Mature Protein Sequence Research Articles

Signal sequence and alanine-rich region of streptococcal protein antigen A of Streptococcus sobrinus can direct localization of alkaline phosphatase to the periplasm of Escherichia coli

Streptococcal protein antigen A (SpaA) of Streptococcus sobrinus is expressed on the surface of cells and extracellularly. Tn phoA which lacks signals for transcription and membrane transport of Escherichia coli alkaline phosphatase was used to analyze the sequences necessary for transport of a SpaA/PhoA fusion protein across the cytoplasmic membrane to the periplasm of E. coli cells. Of 15 alkaline phosphatase-producing isolates analyzed, all were found to localize more than 85% of the SpaA/PhoA hybrid protein to the periplasm of E. coli cells. From DNA sequence analysis, all were found to have Tn phoA inserted into an identical site. The insertion site of Tn phoA was downstream from the coding sequence that generates four tandemly repeated alanine-rich sequences of 82 amino acid residues. These results suggest that in addition to the signal sequence, mature protein sequences containing alanine-rich repeat sequences may play a role in the export of the SpaA protein across a bacterial membrane.

Modulation of the Binding of Matrix Gla Protein (MGP) to Bone Morphogenetic Protein-2 (BMP-2)

Matrix Gla protein (MGP) is an inhibitor of calcification of the arterial wall but the mechanism of inhibition has not been resolved. Since chondrogenesis has been identified in calcified arteries from MPG null mice, we hypothesized that locally produced MGP might inhibit calcification by neutralizing the known effect of bone morphogenetic proteins (BMPs) as promotors of chondrogenesis and bone formation. As the first step to test this hypothesis, we demonstrate that MGP is a binding protein for 125I-BMP-2. Optimal binding is dependent on metals which suggests that the metal binding Gla region in MGP is involved. MGP is shown to undergo a Ca++ induced conformational change despite the presence of the gamma-carboxylase binding site being part of the mature protein sequence. The data propose that MGP matures earlier in the secretory pathway than other vitamin K-dependent proteins. Antibodies were used in an attempt to identify MGP in bovine serum. Conformational specific MGP antibodies were shown to also recognize the Gla region in prothrombin and factor X but did not identify MGP in serum. This finding is supported by electrophoresis data which demonstrate the absence of MGP among Ba-citrate absorbed vitamin K-dependent serum proteins. We conclude that MGP does not exist in normal bovine serum.

A search within the IL-1 type I receptor reveals a peptide with hydropathic complementarity to the IL-1β trigger loop which binds to IL-1 and inhibits in vitro responses

In previous research, we were able to demonstrate that a seven amino acid residue peptide (VITFFSL), designed as an antisense peptide of the β-bulge trigger loop region of interleukin 1β (IL-1β) (QGEESND; residues 48–54 [mature protein sequence]), was able to interact with IL-1 specifically and inhibit the response to IL-1 in an in vitro bioassay. The evidence was consistent with a specific interaction ocurring between antisense peptide and the trigger loop region. On the basis that antisense peptides are able to interact with their corresponding sense peptide sequences as a result of their mutually complementary hydropathic profiles (Fassina G., Verdoliva, A., Cassani, G., Melli, M., 1994. Binding of type I IL-1 receptor fragment 151–162 to IL-1. Growth Factors 10, 99–106; Maier, C.C., Moseley, H.N.B., Zhou, S., Whitaker, J.N., Blalock, J.E., 1994. Indentification of interactive determinants on idiotypic-anti-idiotypic antibodies through comparison of their hydropathic profiles. Immunomethods 5, 107–113), we devised a computer program (FINDH) to search the amino acid residue sequence of interleukin-1 type 1 receptor (IL-1 R1) for peptide motifs possessing hydropathic complementarity to the trigger loop sequence. The most complementary “best-fit peptide” motif (LITVLNI) was located in the third extracellular domain of IL-1 R1. A best-fit peptide corresponding to this motif was synthesised and found to bind to IL-1β as well as inhibit the response to IL-1 in two independent in vitro bioassays (monitoring IL-1 dependent serum amyloid A synthesis and IL-1 dependent alkaline phosphatase activity, respectively). A second peptide motif (VIEFITL) was identified and the corresponding peptide synthesised along with a reordered version (LTILINV) of the best fit peptide. Both failed to bind measurably with IL-1β or inhibit the response to IL-1 in the two bioassays. This best fit peptide behaved very similarly, in terms of IL-1 binding and inhibition behaviour, to the original trigger loop antisense peptide. Reference to the recently released X-ray crystal structure of IL-1β and the IL1-R1 extracellular domain shows that the best fit peptide motif in IL-1 R1 is not apparantly interacting with the IL-1 trigger loop, although both are close in space. The intriguing possibility exists that the best fit peptide motif could represent an alternative site for IL-1β receptor interaction which has not thus far been identified.

Expression of multiple forms of an adhesive plaque protein in an individual mussel, Mytilus edulis

Individual blue mussels, Mytilus edulis L., can express at least 20 variants of a small protein known as M. edulis foot protein 3 or Mefp3. Mefp3 has been shown to be a component of the adhesive plaque of the byssus, the structure securing mussels to solid substrata. The cDNAs and deduced fp3 protein sequences display more variation at the carboxy-terminus than at the N-terminus, although there is some variation present throughout the protein. This indicates that there most likely are multiple copies of the gene encoding this protein. Each protein sequence contains a signal peptide, 24 to 25 residues in length, and a mature protein sequence of 44 to 54 residues. Gly is the most common amino acid in the mature protein at 20 to 25 mol%. Tyr and Arg follow closely at 20 to 23 and 16 to 21 mol%, respectively. Both of these amino acids were previously shown to be post-translationally modified to 3,4-dihydroxyphenylalanine (Dopa) and 4-hydroxyarginine, respectively, in this protein. MALDI-TOF (matrix-assisted laser desorption ionization with time-of-flight) mass spectrometric analysis of the underside of adhesive plaques reveals the presence of Mefp3-like proteins. Curiously, only four or five out of 20 possible fp3 variants are detectable in plaques deposited on glass or plastic. This would suggest that selection of protein variants for deposition onto surfaces is determined at the level of translation.

Cloning of a galactose-binding lectin from the venom of Trimeresurus stejnegeri

A galactose-binding lectin isolated from the venom of Trimeresurus stejnegeri is a homodimer C-type lectin. The cloned cDNA encoding the monomer of Trimeresurus stejnegeri lectin (TSL) was sequenced and found to contain a 5'-end non-coding region, a sequence which encodes 135 amino acids, including a typical 23 amino acid signal peptide followed by the mature protein sequence, a 3'-end non-coding region, a polyadenylation signal, and a poly(A) region. To completely characterize the deduced amino acid sequence, on-line HPLC-MS and tandem MS were used to analyse the intact monomer and its proteolytic peptides. A modified peptide fragment was also putatively identified by HPLC-MS analysis. The deduced amino acid sequence was found to contain a carbohydrate-recognition domain homologous with those of some known C-type animal lectins. Thus TSL belongs to group VII of the C-type animal lectins as classified by Drickamer [(1993) Prog. Nucleic Acid Res. Mol. Biol. 45, 207-232]. At present, a number of C-type lectins have been purified from snake venom, but most of them have been characterized only at the protein level. To our knowledge, this is the first known cDNA sequence of a true C-type lectin from snake venom.

Mechanisms for GroEL/GroES-mediated Folding of a Large 86-kDa Fusion Polypeptide in Vitro

Our understanding of mechanisms for GroEL/GroES-assisted protein folding to date has been derived mostly from studies with small proteins. Little is known concerning the interaction of these chaperonins with large multidomain polypeptides during folding. In the present study, we investigated chaperonin-dependent folding of a large 86-kDa fusion polypeptide, in which the mature maltose-binding protein (MBP) sequence was linked to the N terminus of the alpha subunit of the decarboxylase (E1) component of the human mitochondrial branched-chain alpha-ketoacid dehydrogenase complex. The fusion polypeptide, MBP-alpha, when co-expressed with the beta subunit of E1, produced a chimeric protein MBP-E1 with an (MBP-alpha)2beta2 structure, similar to the alpha2 beta2 structure in native E1. Reactivation of MBP-E1 denatured in 8 M urea was absolutely dependent on GroEL/GroES and Mg2+-ATP, and exhibited strikingly slow kinetics with a rate constant of 376 M-1 s-1, analogous to denatured untagged E1. Chaperonin-mediated refolding of the MBP-alpha fusion polypeptide showed that the folding of the MBP moiety was about 7-fold faster than that of the alpha moiety on the same chain with rate constants of 1.9 x 10(-3) s-1 and 2.95 x 10(-4) s-1, respectively. This explained the occurrence of an MBP-alpha. GroEL binary complex that was isolated with amylose resin from the refolding mixture and transformed Escherichia coli lysates. The data support the thesis that distinct functional sequences in a large polypeptide exhibit different folding characteristics on the same GroEL scaffold. Moreover, we show that when the alpha.GroEL complex (molar ratio 1:1) was incubated with GroES, the latter was capable of capping either the very ring that harbored the 48-kDa (His)6-alpha polypeptide (in cis) or the opposite unoccupied cavity (in trans). In contrast, the MBP-alpha.GroEL (1:1) complex was capped by GroES exclusively in the trans configuration. These findings suggest that the productive folding of a large multidomain polypeptide can only occur in the GroEL cavity that is not sequestered by GroES.

Complete Mapping of Divergent Amino Acids Responsible for Differential Ligand Binding of Folate Receptors α and β

The folate receptor (FR) type alpha may be distinguished from FR-beta by its higher affinity for the circulating folate coenzyme, (6S)-5-methyltetrahydrofolate (5-CH3H4folate), and its opposite stereospecificity for reduced folate coenzymes. Previous studies showed that a single leucine to alanine substitution at position 49 of the mature protein sequence is responsible for the functional divergence of FR-beta (Shen, F., Zheng, X., Wang, H., and Ratnam, M. (1997) Biochemistry 36, 6157-6163); however, the results also indicated that the minimum requirement for conversion of FR-beta to the functional equivalent of FR-alpha should include amino acid substitution(s) downstream of residue 92 in addition to mutation of L49A. To pinpoint those residues, chimeric FR-betaL49A/FR-alpha constructs including progressively shorter segments of FR-alpha downstream of position 92 as well as selected point mutants were studied. Simultaneous substitution of Leu-49, Phe-104, and Gly-166 in FR-beta with the corresponding FR-alpha residues Ala, Val, and Glu, respectively, reconstituted the ligand binding characteristics of FR-alpha. The results also exclude a role for other residues in FR-alpha in determining its functional divergence. A homology model of FR-alpha based on the three-dimensional structure of the chicken riboflavin-binding protein is used to show the position of residues 49, 104, and 166 in relation to the hydrophobic cleft corresponding to the riboflavin-binding pocket.

Isolation and characterization of cDNA clones encoding a 32-kDa dense-granule antigen of Sarcocystis muris (Apicomplexa).

A monoclonal antibody (2F4) directed against a 32-kDa dense-granule antigen of Sarcocystis muris cyst merozoites (bradyzoites) was used to screen a lambda ZAP cDNA expression library. A clone with an insert of 1.4 kb in length (DG 32/1) was isolated. A fusion protein derived from bacteria harbouring the recombinant plasmid DG 32/1 reacted with monoclonal antibody (mAb) 2F4. Southern blot hybridization suggests that the gene is present as a single copy. On Northern blots, a single mRNA species of 1.8 kb was detected by a cDNA-derived probe. In addition, we isolated a full-length clone (DG 32/PH1) by screening the cDNA library with a non-radioactive-labelled cDNA probe. The nucleotide sequence of DG 32/PH1 comprises 1.57 kb. It contains an open reading frame of 882 bp with a coding capacity of approximately 32 kDa. The hypothetical polypeptide consists of a putative N-terminal signal peptide and the mature protein sequence. The occurrence of an N-terminal signal sequence is consistent with the observation that the 32-kDa protein of S. muris is secreted from the dense granules.

Molecular Cloning and Functional Characterization of the Pig Analogue of CD59: Relevance to Xenotransplantation

In this work, we report the cloning of the cDNA for the porcine analogue of human CD59. Degenerate primers, derived from the N-terminal sequence of pig erythrocyte CD59, were used to obtain the corresponding cDNA sequence. From this sequence, gene-specific primers were designed and used to amplify the 3' and 5' ends of the cDNA using the rapid amplification of cDNA ends (RACE) method. The complete 768-bp cDNA so obtained consisted of a 84-bp 5' untranslated region, a 26-amino-acid NH2-signal peptide, a 98-amino-acid coding region, including putative N-glycosylation sites and a glycosylphosphatidylinositol-anchoring signal, and a 312-bp 3' untranslated region. The mature protein sequence was 48% identical to human CD59 at the amino acid level. Northern blot analysis revealed several distinct CD59 transcripts, and a variability in expression levels of the different transcripts in the panel of tissues screened. Stable expression of pig CD59 in a CD59-negative human cell line conferred protection against lysis by complement from pig and several other species. Separate expression of pig and human CD59 at similar levels in the same cell line allowed a direct functional comparison between these two analogues. Pig CD59 and human CD59 showed similar activity in inhibiting lysis by complement from all species tested; in particular, expressed pig CD59 efficiently inhibited lysis by human complement. The relevance of these data to current work in the engineering of pig organs for xenotransplantation is discussed.

Acetyl xylan esterase II from Penicillium purpurogenum is similar to an esterase from Trichoderma reesei but lacks a cellulose binding domain

Penicillium purpurogenum produces at least two acetyl xylan esterases (AXE I and II). The AXE II cDNA, genomic DNA and mature protein sequences were determined and show that the axe 2 gene contains two introns, that the primary translation product has a signal peptide of 27 residues, and that the mature protein has 207 residues. The sequence is similar to the catalytic domain of AXE I from Trichoderma reesei (67% residue identity) and putative active site residues are conserved, but the Penicillium enzyme lacks the linker and cellulose binding domain, thus explaining why it does not bind cellulose in contrast to the Trichoderma enzyme. These results point to a possible common ancestor gene for the active site domain, while the linker and the binding domain may have been added to the Trichoderma esterase by gene fusion.

Identification of the Cysteine Residues Involved in Redox Modification of Plant Plastidic Glucose-6-phosphate Dehydrogenase

The cDNA sequences encoding cytosolic and light-modulated plastidic glucose-6-phosphate dehydrogenase (G6PDH) from potato were modified by polymerase chain reaction and subsequently overexpressed in Escherichia coli. Characterization of the recombinant enzymes showed that they closely resembled their native counterparts. Treatment with reduced dithiothreitol or glutathione led to inactivation of plastidic G6PDH, whereas the activity of the cytosolic isoenzyme was not influenced by reduction. As for the native enzyme, inactivation of recombinant plastidic G6PDH was accelerated by thioredoxin m and could be fully reversed by subsequent addition of oxidant. To identify the residues which are involved in redox regulation of plastidic G6PDH, each of the six cysteines in the mature protein sequence was exchanged separately for serine by site-directed mutagenesis. Two mutant proteins exhibited characteristics of the reduced wild-type enzyme. Exchange of either Cys149 or Cys157 to serine abolished the regulatory properties, suggesting that these cysteine residues are the sites responsible for redox-mediated inactivation of plastidic G6PDH.

Molecular Cloning and Characterization of the Human Mitochondrial NADH:Oxidoreductase 10-kDa Gene (NDUFV3)

The human gene for the 10-kDa flavoprotein subunit of the mitochondrial NADH:ubiquinone oxidoreductase (Complex I) was completely cloned and sequenced. The so-called NDUFV3 gene contains three exons, spanning 20 kb. The open reading frame contains a 34-codon import sequence and a 74-codon mature protein sequence. A database search revealed close homology to bovine and rat protein sequence but not to any other known protein. Northern blot analysis showed that the NDUFV3 gene is ubiquitously expressed. The NDUFV3 gene was assigned by FISH to a single location on chromosome 21q22.3 and might contribute to the Down syndrome phenotype.

Purification, characterization and cloning of a novel variant of the superantigen Yersinia pseudotuberculosis-derived mitogen

The novel superantigen designated as Yersinia pseudotuberculosis-derived mitogen typeB (YPMb) was purified from the cell lysate of Y. pseudotuberculosis O:6 strain (R-104). Proliferative response of human peripheral blood mononuclear cells to the purified YPMb was detectable at a concentration of as low as 1 pg/ml, that is comparable to the previously documented YPM (YPMa). The Vβ repertoire specificity (3, 9, 13.1 and 13.2) of YPMb was also the same as that of YPMa. A gene ( ypmB) encoding YPMb was cloned and its nucleotide sequence was determined. The open reading frame (ORF 453 bp) of the ypmB encodes a protein with 150 amino acid residues, though the precursor protein of YPMa consists of 151 amino acid residues. There is a nucleotide sequence homology of 88.9% between ypmB and ypmA. The low homology in the downstream of the structural gene between ypmB and ypmA and the difference in the GC content in the ORF of ypmB and ypmA from that of the base usage of Y. pseudotuberculosis suggest that the ypm gene originates from another organism. The alignment of the amino acid sequences of mature proteins of YPMb and YPMa revealed that there is 83% homology (108 amino acid residues are identical). Between YPMa and YPMb, the central region is less homologous than the N- and C-terminal regions. Based on the functional similarity of two superantigen molecules, the less homology suggested that the central region is less important for the function of both YPM molecules.

Identification of Functional Domains in Atp11p: PROTEIN REQUIRED FOR ASSEMBLY OF THE MITOCHONDRIAL F1-ATPase IN YEAST

The Atp11p protein of Saccharomyces cerevisiae is required for proper assembly of the F1 component of the mitochondrial ATP synthase. The mutant atp11 genes were cloned and sequenced from 12 yeast strains, which are respiratory-deficient due to a defect in Atp11p function. Four of the mutations mapped to the mitochondrial targeting domain (amino-terminal 39 amino acids) of Atp11p. All the genetic lesions found in the mature protein sequence were shown to be nonsense mutations. This result is consistent with the idea that Atp11p activity is provided, principally, by the overall structure of a functional domain, and not by specific amino acid residues in a localized active site. Amino-terminal (Edman) sequence analysis of fragments derived from limited proteolysis of purified Atp11p, and in vivo functional characterization of deletion mutants, were employed to locate the position of the active region in the protein. Three domains, separated by proline-rich sequences, were identified in the mature protein. The active domain of Atp11p was mapped to the sequence between Phe-120 and Asn-174. The domains proximal (Glu-40 through Ser-109) and distal (Arg-183 through Asn-318) to the active region were found to be important for the protein stability inside mitochondria.

CDNA Cloning of IX/X-BP, a Heterogeneous Two-Chain Anticoagulant Protein from Snake Venom

IX/X-bp is an anticoagulant protein isolated from the venom of the habu snake (Trimeresurus flavoviridis). It is a heterogeneous two-chain protein linked by an interchain S–S bond. We prepared a cDNA library from the venom gland of the habu snake in the vector pSPORT1. cDNA clones containing the coding sequences for IX/X-bp were isolated and sequenced to determine the structure of the proprotein of IX/X-bp. All cDNA clones containing coding sequences of either chain of IX/X-bp consisted of the 5′-end noncoding bases, the first ATG codon, a typical signal peptide sequence that was immediately followed by a mature protein sequence that corresponded to one of the chains, a stop codon, the 3′-end noncoding bases, a polyadenylation signal, and a poly(A)+region. These data indicate that the gene for each chain of the two-chain protein is transcribed and translated separately.

Isolation and characterisation of full-length cDNA clones of the giant taro (Alocasia macrorrhiza) trypsin/chymotrypsin inhibitor

A full-length cDNA encoding the 206 amino acid open reading frame of a trypsin/chymotrypsin inhibitor abundant in the corms of giant taro (Alocasia macrorrhiza) was isolated. An internal fragment was cloned using degenerate primers corresponding to a region of the mature protein sequence and the 'rapid amplification of cDNA ends' (RACE) method used to generate a composite cDNA sequence. The length of the cDNA was close to the predicted size of the corresponding transcript deduced from northern blot analysis of corm mRNA. The inhibitor was expressed strongly in the mature corm, at low levels in leaf blades and petioles but not in roots. Southern blot analysis of the giant taro DNA indicated that this inhibitor is encoded by a small multigene family and this was further supported by the isolation of two different sequence classes from corm cDNA using primers to the ends of the composite sequence.

Molecular Cloning of Human Eotaxin, an Eosinophil-selective CC Chemokine, and Identification of a Specific Eosinophil Eotaxin Receptor, CC Chemokine Receptor 3

The CC chemokine eotaxin is a selective chemoattractant for guinea pig eosinophils, first purified from bronchoalveolar lavage fluid in a guinea pig model of allergic airway inflammation. We have now isolated the gene and cDNA for a human counterpart of eotaxin. The gene maps to chromosome 17 and is expressed constitutively at high levels in small intestine and colon, and at lower levels in various other tissues. The deduced mature protein sequence is 66% identical to human monocyte chemoattractant protein-1, and 60% identical to guinea pig eotaxin. Recombinant human eotaxin produced in insect cells induced a calcium flux response in normal human eosinophils, but not in neutrophils or monocytes. The response could not be desensitized by pretreatment of eosinophils with other CC chemokines, suggesting a unique receptor. In this regard, we show that human eotaxin is a potent and highly specific agonist for CC chemokine receptor 3, a G protein-coupled receptor selectively expressed in human eosinophils. Thus eotaxin and CC chemokine receptor 3 may be host factors highly specialized for eosinophil recruitment in inflammation, and may be good targets for the development of selective drugs for inflammatory diseases where eosinophils contribute to pathogenesis, such as asthma.

Genomic recombination of the mitochondrial atp6 gene in Arabidopsis thaliana at the protein processing site creates two different presequences.

In the mitochondrial genome of the flowering plant Arabidopsis thaliana the atp6 open reading frame is located on the border of one of the repeats resulting in two copies with different presequence extensions. The two presequences of 135 and 97 amino acids respectively show no similarity to each other, while the mature protein sequences are identical. Both preproteins are most likely synthesized in Arabidopsis mitochondria from promoter elements upstream of each copy. The presence of two arrangements in the mitochondrial genome of fertile Arabidopsis plants suggests this recombination to be unrelated to a cytoplasmic male sterile phenotype. This recombination precisely at the mature protein terminus is reminiscent of the domain shuffling model in protein evolution.

Cloning and sequencing of a cDNA encoding acetylcholinesterase in colorado potato beetle, Leptinotarsa decemlineata (say)

A cDNA encoding acetylcholinesterase (AChE, EC 1.1.1.7) was cloned from a cDNA library constructed from an insecticide-susceptible strain of Colorado potato beetle, Leptinotarsa decemlineata (Say). The complete amino acid sequence of AChE deduced from the cDNA consisted of 29 residues for the putative signal peptide and 600 residues for the mature protein with a predicted molecular weight of 67,994. Northern blot analysis of poly(A) RNA showed an approx 13.1-kb transcript. The mature protein sequence had 57 and 61% of amino acid residues identical to those of Drosophila melanogaster and Anopheles stephensi, respectively, and produced a remarkably similar hydropathy profile when compared to those of the two dipterous species. The three residues (Ser, Glu and His) that putatively form the catalytic triad and the six Cys that form intra-subunit disulfide bonds were completely conserved when compared to the other seven AChEs from a broad range of animal species reported to date. Other properties of the deduced protein of AChE, including molecular weight and amino acid composition, agreed well with those of a previously reported study on the purified AChE from the same insect species. All these features firmly established that the cloned cDNA encodes AChE in Colorado potato beetle.

The thylakoid-targeting domain of the chloroplast Rieske iron-sulfur protein is located in the N-terminal hydrophobic region of the mature protein

The thylakoid-targeting domain of the Rieske FeS protein has been located in the N-terminal 55 amino acids of the mature protein by importing chimeric and truncated precursor proteins into isolated pea chloroplasts. A chimeric protein consisting of the presequence of ribulose-bisphosphate carboxylase/oxygenase (Rubisco) small subunit fused to the mature Rieske protein sequence was imported by chloroplasts, processed in the stroma, and translocated into the thylakoids, indicating that the thylakoid-targeting information was located within the mature Rieske protein. A truncated Rieske protein precursor consisting of the presequence and the N-terminal 55 amino acids of the mature protein was imported by chloroplasts and routed to the thylakoids, indicating that the thylakoid-targeting domain of the Rieske protein is located within the predominantly hydrophobic N-terminal region of the mature protein. Chimeric proteins consisting of this truncated Rieske protein precursor fused to the mature Rubisco small subunit and to plastocyanin were efficiently imported by chloroplasts and translocated across the thylakoid membrane. A proton-motive force was necessary for thylakoid translocation of the mature Rieske protein, the truncated Rieske protein, and the fusion protein consisting of the truncated Rieske protein and the mature Rubisco small subunit. In contrast, plastocyanin and the fusion of the truncated Rieske protein with plastocyanin were translocated across the thylakoid membrane in the presence of nigericin and valinomycin, indicating that the energy requirement for thylakoid translocation was conferred by the passenger protein and not by the thylakoid-targeting sequence.