Anton Karabinos,1,* Debashish Bhattacharya,2 Hartmut D. Kratzin, 1 Norbert Hilschmann1
1 Department of Immunochemistry, Max Planck Institute for Experimental Medicine, Hermann-Rein-Straße 3, 37075 Go¨ttingen, Germany2 Department of Biological Sciences, University of Iowa, 138 Biology Building, Iowa City, IA 52242-1324, USA
Received: 20 February 1997 / Accepted: 28 July 1997
Abstract. The human protein NEFA binds calcium,contains a leucine zipper repeat that does not form ahomodimer, and is proposed (along with the homologousNuc protein) to have a common evolutionary historywith an EF-hand ancestor. We have isolated and charac-terized the N-terminal domain of NEFA that contains asignal sequence inferred from both endoproteinaseAsp-N (Asp-N) and tryptic digests. Analysis of this N-terminal sequence shows significant similarity to theconserved multiple domains of the mitochondrial carrierfamily (MCF) proteins. The leader sequence of Nuc is,however, most similar to the signal sequences of mem-brane and/or secreted proteins (e.g., mouse insulin-likegrowth factor receptor). We suggest that the divergentNEFA and Nuc N-terminal sequences may have inde-pendent origins and that the common high hydrophobic-ity governs their targeting to the ER. These results pro-vide insights into signal sequence evolution and themultiple origins of protein targeting.
In a previous study, we reported the cloning and char-acterization of an EF-hand/leucine zipper-containing hu-
man protein, NEFA, that was localized in the cytoplasm,on the plasma membrane, and in the culture medium(i.e., secreted, Barnikol-Watanabe et al. 1994). The se-cretion of proteins from cells is a ubiquitous process. Inprokaryotes, proteins are secreted directly across theplasma membrane, whereas in eukaryotes, proteins arefirst translocated across the membrane of the endoplas-mic reticulum (ER), collected in vesicles, and then trans-ported to the plasma membrane for secretion. Similarpathways are used by membrane proteins in eukaryotes,and the translocation of proteins across membranes and/or their integration into membranes is often triggered bycleavable signal sequences (Jungnickel et al. 1994;Schatz and Dobberstein 1996).
The Signal Sequence of NEFA Is Cleaved In Vivo
The N-terminal region of NEFA is thought to function asa signal sequence. To determine if this signal sequence iscleaved in vivo, native NEFA was first extracted from aKM3 cell line according to Barnikol-Watanabe et al.(1994; Fig. 1A). A small amount of the purified proteinwas then blotted onto PVDF membrane and the N-terminus was determined by gas phase sequencing. Theresults of this procedure were ambiguous due to the re-lease of many residues in the first cycle. We think thatthe majority of NEFA might have been blocked since theyield of phenylthiohydantoin (PTH) derivatives did notcorrelate with the amount of material applied to thePVDF membrane; this has also been described for theclosely related mouse Nuc protein (Miura et al. 1992). Inanother attempt to identify the N-terminus of purifiedNEFA, 40mg of the protein was digested separately with
* Present address:Department of Biochemistry, Max Planck Institutefor Biophysical Chemistry, Am Faßberg 11, 37077 Go¨ttingen, Ger-manyCorrespondence to:D. Bhattachary; e-mail: [email protected]
either endoproteinase Asp-N (Asp-N) or trypsin. Of the40 peptides that were subsequently sequenced, peptideA68 (from the Asp-N digest) and peptide T55 (from thetryptic digest) had the identical N-terminal sequence,PIDIDK (Fig. 2). Since this is not the cleavage site foreither enzyme, this sequence likely represents the N-terminus of the mature protein. The molecular mass ofNEFA was determined by matrix-assisted laser desorp-tion ionization (MALDI) to be 47,731 ± 150 (Fig. 1B).The experimentally inferred mass of NEFA was smallerthan that calculated from the amino acid compositionbased on the DNA sequence (50.2 kDa). This mass was,however, slightly larger than that calculated with prolineas the N-terminus (47,255.9 Da). A deviation of about300 Da cannot be due to MALDI inacuracy; it remains tobe determined whether this mass difference is due to thephosphorylation of NEFA (see Fig. 2).
Analysis of the NEFA N-terminal region with theSIGPEP computer program (HUSAR computer package;German Cancer Research Center, Heidelberg) identifiedthe valine at position 25 that directly precedes thePIDIDK N-terminal sequence as the most likely site forsignal sequence cleavage. Taken together, these resultsindicate that the cDNA encoding NEFA contains a maxi-mum of 25 amino acids at the 58-terminus that forms acleavable signal sequence. This signal sequence likely
results in the secretion of NEFA into the culture medium(Barnikol-Watanabe et al. 1994). The protein, Nuc, alsocontains a functional N-terminal signal peptide (Miura etal. 1992). The Nuc signal sequence is thought to be re-sponsible for the targeting of this protein to the lumen ofthe ER (while bound to cyclooxygenase, COX, Ballif etal. 1996).
An evolutionary and functional (see above) relation-ship between the NEFA/Nuc signal sequences is sup-ported by their shared targeting to the cell membraneand/or secretion and the high identity over the remainingsequence of these proteins (61%, Karabinos et al. 1996).Both NEFA and Nuc begin with a signal sequence that ischaracterized by the following features: variable basicN-terminal region, hydrophobic core (interupted in theNuc sequence by polar amino acid serine/S and in theNEFA sequence by three polar amino acids—glutamine/Q, tyrosine/Y, and threonine/T as well as by two cys-teines/C) and a polar C-terminal region (von Heijne1985). Short downstream sequences of these proteins dif-fer significantly (see divergent N-termini in Fig. 3A).Examination of signal sequences from many proteins hasshown that these regions specify not only their accumu-lation in the cytoplasm vs secretion but also determinethe targeting pathway through interactions with cytosolicchaperones or targeting factors. These studies show thatdespite a shared target among many proteins, high simi-larity within the signal sequences is often lacking (re-viewed in Zheng and Gierasch 1996; Schatz and Dob-berstain 1996). It is of interest, therefore, to determinewhether sequences that specify the same target in differ-ent proteins (or different targets in closely related pro-teins) are the result of convergent evolution or domainshuffling. One theory of protein evolution argues thatnew proteins or protein domains such as signal se-quences are more likely to evolve from older, existingdomains rather than be the result of de novo synthesis(Doolittle 1995). Resolving the phylogeny of signal se-quences is therefore expected to provide clues not only tothe origin of intra- and extracellular targeting of proteinsbut also (by searching for the ancient patterns) to thestill-mysterious primary sequence code necessary for thefunctional competence of these domains.
Results of Database Searches Using the NEFA/NucSignal Sequences
The amino acid sequence of the NEFA N-terminus (50residues) was used to query the NCBI nonredundant pro-tein database and the Swissprot/EBI database using theBLITZ (Sturrock and Collins 1993) and BLAST(Altschul et al. 1990) programs. Both of these programsidentified human 2-oxoglutarate-malate carrier protein(OGC), a member of the mitochondrial carrier family(MCF, see Kuan and Saier 1993) of proteins, as sharing
Fig. 1. A SDS-PAGE (right) and corresponding immunoblot (left) ofthe final purification steps for NEFA. Mature NEFA was extractedfrom a KM3 cell line using a published procedure (Barnikol-Watanabeet al. 1994).Lane 1: fraction after isoelectric refocusing;lane 2: frac-tion, eluted from the preparative SDS-PAGE;lane 3:pure NEFA afterRP-HPLC; lane 4: molecular weight marker (LMW, Pharmacia,Freiburg, i. Br., Germany).B Laser desorption/ionization mass spec-trum of the purified protein NEFA. The molecular mass was deter-mined as 47,731 ± 150 Da using a method described elsewhere (Klafkiet al. 1992).
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the highest sequence similarity with NEFA (see Table 1,Fig. 3B). Like other members of the MCF proteins, OGCcontains three repeats; each repeat is of approximatelength 100 residues (depicted here as OGC-1, -2, and -3,Fig. 3C). Each of the OGC repeats contains two trans-membrane spanners (Kuan and Saier 1993). The region
of OGC identified with the amino acid database searchesas being most similar to the NEFA N-terminal region istransmembrane spanner-5 with additional conservedresidues that were located predominantly at the bordersof this and the other homologous transmembrane span-ners-1 and -3 (defined here are the MCF domains 1, 3,
Fig. 2. Amino acid sequence of NEFA deduced from the cDNA(Barnikol-Watanabe et al. 1994) and the sequence of the peptides,determined by Edman degradation. Purified NEFA was specificallycleaved with endoproteinase Asp-N or trypsin and separated by nar-row-bore RP-HPLC using a Vydac 218TP C18 (200 × 2.1 mm) andKromasil 100 C8 column (250 × 4.6 mm) as described elsewhere(Klafki et al. 1992). The amino acid sequences (underlined) were de-termined as in Klafki et al. (1992). The heptad repeat region (markedwith asterisks), hydrophilic region (depicted by ± characters) with a
putative nuclear targeting sequence (NLS), two EF-hand motifs, theacidic region, and the leucine zipper motif have been previously re-ported (Barnikol-Watanabe et al. 1994; Karabinos et al. 1996). Phos-phorylation sites were indicated by screening of the deduced aminoacid sequence of NEFA with the PROSITE program (Bairoch 1989)—four phosphorylation sites were found for protein kinase-C (ST-X-RK),ten for casein kinase-2 (ST-X2-DE), and three for tyrosine kinase [RK-X2(3)-DE-X3(2)-Y]. The functional significance of these sites remainsto be determined.
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and 5 shown in Fig. 3B). These domains together wereused to generate the conserved motif in which the three-fold presence of this sequence is characteristic of allmembers of the MCF with the exception of the singlenonmitochondrial member, the peroxisomal protein POX(Fig. 3B; Kuan and Saier 1993; Jank et al. 1993). A weaksimilarity was also found between the complete NEFAN-terminal domain and the signal sequence of a mousemast cell carboxypeptidase A precursor (Fig. 3B, pre-dicted number4 1.06 × 10−1).
To test the results of the database searches we didadditional queries using only the hydrophobic and hy-drophilic regions of the NEFA N-terminal peptide aswell as these regions from other MCF sequences. Ourcriterion for accepting a match as being biologically sig-
nificant was based on the findings of Thode et al. (1996),who have shown that a string of six identical amino acidsis the minimum match between two protein regions thatcannot be explained by chance alone. These authors alsosuggested that in regions of length less than 20 residues,a minimum match of 60% should also be interpreted asbeing biologically informative (see also Sander andSchneider 1991). There is a match of 7/11 (63.6%) be-tween the hydrophilic regions of the NEFA and humanOGC domain 5 peptide (Fig. 3B). The database searchesusing the hydrophilic or hydrophobic regions of theNEFA and MCF family (Fig. 3B) as query sequencessuggest that the MCF domain can be divided into tworegions, a highly divergent hydrophobic region in whichno conserved motif can be found and a more conserved
Fig. 3. A Comparison of the N-terminal nucleotide and correspond-ing amino acid sequences of the NEFA (human; accession numberX76732) and Nuc (human; accession number M96824) cDNAs. Thenumberingindicates the positions of the amino acids in the NEFA andNuc proteins.Bold lettersdenote identical amino acids in the alignedsequences.B Multiple alignment of the hydrophobic/hydrophilic re-gions of the NEFA and Nuc N-terminal domains with the correspond-ing region of carboxypeptidase A (Cp-A; accession number P15089)and domain 1 (and domains 3 and 5 from OGC and POX) from a
number of MCF members. The positions, taxon name-abbreviations,and accession numbers of aligned MCF sequences were reported else-where (Kuan and Saier 1993).C Hydrophobic moment of the alignedsequences (OGC, NEFA, and Nuc) shown in Fig. 3B (according to theKyte-Doolittle program in the GCG computer package). Note the com-mon charge distribution and the different hydrophobic moment (H/index is for the hydrophilicity index and indicates the hydrophobicitypeaking of the analyzed sequences) in the NEFA/Nuc and OGC pep-tides.
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hydrophilic region that can be used to identify membersof the MCF and related peptides (data not shown).
To determine the origin of the Nuc signal sequence,50 amino acids of the Nuc N-terminus were used toquery the protein database using the BLAST program.This search identified two receptor tyrosine kinases(RTK, Yarden and Ullrich 1996)—mouse insulin-likegrowth factor type II receptor (IGFR) and avian epider-mal growth factor receptor (EGFR)—as sharing thehighest sequence similarity with the Nuc sequence(Table 1). On the other hand, the BLITZ search identi-fied, after the homologous bovine Nuc sequences, IGFR,mouse amyloid-like protein (APLP), and the human an-giotensin-converting enzyme (ACE) as having the high-est sequence similarity with human Nuc (Table 1). Por-tions of the hydrophobic regions of the above-mentionedfive signal sequences were identified by this databasesearch as being most similar to Nuc (Fig. 4A).
Possible Scenarios for the Origin of the NEFA/NucSignal Sequences
Two possible scenarios for the origin(s) of the N-terminal domains of NEFA and Nuc are as follows:
1. The N-terminal domains of NEFA/Nuc trace their an-
cestry to two independent fusion events involving anMCF spanner domain (NEFA) and a RTK-like do-main (Nuc).
2. The N-terminal domain existed in the common ances-tor of the NEFA/Nuc coding regions and has inde-pendently evolved within the gene duplication prod-ucts.
Evidence in support for the single origin of the NEFA/Nuc signal sequences (Scenario 2) comes from the rela-tively high identity within the central hydrophobic andpolar C-terminal regions and the P, L/I, D/E residuesfollowing the shared cleavage site at the N-termini of themature proteins (see alignment in Fig. 3A). The commonancestor of NEFA/Nuc would, in this case, be expectedto have had an N-terminal domain that is similar to thatin Nuc or a recognizable MCF-like domain. Strong se-lection for an altered targeting function for either NEFAor Nuc would have resulted in the divergence of thissequence. In fact, the N-terminal domain of a protein inCaenorhabditis elegansthat shares a high similarity toboth NEFA and Nuc (36% amino acid identity in eachcase) does not contain a MCF-like domain and has alimited sequence similarity to the N-terminal domainfrom IGFR (see boxed region in Fig. 4A). It is difficultto imagine therefore, given a single origin of the NEFA/Nuc N-terminal domains, why the NEFA sequencewould converge to that of a typical MCF-like sequence
Table 1. Results of database searches using the BLAST and BLITZ programs and N-terminal domains of NEFA/Nuc as query sequencesa
ProgramQueryseq. Acc. No. Organism Description P
BLAST NEFA P80303 Homo sapiens NEFA precursor 9.7 × 10−31
Q02978 Homo sapiens Mitochondrial 2-oxoglutarate/malate carrier (OGC) 1.5 × 10−4
APP1_MOUSE Mus musculus Amyloid-like protein 1 5.9 × 10−3
ACE_HUMAN Homo sapiens Angiotensin-converting enzyme 7 × 10−3
a Only the five highest-scoring sequences are listed.P indicates the probability that the best uninterrupted similarity matches found occur by chance(Altschul et al. 1990). Predicted number (Pred. No.) is the number of search results that would, by chance alone, have a score greater than or equalto the score under scrutiny and is derived from the total score distribution (Sturrock and Collins 1993)
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when this protein is not presently targeted to the mito-chondrion (see below). Taken together, these results areconsistent with the secondary, independent origin of theN-terminal domain in NEFA via domain shuffling in-volving an existing MCF-like sequence. This MCF-likesequence appears to have then diverged to allow thetargeting of NEFA to the ER, rather than to the mito-chondrion, prior to secretion.
Pairwise analyses of the N-terminal domains ofNEFA/Nuc with other signal/transmembrane sequencesidentified with our database searches show that the cen-tral hydrophobic (leucine-rich) region is most similaramong these divergent sequences (see Fig. 4B). Exclu-sion of the leucine-rich sequence led to a decrease inidentity in virtually all pairwise comparisons (notableexceptions are the EGFR/IGFR [homologous] andNEFA/OGC sequences). This result suggests that thecentral hydrophobic region is subject to strong selectionresulting in the convergence to conserved amino acids(i.e., leucine) that are shared among these distantly re-lated membrane-spanning and/or secretory proteins. Thesequence identity between the N-terminal domains ofNEFA and the OGC shown with this analysis are furtherunderscored by the finding of conserved cysteine resi-dues within the hydrophobic regions of NEFA, OGC,and phosphate carrier proteins (PIC; cysteines are under-lined in Fig. 3B). The cysteine residue in the PIC proteinhas been shown to have a significant functional role inyeast (Schroers et al. 1997).
These data reveal the dynamic nature of signal se-quences and suggest that domain shuffling may play animportant role in their evolution (Doolittle 1995). Butwhat is the functional meaning of the identified differ-ences between the N-terminal domain of the Nuc proteinand those of NEFA/OGC? Members of the MCF proteinfamily do not contain a cleavable N-terminal signal se-
quence and are targeted to mitochondria as a result of theinternal signal. Analyses of the MCF domains showedthat each of the three repeats may contain its own signalsequence and that the efficiency of mitochondrial deliv-ery is positively correlated with the number of repeats(Gwendolyn et al. 1986; Pfanner et al. 1987; Zara et al.1992). The internal signals do not have the typical am-phiphilic structure of mitochondrial signal sequences(Haucke et al. 1995). Moreover, the peroxisomal proteinPOX, the only nonmitochondrial member of the MCFfamily, contains a divergent MCF domain (see above). Itis possible therefore that the transmembrane region fol-lowed by the conserved (positive charged) hydrophilicregion within the MCF domain (Fig. 3B) could partici-pate in the mitochondrial targeting of these proteins. Thishypothesis is consistent with the novel mechanism ofimport proposed recently for another mitochondrial innermembrane protein, BCS1, that is also translated withoutany cleavable signal sequence (Fo¨lsch et al. 1996). Itwould also explain the finding of Dyer et al. (1996) thatPOX could be localized in mitochondria in the absenceof peroxisomal proliferation. These authors postulate thata competition exists for the targeting of POX to mito-chondria (via some residual mitochondrial targeting in-formation) or peroxisomes. As shown in Fig. 3B, thethird and the fifth MCF domains are still positivelycharged and may therefore contain this residual informa-tion that results in mitochondrial targeting.
If this hypothesis is correct, the existence of the MCF-like domain in the NEFA N-terminal region may reflecta previous mitochondrial targeting of this protein (pre-liminary immunoelectron microscopy using a polyclonalantibody against NEFA has failed, however, to localizethis protein in purified mitochondria [T. Cole, L.A.Awni, and A.K. unpublished results]) that has later beenlost; NEFA contains a divergent MCF-motif due to the
Fig. 4. A Alignment of the amino acid se-quences of theC. elegansNEFA/Nuc homo-logue (C. el.; accession number Z50853), mouseIGFR, chick EGFR, humanNuc, humanACE,mouseAPLP,humanNEFA, and humanOGC3proteins. The full names and accession numbersof aligned sequences are shown in Table 1.Boldlettersdenote identical amino acids between thehuman Nuc and aligned sequences. A region ofsimilarity between the IGFR and C. el. se-quences has beenboxed.B Pairwise amino acididentities for the sequences aligned inA. Thepercentage identities were calculated for the re-gion between thearrowswith and without (boldletters) the central hydrophobic sequence be-tween theasterisks(seeA).
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replacement of a nonpolar residue with aspartic acidwithin a region that is highly conserved in MCF se-quences (shown with the arrowhead in Fig. 3B). Thismutation and/or the higher hydrophobicity of the NEFAsequence (Fig. 3C; necessary for the high-affinity asso-ciation with the signal recognition particle; Ng et al.1996; Zheng and Gierasch 1996) is implicated as thereason for the present accumulation of NEFA in the cellmembrane and in the culture medium (see above). Thissuggests that the hydrophobicity of the Nuc/NEFA N-terminal sequence is the minimal requirement for thetargeting of these proteins to the ER and is consistentwith recent findings that hydrophobicity alone plays adominant role in ER targeting (Ng et al. 1996; Zheng andGierasch 1996).
Another more intriguing explanation is that the MCF-like domain is not limited to mitochondrial-targeted pro-teins but has another yet-unidentified function in NEFAand the MCF proteins (i.e., another region of the MCFrepeats controls mitochondrial targeting in the latter pro-teins). An important experiment would be to replace oneof the spanner domains in a typical MCF protein with therelatively more hydrophobic NEFA OGC-like sequence.The mitochondrial targeting of the MCF protein maythen be compromised with weak accumulation in the ER.Such an altered targeting has already been described withPOX (see above). In conclusion, our study has resulted ina number of interesting hypotheses that are amenable tomolecular genetic analyses. Such analyses are needed toreach conclusions about the complex evolution of target-ing sequences.
Acknowledgments. We thank the Deutsche Forschungsgemeinschaftfor financial support for D.B. (BH 4/1-2); S. Barnikol-Watanabe, H.Gotz, and V. Zavazal for discussion and critical reading of the manu-script; and D. Hesse and R. Merker for technical assistance.
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