserved homology blocks in either class of sequence. mtps are composed of two structurally distinct domains : an amino-terminal domain with amphiphilic a-helical character and a carboxy-terminal domain with different amphiphilic properties. In the latter domain, Arg residues are frequently found either around position -2 or position - 10, counting from the cleavage site. ctps also show signs of a domain structure with an uncharged amino-terminal part, a central nonamphiphilic part lacking acidic residues and a carboxy-terminal part that is predicted to form an amphiphilic 6-strand. We suggest that ctps, unlike mtps, will not spontaneously integrate into lipid bilayers to any significant degree; rather, we favour a model where they bind either to the membrane surface and/or to specific receptors on the chloroplast. Correqmndence to G. von Heijne, Institutionen for Moleky- Iiirbiologi, Huddinge sjukhus, K87, S-14186 Huddinge, Sweden Abbreviations. mtp, mitochondrial targeting peptide; ctp, chloroplast transit peptide. MATERIALS AND METHODS Sequence samples A total of 37 mitochondrial targeting peptides (mtps) with known cleavage sites (matrix protease) were collected from the literature (Table 1). This collection was further subdivided into a sample containing sequences from Saccharomyces and Neurospora, and a sample containing sequences from higher eukaryotes. The two samples were analyzed separately as well as pooled. Likewise, a total of 18 chloroplast transit peptides (ctps) with known cleavage sites (stromal protease) were analyzed (Table 1). For the analysis of the amino-terminal region, an additional eight ctps with unknown cleavage sites were included in the sample (Table 1). Control samples of mature, imported mitochondrial or chloroplast proteins included all soluble or peripheral membrane proteins of known sequence (with their targeting sequences removed) from Table 1. For comparison with the amino-terminal region of imported mitochondrial proteins, residues 1-15 were removed from all sequences in the control sample. Apolar regions from secretory signal peptides were obtained from the SIGPEP database [36]. Residues -13 to -7 from a total of 94 prokaryotic signal peptides and residues - 13 to - 6 from a total of 472 eukaryotic signal peptides, all with known cleavage sites, were pooled. Hydrophobic moment analysis Hydrophobic moment analysis was performed according to [37]. The window size was 11 residues for mtps and 10 residues for ctps. The statistical significance of peaks in the hydrophobic moment vs. 6 plots was assessed by comparison with the hydrophobic-moment profiles for control samples of

cn w (T\ Table 1. Mitochondriul-targeting peptides and chloroplast transit peptides analyzed in this paper Known cleavage sites are indicated by... PS. photosystem; RuBisCO, ribulosc bisphosphate carboxylaseioxygenase; EPSP, 5-enolpyruvylshikimate-3-phosphate synthase Source Protein Sequence Reference Mitochondrial-targeting peptides Sacchuromyces ATPase F, p-subunit ATPase Fo subunit 4 Cytochrome oxidase IV Cytochrome oxidase V Cytochrome oxidase VI Cytochrome oxidase VIII Superoxide dismutase (Mn) Stabilizing factor 9-kDa protein Rieske Fe-S protein C1-tetrahydrofolate synthase Dihydrolipoamide dehydrogenase Neurospora Bovine Chicken Human Maize Rat Cytochrome oxidase IV Cytochrome oxidase V Proteolipid Adrenodoxin Adrenodoxin reductase ATPase inhibitor Cytochrome oxidase IV Cytochrome P-450 (side-chain cleavage) Cytochrome P-450 (1 1 B) ATP synthase F6 Oligomycin sensitivity conferral protein Proteolipid PI 5-Aminoleavulinate syiithase Aspartate aminotransferase ATPase F1 B subunit Lipoamide dehydrogenase Superoxide dismutase (Mn) Ornithine transcarbamylase Pyruvate dehydrogenase a-subunit Pyruvate dehydrogenase /?-subunit Superoxide dismutase (Mn) Carbamyl phosphate synthetase I Malate dehydrogenase Serine: pyruvate arninotransferase Succinyl-CoA syntbetase, mubunit Aldehydedehydrogenase MVLPRLYTATSRAAFKAAK^QSAPLLSTSW MSMSMGVRGLALRSVSKTLFSQGVRCPSMVIGARY-MSSTPEKQTD MLSLRQSIRFFKPATRTLCSSRYLL-QQKPVVKTAQ MLRNTFTRAGGLSRITSVRF^AQTHALSNAA MLSRAIFRNPVINRTLLRARPGAYHATRLTKNTFIQSRKY-SDAHDEETFE MLCQQMIRTTAKRSSNIMTRPllMKRS^VHFKDGVYEN MFAKTAAANLTKKGGLSLLSTTARRT-KVTLPDLKWD MLNRCISRNTRLPVNLRIASRFY-SDGPLGGAGP MLGIRSSVKTCFKPMSLTSKRLISQSLLAS-KSTYRTPNFD MLSRLSLLSNSRAFQQARWRIYRLKVSPTVHASQ-YHILSGRKLA MLRIRSLLNNKRAFSSTVRTL-TINKSHDVVI... RAPALRRSIATTVVRC-NAETKPVPPH MLRTPTVSALVRNVAVRAAKPTMAVRA^ASTMPISNPT MASTRVLASRLASQMAASAKVARPAVRVAQVSKRTTIQTGSPLQTLKRTQMTSIVNATTRQAFQKRA~YSSEIAQAMV MAARLLRVASAALGDTAGWRLLVRPRAGAGGLRGSRGPGLGGGAVATRTLSVSGRAQ~SSSEDKITVH MAPRCWRWWPWSSWTRTRLPPSRSIQNFGQHF~STQEQTPQIC MAATALAARTRQAVWSVWAMQGRGF*GSESGDNVRS MLATRVFSLIGRRAISTSVCVR-AHGSVVKSED MLARGLPLRSALVKACPPILSTVGEGWGHHRVGTGEGAG^ISTKTPRPYS MALWAKARVRMAGPWLSLHEARLL^GTRGAAAAPKA MILQRLFRLSSAVQSAISVSWRRNIGITAVAF^NKELDPVQKL MAALAVSGLSQQVRCFSTSVVRP^FAKLVRPPVQ MQTTGALLISPALIRSCTRGLIRPVSASFLSRPEIQSVQPSYSSGPLQVARREFQTSVVSR~DIDTAAKFIG MEAVVRRCPFLARVSQAFLQKAGPSLLFYAQHCPKMMEAAGQ-QVEETPAAQP MALLQSRLLLSAPRRAAATARA^SSWWSHVEMG MTSLWGKGTGCKLFKFRVAAAPASGALRRLTPSASLPPAQLLLRAVRRRSHPVDYAAQ~TSPSPKAGAA MQSWSRVYCSLAKRGHFNRISHGLQGLSAVPLRTY^ADQPIDADVT MLSRAVCGTSRQLAPAIJGYLGSRQ~KHSLPDLPYD MLFNLRILLNNAAFRNGHNFMVRNFRCGQPLQ*NKVQLKGRDL MRKMLAAVSRVLSGASQKPASRLLVASRN-FANDATFEIK MAAVSGLVAETPSEVSGLLKRRFHWTAPAAAVQVTVRDAIN MALRTLASKKVLSFPFGGAGRPLAAAASARG^VTTVTTVTLPDLS MTRILTACKVVKTLKSGFGLANVTSKRQWDFSRPGIRL^LSVKAQTAHI MLSALARPVGAALRRSFSTSAQ"^AKVAVLGASG MFRMLAKASVTLGSRAASWVRNMG^SHQLLVPPPE MVSGSSGLAAARLLSRTFLLQQNGIRH-GSYTASRKNI MLRAALSTARRGPRLSRLL-SAAATSAVPA

[771 ~781 [821 ~ 3 1 ~ 5 1 Spinach Chloroplast transit peptides 10-kDa protein ATPase &subunit Ferrodoxin oxidoreductase Light-harvesting-complex protein I1 MATSVMSSLSLKPSSFGVDTKSAVKGLPSLSRSSASFTVRA^SGVKKIKVDK MAALQNPVALQSRTTTAVAALSTSSTTSPPKFSLSFSSSTATFNPLRLKILTASKLTAKPRGGALGTRM-VDSTASRYAS MTTAVTAAVSFPSTKTTSLSARSSSVISPDKISYKKVPLYYRNVSATGKMGPIRA-QIASDVEAPP MASSTMALSSPSLAGKAVKLGPTASEIIGEGRIT^MRKTAGKPKT 1691 [331 [701 Wedel, personal Rieske Fe-S protein Small subunit of RuBisCO RuBisCO activase Nitrite reductase Acyl carrier protein I PS 1-3 PS 1-5 PS 1-6 Glutamine synthase 2 Thioredoxin F PS I1 20-kDa protein communication MIISIFNQLHLTENSSLMASFTLSSATPSQLCSSKNGMFAPSLALAKAGRVNVLISKERIRGMKLTCQ~ATSIPADNVP [711 MASSVLSSAAVATVSRTPAQASM~APFTGLKSTVGFPATKNDDITSLASNGGRVQC-MKVWPTQ~MK [721 MATAVSTVGAATRAPLNLNGSSAGASVPTSGFLGSSLKKHTNVRFPSSSRTTSMTVKA~AENEEKNTDK [731 MASLPVNKIIPSSTTLLSSSNNNRRNNSSIR-CQKAVSPAAE [741 MASLSATTTVRVQPSSSSLHKLSQGNGRCSSIVCLDWGKSSFPTLRTSRRSFISA-AKKETIDKVC [751 MASIASSVAVRLGLTQVLPNKNFSSPRSTRLVVR^AAAPA unpublished results MAAATASLSSTLLAPCSSKQPQPQQQHQHQQLKCKSFSGLRPLKL~ISSNNSSSSLSMSSARRSMTCRA-ELSPSLVISL unpublished ~761 MASLATLAAVQPTTLKGLAGSSIAGTKFTSARRQSFKL~VRSGAIVA-KYGDKSVYFD results MAQILAPSMQCQMKLSKGLTSSMTPSPWTSILLKQGQKGSIKCSTKFRVCAS~QSDHG unpublished results MALHLSLSHQSWTTPAHPITSSDPTRSSVPGTGLSRRVDFLGSCKINGVFVVKRKDRRRMRGGE unpublished results MAAATSATAIVNGFTSPFLSGGKKSSQSLLFVNSKVGAGVSTTSRKLVVVAAAAAPKKSWIPAVKGGGNFL unpublished results Silene Ferredoxin Pea Early light-induced protein Heat-shock protein Gin synthase Tomato PS 1-2 PS I1 Chlorophyll ah apoprotein Light-harvesting-complex protein I Brassica Acyl carrier protein Chlamydomonas Small subunit of RuBisCO Arabidopsis EPSP Petunia EPSP MASTLSTLSVSASLLPKQQPMVASSLPTNMGQALFGLKAGSRGRVTAM~ATYKVTLITK MAVSSCQSIMSNSMTNISSRSRVNQFTNIPSVYIPTLRRNVSLKVRSMAEGEPKEQS MAQSVSLSTIASPILSQKPGSSVKSTPPCMASPLRRQLPRLGLRNVRAQAGGDGDN MSSLSDLINFNLSDSTEKIIAEYIWVGGSGIDIRSKARTLPGPVSDPAKLPKWNY MAMATQASLFTPPLSVPKSTTAPWKQSLVSFSTPKQLKSTVSVTRPIRAM-AEEAPAATEE MATSMALSSSTFAGKAVKLSPSSSEITGNGRVTMRKTATKAKPASSGSPWYGPDRVKY MASNTLMSCGIPAVCPSFLSSTKLSKFAAAMPVSVGATNSMSRFSM MSTTFCSSVSMQATSLAATTRISFQ~PALVSTTNLSFNLRRSIPTRFSISC-AAKP~TVEKV ~ 4 1 MAVIAKSSVAAVARPARSSVRPMAALKPAVKAAPVAAPAEAND-MMVWTPVNNK [791 [801 [811 [861 1861

538 100 randomly scrambled copies of each wild-type 11- or 10- residue sequence using a one-sided t-test. An amphiphilic a-helix is characterized by a strong hydrophobic-moment component for an angle 6 = 90-100" between successive residues and an amphiphilic B-sheet has a corresponding strong component for 6 = 160-180" [7]. Amino-acid-frequency analysis Overall amino acid distributions in the various samples were compared using a 1' test (19 degrees of freedom). When two distributions were found to be significantly different (P < by this method, the counts for individual amino acids were compared again using a 1' test (one degree of freedom). The statistical significance of peaks in the position-specific distribution of individual amino acids was assessed by calculating, from a binominal distribution, the probability that an equal or greater number of the residue in question would be found when n residues (n = number of sequences in the sample) were drawn at random from a pool of amino acids with average frequencies the same as those found in the full mtp or ctp samples. RESULTS Mitochondria1 targeting peptides In a previous study, we showed that mtps are enriched for Arg, Leu and Ser, contain very few Asp and Glu and have the potential to fold into amphiphilic a-helices [2]. We have now extended this study to include a total of 37 mtps with known cleavage sites (Table 1). mtps from lower eukaryotes (Saccharomyces and Neurospora, 14 entries) have been analyzed separately from mtps from higher eukaryotes (mainly mammals, 23 entries). Overall amino acid composition qf mtps With the larger and more well-defined collection now available, we have re-evaluated the amino acid preferences found in our earlier study. As shown in Table 2, mtps contain significant more Ala, Leu, Arg and Ser and less Asp, Glu, Ile and Lys residues than the control sample of imported, soluble mitochondria1 proteins (P < in all cases). The reduction in Ile and Lys is only apparent in mtps from higher eukaryotes. mtps from lower eukaryotes are also significantly enriched for Thr (data not shown). The C-terminal 10-12 residues have difierent umphiphilic properties than the N-terminal domain Since positively charged, amphiphilic a-helices have been suggested to be important for the function of mtps, we first wanted to know whether these amphiphilic sections were distributed throughout the mtps, or confined to a smaller part. The mtps in our sample were aligned with coincident cleavage sites, and the mean a-helical hydrophobic moment (6 = 95") for successive 11-residue segments was determined (Fig. 1 A). As a control, the amino acids in each 11-residue segment in each sequence were scrambled and reassembled into 100 random segments and the same analysis was performed on this randomized sample. From the figure it is clear that the 10-15 carboxy-terminal residues do not consistently fold into cr-helices with a higher hydrophobic moment than the Table 2. Amino ucid,frequencies Statistically significanl differences (P < text for description of peptidcs analyzed Amino Mitochondrial Chloroplast pcptides acid peptidcs A C D E F G H I K L M N P Q R S T V W Y Total are indicated by *. See mtp mature ctp -lo/-1 N 3-10 mature 0.141* 0.084 0.016 0.013 0.003* 0.048 0.008* 0.068 0.036 0.039 0.064 0.081 0.011 0.024 0.032* 0.061 0.034* 0.067 0.122* 0.091 0.016 0.024 0.025 0.038 0.046 0.050 0.042 0.040 0.135* 0.047 0.112* 0.058 0.065 0.054 0.064 0.075 0.016 0.009 0.011 0.028 1138 9543 0.114 0.128 0.013 0.022 0.007* 0.006 0.007* 0.022 0.034 0.017 0.046 0.072 0.007 0.000 0.037 0.067 0.062 0.022 0.093 0.022* 0.023 0.044 0.042 0.039 0.060 0.039 0.041 0.017 0.058 0.167* 0.192* 0.150 0.089* 0.089 0.066 0.078 0.003 0.000 0.004* 0.000 945 180 0.139 0.019 0.005 0.000 0.01 9 0.019 * 0.014 0.062 0.010* 0.111 0.038 0.038 0.014* 0.053 0.005 * 0.255 0.106 0.091 0.000 0.000 208 0.077 0.019 0.065 0.077 0.034 0.074 0.014 0.055 0.070 0.083 0.029 0.043 0.048 0.033 0.045 0.055 0.053 0.083 0.012 0.028 2235 controls. This was confirmed using shorter windows of 8-10 residues (not shown). Residues -24 to -15, on the other hand, have a high mean a-helical hydrophobic moment. In order to characterize the carboxy-terminal region further, we calculated the mean hydrophobic moment of the samples as a function of 6 for residues - 1 I to - 1 (Fig. 1 B). A significant peak was apparent around 6 = 75 ' (P < lo-'). Also shown in Fig. 1 B is the hydrophobic-moment profile for residues -18 to -8, with a typical amphiphilic a-helix peak at 6 = 95" (P < lop3). The transition between the two regions is readily apparent in the 6 = 75" and 6 = 95" profiles in Fig. 1 A. Similar results were obtained when the mtps from lower and higher eukaryotes were analyzed separately (not shown). These observations suggest that the carboxy-terminal 10-15 residues in mtps do not, as a rule, tend to form classical amphiphilic a-helices. The possible significance of the 6 = 75" structure is discussed further below. Arg is enriched around positions - 2 and - 10 Since the cleavage of matrix-targeting mtps from mature proteins is highly specific, it seemed likely that distinct patterns of individual amino acids might be found in the region close to the cleavage site. We thus calculated the incidence of each of the 20 amino acids as a function of position relative to the cleavage site (located between positions - 1 and + 1). Since the data base is still fairly small, only very strong position-specific preferences can be found at this stage. Nevertheless, a significant enrichment of Arg residues is found at position -2 (P < lop3) and at position -10 (P < Fig.2). Less significant peaks are found in the neighboring positions, - 3 and - 11 (P < 0.05). The - 2 peak is apparent

539 Fig. 1. Mean normalized hydrophobic moment of mtps as a function of 6 and of position relative to the cleavage site. (A) Mean normalized hydrophobic moment of mtps as a function of position relative to the cleavage site (located between positions -1 and +I). The mean hydrophobic moment (p) for an 1 I-residue window starting at the indicated position was normalized by first subtracting the mean value (p)5cr calculatcd for a control sample consisting of 100 randomly scrambled copies of each of the wild-type 11-residuc stretches contributing to a given point and then dividing by the standard deviation u of the calculated (p) value. The shaded area indicates 2 standard deviations in the determination of (p);(0) 6 = 95"; (+) 6 = 75". (B) Mean normalized hydrophobic moment of mtps as a function of 6 for segments - 11 to - 1 (0) and - 18 to -8 (W). Normalization was carried out as in A. The shaded area indicates f 2 standard deviations in the determination of (p) 0.5 0.4 0.3 0.2 0.1 Table 1 were compared with the composition of a sample including all remaining residues, i.e. from residue 16 to the carboxy terminus (excluding the integral membrane proteins, see Materials and Methods). Two differences were observed : Pro and Ser are significantly more abundant in the aminoterminal region of the mature proteins (So/, vs 5% Pro and 9% vs 6% Ser, P < for both). In summary, at least two domains can be identified in mtps. The adjoining amino-terminal region of the mature proteins also seems to have a distinct amino acid composition. 0.0-25 -20-15 -10-5 +I +6 +11 +I6 +21 Position Fig. 2. Positional distrihufion of Arg residues in nztps. The cleavage site is between positions - 1 and + 1 (arrow) Chloroplasl targeting peptides A total of 18 stroma-targeting ctps with known cleavage sites and an additional eight sequences where the stromal cleavage sites have not been determined (Table 1) were analyzed using the same methods as above. in mtps from both lower and higher eukaryotes, whereas the - 10 peak is much more prominent in the sample from higher eukaryotes (not shown). Interestingly, mtps with Arg at position - 2 do not seem to be enriched for Arg at position - 10 and vice versa (data not shown). Furthermore, out of 16 sequences with Arg in position - 10, all but one have an apolar residue in position -8 (mostly Phe, Ile or Leu). Arginine residues elsewhere in the rntps do not show this bias towards apolar residues in their downstream next-nearest-neighbor position and mtps that do not have Arg in position -10 are not enriched for apolar residues in position - 8. We also compared the overall amino acid composition of the amphiphilic amino-terminal regions (up to and including residue - 17) and the carboxy-terminal regions (residues - 13 to - 1). No significant differences were observed. The,first 10-15 residues of' mature, imported mitochondria1 proteins are rich in Pro and Ser The amino acid composition of the 15 amino-terminal residues of the mature *mitochondria1 proteins shown in CTPS contain.few acidic residues, but many Ser and A h The overall amino acid composition of the ctps were compared with the composition of mature chloroplast proteins (Table 2). Significant differences (P < lop3) were found for Ser and Thr, which are more abundant in the ctps (19% vs 6% for Ser, 9% vs 5% for Thr) and for Asp, Glu and Tyr which are almost completely absent from the ctps. In contrast to the mtps, however, Arg and Leu were not found to be enriched in the chloroplast transit peptides. It has been suggested that ctps contain three fairly wellconserved 'homology blocks' [4]: an amino-terminal block with the consensus sequence MAXSXMXSS (where X denotes the possible presence of an undefined residue), a central block PXFXGXK, and a carboxy-terminal block GXGRV just before the cleavage site. These blocks were found by analyzing six homologous ctps from light-harvesting complex protein I1 polypeptides, six homologous ctps from ribulose-bisphosphate carboxylase/oxygenase small subunit polypeptides, one ferredoxin, and one plastocyanin ctp. We have searched our collection of nonhomologous ctps and found no significant conservation of any of these blocks except for the-amino-terminal MA dipeptide. Neither have we

540 Fig. 3. Mean normulized hydrophobic moment of chlorophst transit peptides. (A) Shows the mean normalized hydrophobic moment as a I'unction of 6 for the rcgion - 10 to -1, and (B) is the positional plot for 6 = 165" (cf. Fig.1) found any evidence that a critical serine at position - 8 relative to the cleavage site [6] is conserved. We conclude that ctps do not contain regions of highly conserved amino acids. The amino-terminal 10 residues,form a distinct domain The amino-terminal region of the ctps in our sample is significantly different (P < from the remainder of these sequences in overall amino acid composition: there are very few charged amino acids (Arg, Lys, Asp, Glu) and very few Pro and Gly among the first 10 residues (Table 2). In addition, the residue next to the initiator Met is almost invariably an alanine (22 cases out of 26). Serine, which is present at a level of around 20% in thc ctps, is not specifically enriched in any one position in the amino-terminal region. Hydrophobicmoment analysis does not reveal any amphiphilic structures when applied to the sample of N-terminal-aligned ctps. The region next to the cleavage site is a potential amphiphilic P-strand Hydrophobic moment analysis reveals that the last 10 residues of stroma-targeting ctps have a very pronounced peak (P < lo-') in the hydrophobic moment plot for an angle 6 = 165" between successive residues (Fig.3A). This value of 6 corresponds to the slightly curved amphiphilic P-strand normally found in P-sheets [7]. As shown in Fig. 3 B, the 6 = 165" structure is only found in the carboxy-tcrminal region. No other amphiphilic structure is apparent in the C-termiaalaligned sample (data not shown). It is interesting to note that thylakoid-targeting ctps (see below) often seem to have an intermediate cleavage site close to a similar stretch of amphiphilic P-strand [8]. It is thus possible that the same stromal protease cleaves both kinds of ctps. The amino acid composition of the carboxy-terminal 10 residues is also different from the more amino-terminal parts. Specifically, Arg is enriched in this region (14% vs 3%, P < and Leu is found only infrequently (2% vs lo%, P < Table 2). Although the ctp sample is rather too small for the reliable identification of position-specific amino acid preferences, it is apparent that turn-inducing residues (Gly, Ser, Asp, Asn, Pro) [9] are largely absent from positions - 3 to + 1, i.e. from the immediate vicinity of the stromal cleavage site. Ala, on the other hand, occurs rather frequently in this region (data not shown). Targeting to the mitochondria1 intermembrane space and to the thylakoid lumen depends on mosaic targeting peptides with signal-peptide-like extensions Both the mtps and ctps analyzed above target proteins for import through two membranes into the matrix of mitochondria or the stroma of chloroplasts. However, some proteins are routed to other suborganellar locations such as the mitochondrial intermembrane space or the chloroplast thylakoid-membrane system. It has been proposed that intermembrane-space proteins are first imported into the matrix compartment and then re-exported through the inner membrane [lo]. A similar model may be applicable to proteins of the thylakoid lumen and to some thylakoid-membrane proteins: they are first imported into the stromal compartment and then further routed into the thylakoid [I 11. A collection of targeting signals from such proteins is shown in Fig. 4. It is immediately obvious that these signals are mosaic structures with a typical amino-terminal matrix-targeting intp or stroma-targeting ctp attached to a carboxy-terminal apolar domain. Indeed, this latter region is very similar to known secretory signal peptides involved in routing proteins into the secretory pathway of both prokaryotic and eukaryotic cells [12]. Signal peptides are characterized by a short aminoterminal positively charged region, a central apolar region some 7-15 residues long, and a carboxy-terminal region typically 5 - h residues long and with small, uncharged amino acids in positions -3 and -1, counting from the cleavage site. Assuming that the mosaic targeting peptides are first cleaved just before the apolar stretch upon import into the matrix or stroma [8], bona fide amino-terminal signal peptides would be generated. In the case of the thylakoid-targeting peptides this analogy extends all the way up to and including the final cleavage site (A-X-AJ), whereas the mitochondrialintermembrane-space-targeting peptides seem to be cleaved at a final site which does not always follow the (-3, - 1) pattern (Fig. 4). From preliminary data (obtained in collaboration with Dr C. Robinson) it appears that at least some thylakoid-targeting ctps are first cleaved in, or close to, a potential amphiphilic P-strand similar to that found in stroma-targeting ctps, thus exposing the signal-peptide-like structure. It is interesting to note that the apolar region of the intraorganellar signals differs significantly in amino acid composition from the corresponding regions in both prokaryotic and eukaryotic signal peptides. The typical apolar region of

Yeast cytochrome c1 [87] MFSNLSKRWAQRTLSKSFYSTATGAASKSGKLTQKLVTAGVAAAGITASTLLYADSLTAEA#MTAAEHGLHA Yeast cytochrome hz [88] MLKYKPLLKISKNCEAAILRASKTRLNTIRAYGSTVPKSKSFEQDSRKRTQSWTALRVGAILAATSSVAYLNWHNGQIDN#EPKLDMNKQKN Yeast cytochrome c peroxidase [89] MTTAVRLLPSLGRTAHKRSLYLFSAAAAAAAAAATFAYSQSHKRSSSSPGGGSNHGWNNWGKAAALAS#TTPLVHVASV Human cytochrome c1 [90] MAAAAASLRGVVLGPRGAGLPGARARGLLCSARPGQLPLRTPQAVALSSKSGLSRGRKVMLSALGMLAAGGAGLAVALHSAVSA#SDLEVHPPSY Spinach 16-kDa protein [91] MAQAMASMAGLRGASQAVLEGSLQISGSNRLSGPTTSRVAVPKMGLNIRAQQVSAEAETSRRAMLGFVAAGLASGSFVKAVLA#EARPIVVGPP Spinach 23-kDa protein [91] MASTACFLHHHAAISSPAAGRGSAAQRYQAVSIKPNQIVCKAQKQDDNEANVLNSGVSRRLALTVLIGAAAVGSKVSPADA#AYGEAANVFG Spinach 33-kDa protein [8] MAASLQASTTFLQPTKVASRNTLQLRSTQNVCKAFGVESASSGGRLSLSLQSDLKELANKCVDATKLAGLALATSALIASGANA#EGGKRLTYDE Spinach plastocyanin [92] MATVASSAAVAVPSFTGLKASGSIKPTTAKIIPTTTAVPRLSVKASLKNVGAAVVATAAAGLLAGNAMA#VEVLLGGGDGSLAFLPGDFS Spinach CFo-2 [Steppuhn et al., unpublished results] MANMLVASSSKTLPTTTTTTITPKPKFPLLKTPLLKLSPPQLPPLKHLNLSVLKSAAITATPLTLSFLLPYPSLA#EEIEKASL~D Spinach photosystem 1-4 protein [76] MSFTIPTNLYKPLATKPKHLSSSSFAPRSKIVCQQENDQQQPKKLELAKVGANAAAALALSSVLLSSWSVAPDAAMA#DIAGLTPCKE Chlanzydomonas reinhardtii oxygen-evolving complex 2 (33 kda) [93] MATALCNKAFAAAPVARPASRRSAVVVRASGSDVSRRAALRGFAGAAALVSSSPANA#AYGDSANVFG Chlamydomonas reinhardtii Cu(1I)-repressible cytochrome c [21] MLQLANRSVRAKAARASQSARSVSCAAAKRGADVAPLTSALAVTASILLTTGAASASA#ADLALGAQVF Pea cytochrome f [94] MQLTRNAFSWIKKEITRSISVLLMIYIITRAPISNA#YPIFAQQGYE Fig.4. A collection ofintru-organellar sorting signals. The location of the photosystem 1-4 protein is not known, but the structure of its transit peptide suggests that it is lumenal. Final cleavage sites are indicated by #. References are given in square brackets, apolar regions are underlined 541 0.5 0.4 '0 0.2.- w 0 g 0.2 0.1 0.c A C D E F G H I K L M N P O R S T V W Y Residue Fig. 5. Amino acid content of the apolur regions underlined in Fig.4. Intra-organellar sorting signals (black bars); bacterial secretory signal peptides (white bars) and eukaryotic secretory signal peptides (grey bars) a eukaryotic signal peptide has some 40% Leu, 10% Ala and 40% other apolar residues; that of a prokaryotic signal peptide has 25% Leu, 20% Ala and 40% other apolar residues; and the apolar regions of the intra-organellar signals have 20% Leu, 35% Ala and 20% other apolar residues (Fig. 5). DISCUSSION Mitochondria1 and chloroplast targeting peptides contrasted As shown above, neither mtps nor ctps contain stretches of highly conserved sequence; rather, they are remarkably variable in their amino acid sequence. Nevertheless, they seem to be built from two (mtp) or three (ctp) structurally distinct domains and they have overall amino acid compositions that are very different from average soluble proteins. In terms of gross amino acid content, both mtps and c' 1 1's lack acidic residues, but beyond this they are different: mtps are mainly enriched in Arg (14%), Ser (11Y0) and Ala (14%), whereas ctps are characterized by a high percentage of Thr (9%) and an extremely high content of Ser (19%). In fact, it is possible to discriminate efficiently between mtps and ctps by simply checking whether the fraction of Ser in the targeting peptide,f,,,, is smaller or greater than 0.07 + 1.4 xfarg. More than 90% of the mtps can be told apart from more than 90% of the ctps in this way. In mtps, two domains can be discerned: an amino-termind region, that can potentially fold into an amphiphilic a- helix (6 = 95"), and a carboxy-terminal domain of 10-15 residues that is characterized by a strong component of the hydrophobic moment for 6 = 75". Within this latter domain, Arg is very often found at positions -2 and -10. The 6 = 75" structure could correspond to an a-helix with a clockwise spiralling ridge of apolar (or polar) amino acids

542 A B Fig.6. Helical net plots of residues - 18 to - 1 of the mtps from the Saccharomyces Rieske Fe-S protein (A) and Neurospora rytochrornc. oxidase IV (B). Putative apolar i, i + 5 ridges are shaded and positively charged residues are highlighted. The cleavage site is at the top of the diagrams. Note that two identical copies of the helical net have been placed side by side to allow the apolar i, i + 5 spirals to be seen more clearly involving residues i, i + 5, i + 10, etc. In fact, such a ridge can be seen on helical net plots of the carboxy-terminal region in many mtps (Fig. 6) and could be an indication that helix/ helix packing [I31 might take place between mtps and the matrix protease responsible for cleavage. It is also noteworthy that the amino-terminal regions of mature imported mitochondrial proteins are rich in Pro and Ser. Conceivably, this could be related to the cleavage specificity (see below). Stroma-targeting ctps seem to have at least three distinct regions. At their amino-terminal they have a stretch of some 10 amino acids that is mostly uncharged and also contains few Pro and Gly residues (there is no corresponding domain in mtps). The residue next to the initiator Met is almost invariably Ala (in mtps this residue is often Leu, 40% of all sequences, but almost never Arg, 3% of all sequences). This probably means that the initiator Met is normally removed in ctps but not in mtps [14, 151. The central region is of variable length, is rich in Ser, but contains few if any acidic residues. Finally the carboxy-terminal region (8-10 residues), contains an increased proportion of Arg residues and can be folded into an amphilic /?-strand. The number of nonhomologous sequences with known cleavage sites is not yet sufficiently large to draw any strong conclusions regarding positional amino acid preferences in the vicinity of the cleavage site, but turn-promoting residues seem to be avoided at positions - 3 to + 1. We also note that the amino- and carboxy-terminal regions of ctps seem to be more sensitive to deletions than the central region [6, 161. The CdrbOXy-terminal amphiphilic /?-strand would seem likely to be involved in interactions with the stromal protease rather than with the chloroplast envelope and, except for the uncharged but not very hydrophobic amino-terminal domain, there are no other indications from the sequence data that ctps can form structures that might partition into lipid bilayers. mtps, on the other hand, due to their amphiphilic character, interact strongly with lipid monolayers and bilayers in model systems [l, 171. Possibly, the net positive charge of ctps could induce a loose association with the surface of a negatively charged membrane, but in addition some kind of receptor-mediated mechanism must be imagined. The absence of any significant overall enrichment in positively charged residues (in contrast to the high incidence of Arg in mtps) may be related to the fact that, unlike mitochondria, protein import into chloroplasts does not require an electrochemical gradient across the inner envelope membrane [I 81. Another possibility is that Arg residues in mtps may bind specifically to cardiolipin present in the mitochondrial but not the chloroplast envelope [19, 201. A remarkable exception to these clear-cut differences between mtps and ctps is provided by the Chlamydomonas Cu(I1)-repressible cytochrome c ctp (Fig.4). This is a mosaic sorting signal targeting the protein to the thylakoid lumen [21]. However, the amino-terminal, presumably stroma-targeting, part would be classified with the matrix-targeting mtps by all criteria discussed above: high Arg and modest Ser content, Met-Leu amino-terminal sequence and a high hydrophobic moment for 6 = 95" but not for 6 = 165" (data not shown). It should be interesting to study the behavior of this protein in in vitro import assays. Cleavage sites in mtps and ctps Our results show that the specifications of the cleavage sites in mtps and ctps do not reside in highly conserved consensus patterns of amino acids. The amphiphilic b = 75" structure found just before the cleavage site in mtps and the 6 = 165" structure found in a similar position in ctps most probably contribute to cleavage-site recognition. The high content of Pro and Ser in the region immediately downstream of the mtp cleavage site suggests that the possibly a-helical mtp is normally followed by a rather loosely structured piece of chain; the transition point could be a recognition element for a matrix protease. Arg residues around positions -2 and - 10 also seem to play an important role in defining the precise cleavage site in mtps (the prevalence of Arg in position -2 has been noted before [22]). It has recently been shown that human mitochondria contain two distinct matrix-localized processing activities, proteases I and I1 [23]. Protease I seems to cleave all matrix-targeting mtps, whereas protease 11 cleaves only some mtps at a second site, subsequent to cleavage by protease 1. Protease I thus generates either the mature protein or an intermediate that is further processed by protease 11. In the

543 two cases where a protease I intermediate has been documented (rat ornithine transcarbamylase and malate dehydrogenase), the first cleavage takes place between residues -9 and - 8 (Arg-AsnJPhe) for ornithine transcarbamylase or between - 10 and -9 (Arg-ArgJSer-Phe) for malate dehydrogenase [24, 251. In both cases, residue -10 is an Arg and residue -8 is an apolar Phe. Neither sequence has Arg in position -2 relative to the amino terminus of the mature protein (Val-GlnJSer and Asn-AsnlAla). This fits quite well with the findings reported here: almost all mtps have either an Arg in position -2 or an Arg in position -10 followed by an apolar residue in position -8. Few have Arg in both positions - 10 and -2, and those that lack an Arg in position - 10 also tend to lack an apolar residue in position - 8. Possibly, most or all mtps with an Arg in position -10 followed by an apolar residue in position - 8 and lacking an Arg in position - 2 are cleaved in two steps: first, by protease 1 after residue -9 or - 10 (generating an eight- or nine-residue intermediate) and then by protease 11. mtps with Arg in position -2 but not at - 10 are likely to be cleaved only by protease I. Good candidates for two-step processing would thus be, for example, bovine adrenodoxin reductase, cytochrome oxidase IV and proteolipid PI and human pyruvate dehydrogenase b-subunit. The yeast cytochrome oxidase IV mtp and the Neurospora Rieske Fe-S and ATPase-subunit-9 mtps are also cleaved in two steps. The cytochrome oxidase IV mtp is cleaved between positions - 9 and - 8 (Arg-ThrlLeu) by the matrix protease isolated from yeast and cleaved at the final cleavage site (Leu- LeulGln) upon import into intact mitochondria [26]. When the final site is deleted, only the first cleavage takes place upon import. The Rieske Fe-S mtp is also cleaved first between positions - 9 and - 8 (Arg-AlaJLeu) and then at the final site (Leu-GlnlGly) [27]. Both mtps thus have Arg in position - 10 followed by an apolar Leu in position - 8 and neither has Arg in position -2 (it should be noted, though, that the second cleavage of the Rieske Fe-S mtp might be related to translocation of the protein back through the inner membrane, and that it is not known whether this cleavage takes place in the matrix or the intermembrane space). The ATPasesubunit-9 mtp, on the other hand, is cleaved initially between residues - 32 and ~ 31 (Arg-ThrJIle) and then at the final site (Arg-AlajTyr) [28]; in this case it would seem that protease I is responsible for both cuts. Whether two distinct proteases exist in Neurospora and yeast is unknown but seems likely in view of these results. In summary, part of the protease-i-recognition sequence seems to be an Arg in position - 2 relative to the cleavage site. Protease I1 seems to have the peculiar property of removing precisely eight or nine residues from the amino terminus of a precursor protein; it seems to be an octapeptidyl peptidase. The reason(s) for two-step processing of some mtps can only be speculated upon; an attractive idea is that the short extension on the intermediate may be required for correct folding of the mature protein, or for its assembly into larger multisubunit complexes. Whether multiple cleavage enzymes can take part in the processing of stroma-targeting ctps is not known. If proper folding or assembly of the mature protein can be controlled by amino-terminal extensions, chloroplasts may also have envolved multistep processing of targeting peptides. Processing intermediates have been found for pea ribulose bisphosphate carboxylase/oxygenase small subunit [29, 301 and for the L18 ribosomal protein from Chlamydomonas [31], but the intermediate cleavage sites were not determined. The plastid-encoded CFo-I and CFo-IV subunits are synthesized with polar amino-terminal extensions of 17 and 18 residues, respectively [32, 331. It is thus not unlikely that chloroplasts will be found to be similar to mitochondria in this respect. Intra-organellar sorting signals: remnants of a secretory pathway? The remarkable similarity between the intra-organellar sorting signals (targeting proteins to the mitochondria1 intermeinbrane space and the thylakoid lumen) and secretory signal peptides is a strong indication that the secretory machinery of the bacterial precursors to mitochondria and chloroplasts has been retained in the present day organelles. Indeed, the cleavage sites in the precursors of proteins of the thylakoid lumen are of the same type as in bacterial signal peptides (A-X-AJ), whereas only some of the cleavage sites in precursors of intermembrane-space proteins follow this pattern. There is a marked gradation in the overall amino acid composition of the apolar regions in the eukaryotic, prokaryotic and intra-organellar signal peptides (Fig. 5). Although the content of Ala + Leu remains fairly constant, eukaryotic signal peptides have a strong preference for Leu, prokaryotic signal peptides contain equal numbers of Ala and Leu residues and the intra-organellar signals have a strong bias towards Ala. Thus, although similar in basic design, eukaryotic signal peptides are markedly more hydrophobic than the intra-organellar sorting signals. It is conceivable that the latter could be erroneously recognized as secretory signal peptides if they contained apolar regions of a mean hydrophobicity comparable to that of true eukaryotic signal peptides [34]. In line with this speculation, the apolar region of the thylakoid-targeting signal peptide of the chloroplastencoded cytochromef protein contains no Ala but many Leu, Ile and Val residues (Fig. 4). 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