Kinetics of the Reconstituted Tricarboxylate Carrier from Eel Liver Mitochondria

The tricarboxylate carrier from eel liver mitochondria was purified by chromatography on hydroxyapatite and Matrix Gel Blue B and reconstituted into liposomes by removal of the detergent with Amberlite. Optimal transport activity was obtained by using a phospholipid concentration of 11.5 mg/ml, a Triton X-114/phospholipid ratio of 0.9, and ten passages through the same Amberlite column. The activity of the carrier was influenced by the phospholipid composition of the liposomes, being increased by cardiolipin and phosphatidylethanolamine and decreased by phosphatidylinositol. The reconstituted tricarboxylate carrier catalyzed a first-order reaction of citrate/citrate or citrate/malate exchange. The maximum transport rate of external [¹⁴C]citrate was 9.0 mmol/min per g of tricarboxylate carrier protein at 25°C and this value was virtually independent of the type of substrate present in the external or internal space of the liposomes. The half-saturation constant (K _m) was 62 M for citrate and 541 M for malate. The activation energy of the citrate/citrate exchange reaction was 74 kJ/mol from 5 to 19°C and 31 kJ/mol from 19 to 35°C. The rate of the exchange had an external pH optimum of 8.     

Detergent binding to unmyristylated protein kinase A—Structural implications for the role of Myristate

Myristylation often governs the targeting of protein kinases to the plasma membrane. It is now known that a key member of the src family of protein tyrosine kinases, pp60^v-src, binds to the lipid bilayer of the plasma membrane via a myristylated amino terminal sequence. The mechanism of this interaction is not known; however, myristic acid (Myristic acid may also be referred to as Myristate) and residues 2 through 14 are also absolutely required (Resh and Ling, 1990). This review presents an analysis of crystal structures of detergent-modified recombinant and myristylated mammalian catalytic subunit of protein kinase A. Crystals of unmyristylated recombinant catalytic subunit of protein kinase A are grown in the presence of Mega 8, a glucamide-type of detergent, and only this detergent binds, which results in a resolution extension (Knightonet al., 1991a). Comparisons of these two structures reveal that the detergent association with the recombinant enzyme binds in exactly the same hydrophobic pocket of the protein occupied by myristic acid in the mammalian protein (Karlssonet al., 1993; Zhenget al., 1993a). Removal of the detergent through soaking results in the local unwinding of the first helix, helix A, and disorder of the canonical recognition sequence of the phosphorylation site, Ser 10 (Zhenget al., 1993b). These results suggest that anchoring the myristic acid inside the protein results in formation of a stable structural template, which includes the myristylated amino terminal sequence important for the recognition by protein kinases. This inside out motif might provide a structural paradigm for the recognition of myristylated proteins, including pp60^v-src.     

Covalent structure of botulinum neurotoxin type A: Location of sulfhydryl groups, and disulfide bridges and identification of C-termini of light and heavy chains

Botulinum neurotoxin Type A is synthesized byClostridium botulinum as a 150 kD single chain polypeptide. The posttranslational processing of the 1296 amino acid residue long gene product involves removal of the initiating methionine, formation of disulfide bridges, and limited proteolysis (nicking) by the bacterial protease(s). The mature dichain neurotoxin is made of a 50-kD light chain and a 100-kD heavy chain connected by a disulfide bridge. DNA derived amino acid sequencepredicted a total of 9 Cys residues (Binzet al., 1990,J. Biol. Chem. 265, 9153–9158; Thompsonet al., 1990,Eur. J. Biochem. 189, 73–81). Treatment of the dichain neurotoxin, dissolved in 6 M guanidine. HCl, with 4-vinylpyridine converted 5 Cys residues into S-pyridylethyl cysteine residues; but alkylation after mercaptolysis converted all 9 Cys residues in the S-pyridylethylated form. After confirming the predicted number of Cys residues by amino acid analysis, the positions of the 5 Cys residues carrying sulfhydryl groups and the 4 involved in disulfide bridges were determined by comparing the elution patterns in reversed-phase HPLC of the cyanogen bromide mixtures of the exclusively alkylated and the mercaptolyzed-alkylated neurotoxin. The chromatographically isolated components were identified by N-terminal amino acid sequence analysis. The HPLC patterns showed characteristic differences. The Cys residuespredicted in positions 133, 164, 790, 966, and 1059 were found in the sulfhydryl form; Cys 429 and 453 were found disulfide-bridged connecting the light and heavy chains, and Cys 1234 and 1279 were found in an intrachain disulfide-bridge near the C-terminus in the heavy chain. Ten amino acid residues, Thr 438-Lys 447,predicted to be present in the single chain neurotoxin were not found in the dichain neurotoxin. Nicking of single-chain neurotoxin by the protease(s) endogenous to the bacteria therefore appears to excise these 10 amino acid residues from the nicking region which leaves Lys 437 as the C-terminus of the light chain and Ala 448 as the N-terminus of the heavy chain. The N-terminal Pro 1 and C-terminal Leu 1295,predicted from the nucleotide sequence, remain conserved after nicking. Residues Pro 1-Lys 437 and Ala 448-Leu 1295 constitute the light and heavy chains, respectively. The C-termini were determined by isolation of short C-terminal peptide fragments and subsequent sequence analysis by Edman degradation. About 20% of the amino acid sequence predicted from DNA analysis was confirmed in these studies by protein-chemical methods.     

Identification and Kinetic Characterization of HtDTC, The Mitochondrial Dicarboxylate–Tricarboxylate Carrier of Jerusalem Artichoke Tubers

Jerusalem artichoke (Helianthus tuberosus L.) tubers were reported to be tolerant to cold and freezing. The aim of this study was to perform a kinetic characterization of the mitochondrial dicarboxylate–tricarboxylate carrier (HtDTC) and to assess a possible involvement of this carrier in the cold tolerance of tubers. The HtDTC was purified from isolated mitochondria by sequential chromatography on hydroxylapatite/celite and Matrex Gel Orange A. SDS gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent molecular mass of 31.6 kDa. A polyclonal antibody raised against the tobacco DTC cross-reacted with the purified protein on Western blot analysis. In gel trypsin, digestion of the purified HtDTC yielded peptides that exhibited strong amino acid sequence similarity to previously identified plant DTCs. Furthermore, using degenerate primers, a portion of the Htdtc cDNA was amplified and sequenced; this cDNA encoded for a protein with high sequence similarity to known plant homolog DTCs. When reconstituted in liposomes loaded with dicarboxylate (2-oxoglutarate, malate, malonate, succinate, and maleate) or tricarboxylate anions (citrate, trans-aconitate, and isocitrate), the purified HtDTC transported all these anions in exchange with external [¹⁴C]2-oxoglutarate. A kinetic characterization of HtDTC was performed: (a) the half-saturation constant K _m and the V _max at 25^∘C of the 2-oxoglutarate/2-oxoglutarate exchange by reconstituted HtDTC were found to be 360 μM and 10.9 μmol/(min mg protein), respectively; (b) the activation energy E _a of the succinate/2-oxoglutarate exchange by the reconstituted HtDTC was found to be 50.7 kJ/mol constant between −5 and 35^∘C. Similarly, the activation energy E _a of succinate respiration of isolated Jerusalem artichoke mitochondria, measured between −2 and 35^∘C, was shown to be constant (65.3 kJ/mol). The physiological relevance of kinetic properties and temperature dependence of transport activities of HtDTC is discussed with respect to the cold tolerance ability of Jerusalem artichoke tubers.     

Characterization and sequencing of cDNA clone encoding the phloem protein PP2 of Cucurbita pepo

Direct N-terminal amino acid sequencing of the phloem protein 2 (PP2) from 3-month old Cucurbita pepo L. (pumpkin), purified by SDS-PAGE and blotted onto PVDF membrane, showed that the protein had a blocked N-terminus. However, after in situ cleavage of the polypeptide in a gel slice by cyanogen bromide, 75 residues of sequence on two cyanogen bromide fragments were determined. An oligonucle-otide probe based on this amino acid sequence was used to screen a cDNA library, constructed from mRNA of 3–5-day old seedling hypocotyls, in ZAP II. A cDNA clone (p11A) predicted an amino acid sequence of 218 residues, in full agreement with the sequences determined for two CNBr fragments of PP2, and suggests that the N-terminus of the protein is a blocked methionine residue which is cleaved off by CNBr. Two additional cDNA clones were sequenced but no heterogeneity in the PP2 sequence was found. The deduced amino acid sequence of C. pepo differs in nine residues from the recently published sequence of Cucurbita maxima (Bostwick et al., Plant Cell 4 (1992) 1539–1548). Southern blot showed that PP2 is encoded by a gene family with a relatively large number of members (estimated as 7–15 per haploid genome).     

Purification of (1→3)-β-glucan endohydrolase isoenzyme II from germinated barley and determination of its primary structure from a cDNA clone

A (13)--D-glucan 3-glucanonydrolase (EC 3.2.1.39) of apparent M _r 32 000, designated GII, has been purified from germinated barley grain and characterized. The isoenzyme is resolved from a previously purified isoenzyme (GI) on the basis of differences in their isoelectric points; (13)--glucanases GI and GII have pI values of 8.6 and 10.0, respectively. Comparison of the sequences of their 40 NH₂-terminal amino acids reveals 68% positional identity. A 1265 nucleotide pair cDNA encoding (13)--glucanase isoenzyme GII has been isolated from a library prepared with mRNA of 2-day germinated barley scutella. Nucleotide sequence analysis of the cDNA has enabled the complete primary structure of the 306 amino acid (13)--glucanase to be deduced, together with that of a putative NH₂-terminal signal peptide of 28 amino acid residues. The (13)--glucanase cDNA is characterized by a high (G+C) content, which reflects a strong bias for the use of G or C in the wobble base position of codons. The amino acid sequence of the (13)--glucanase shows highly conserved internal domains and 52% overall positional identity with barley (13, 14)--glucanase isoenzyme EII, an enzyme of related but quite distinct substrate specificity. Thus, the (13)--glucanases, which may provide a degree of protection against microbial invasion of germinated barley grain through their ability to degrade fungal cell wall polysaccharides, appear to share a common evolutionary origin with the (13, 14)--glucanases, which function to depolymerize endosperm cell walls in the germinated grain.     

Sequence analysis of an actin isoform in the flatworm Diphyllobothrium dendriticum (Cestoda)

We have examined actin cDNA of the flatworm Diphyllobothrium dendriticum (Cestoda). Actin is a contractile protein that has been implicated in a variety of developmental and cellular processes. It is highly conserved and present in all eukaryotic cells. It is of particular interest to analyze evolutionary preserved genes in flatworms, because ancestral flatworms are regarded to play a central role in the evolution of the metazoans (Barnes et al., 1998). Screening a cDNA library of D. dendriticum (UniZap XR, Stratagene) with a human -actin probe resulted in several positive clones. One of the cDNA inserts, Didactl, consisting of 1392 bp was completely sequenced. The established nucleotide sequence revealed a 5 untranslated region of 33 bp, the entire open reading frame of 1128 bp and a 3 untranslated region of 231 bp which ends in a stretch of 21 A residues. The potential polyadenylation signal (AATAAA) is located 14 bp upstream of the poly (A) tail. The deduced amino acid sequence of Didactl is 376 amino acids long. It is a typical invertebrate actin (Fyrberg et al., 1981) resembling more the cytoplasmic than the muscular isoforms of vertebrate actins. Didactl is for example 96% homologous to human cytoplasmic -actin but only 92.6% identical with human smooth muscle -actin. The actin proteins are generally encoded by a multigene family which differs in size from species to species. Most organisms have four to eight genes coding for actin in their genome, but the number of actin genes can also be over 20 (Hamelin et al., 1988). Sequence comparisons of Didactl and the partly sequenced cDNA clones indicate that D. dendriticum has at least four different genes coding for actin in its genome.     

Chicken smooth muscle myosin light chain kinase is acetylated on its NH2-terminal methionine

The reported cDNA structrre, of chicken smooth muscle myosin light chain kinase (smMLCK) encodes a protein of 972 residues (Olsonet al. Proc. Natl. Acad. Sci USA, 87: 2284–2288, 1990). The calculated Mr is 107, 534 whereas the estimate by SDS-PAGE is approximately 130, 000. Gibson and Higgins (DNA Sequence (in press)) have recently reported the possibility of errors, in the cDNA sequence for non-muscle MLCK and that the NH₂-terminus of both it and smMLCK may extend beyond the reported coding region. The native smMLCK is NH₂-terminally blocked. A CNBr peptide derived from smMLCK contains the NH₂-terminal sequence Asp-Phe-Arg-Ala corresponding to residues 2 to 4 in the smMLCK sequence indicating, that Met-1 is present. Using a limited thermolysin digest we isolated an NH₂-terminally blocked peptide by reversed-phase HPLC. This thermolytic peptide had a mass of approximately 797 by time of flight mass spectrometry. Amino acid analysis and Edman sequencing of a CNBr-subfragment of the thermolytic peptide indicated that it had the composition and sequence, (Met)-Asp-Phe-Arg-Ala-Asn, with a calculated mass of 753. The difference in mass corresponds to the NH₂-terminal Met being blocked by actylation. The results demonstrate that the NH₂-terminal sequence of smMLCK inferred from the reported cDNA sequence is correct and that the proposed initiating, Met is not removed, but modified by -NH₂ acetylation of the translation product.     

Nucleotide sequence of cDNA clones encoding the β subunit of mitochondrial ATP synthase from the green alga Chlamydomonas reinhardtii: The precursor protein encoded by the cDNA contains both an N-terminal presequence and a C-terminal extension

cDNA and genomic clones encoding the subunit of mitochondrial ATP synthase from Chlamydomonas reinhardtii have been isolated using heterologous DNA probes from the photosynthetic bacterium Rhodospirillum rubrum. The protein encoded by the cDNA is 79–83% identical to corresponding proteins from higher-plant and mammalian mitochondria, and 75% identical to the R. rubrum protein. It contains both an N-terminal presequence and a unique C-terminal extension. The presequence, which is the first mitochondrial presequence determined in C. reinhardtii, is similar in structure to mitochondrial presequences from other organisms. As chloroplast presequences from C. reinhardtii also share features with mitochondrial presequences from other organisms (L.-G. Franzén et al., FEBS Lett 260 (1990) 165–168), this raises interesting questions about protein targeting to chloroplasts and mitochondria in C. reinhardtii. The possibility that the C-terminal extension is involved in targeting the protein to the mitochondrion is discussed. Southern blot analysis indicates that the protein is encoded by a single-copy gene.     

Homology between chitinases that are induced by TMV infection of tobacco

Recently, four chitinases have been detected in tobacco mosaic virus (TMV) infected tobacco: two acidic chitinases that were identified as pathogenesis-related (PR) proteins P and Q and two basic chitinases (Legrand et al., Proc.Natl. Acad. Sci. USA, in press). Here, it was shown that P and Q are closely serologically related but not related to other known acidic tobacco PR proteins. Antisera to P and Q were used to characterize translation products of TMV-induced mRNAs that were hybrid-selected with cDNA clones described previously (Hooft van Huijsduijnen et al., EMBO J 5: 2057–2061, 1986). In this way cDNA clones corresponding to the acidic and basic chitinases were identified. The partial amino acid sequences of the acidic and basic tobacco chitinases that were represented in the clones, showed an approximately 70% homology to each other and to the sequence of a bean chitinase. Although the acidic and basic chitinases differ in apparent molecular weight, they were found to have homologous C-termini.Hybridization of cDNA probes to genomic blots indicated that the acidic and basic chitinases are each encoded by two to four genes in the amphidiploid genome of Samsun NN tobacco. A similar complexity was found for the genes encoding the tobacco PR protein that is homologous to the sweet-tasting protein thaumatin and to the bifunctional trypsin/-amylase inhibitor from maize.     

Binding of sulfosuccinimidyl fatty acids to adipocyte membrane proteins: Isolation and ammo-terminal sequence of an 88-kD protein implicated in transport of long-chain fatty acids

Summary We recently reported (Harmon et al., J. Membrane Biol. 124:261–268, 1991) that sulfo-N-succinimidyl derivatives of long-chain fatty acids (SS-FA) specifically inhibited transport of oleate by rat adipocytes. These compounds bound to an 85–90 kD membrane protein which was also labeled by another inhibitor of FA transport [³H]DIDS (4,4-diisothiocyanostilbene-2-2-sulfonate). These results indicated that the protein was a strong candidate as the transporter for long-chain fatty acids. In this report we determined that the apparent size of the protein is 88 kD and its isoelectric point is 6.9. We used [³H]SS-oleate (SSO), which specifically labels the 88-kD protein, to isolate it from rat adipocyte plasma membranes. Identification of 15 amino acids at the N-terminus region revealed strong sequence homology with two previously described membrane glycoproteins: CD36, a ubiquitous protein originally identified in platelets and PAS IV, a protein that is enriched in the apical membranes of lipidsecreting mammary cells during lactation. Antibody against PAS IV cross-reacted with the adipocyte protein. This, together with the N-terminal sequence homology, suggested that the adipocyte protein belongs to a family of related intrinsic membrane proteins which include CD36 and PAS IV.     

Structure,function and regulation of the tricarboxylate transport protein from rat liver mitochondria

Recent progress is summarized on the structure, function, and regulation of the tricarboxylate (i.e., citrate) transport protein (CTP) from the rat liver mitochondrial inner membrane. The transporter has been purified and its reconstituted function characterized. A cDNA clone encoding the CTP has been isolated and sequenced, thus enabling a deduction of the complete amino acid sequence of this 32.6 kDa transport protein. Dot matrix analysis and sequence alignment indicate that based on structural considerations the CTP can be assigned to the mitochondrial carrier family. Hydropathy analysis of the transporter sequence indicates six putative membrane-spanning -helices and has permitted the development of an initial model for the topography of the CTP within the inner membrane. The questions as to whether more than one gene encodes the CTP and whether more than one isoform is expressed remain unanswered at this time. Studies documenting a diabetes-induced alteration in the function of several mitochondrial anion transporters, which can be reversed by treatment with insulin, provide a physiologically/pathologically relevant experimental system for studying the molecular mechanism(s) by which mitochondrial transporters are regulated. Potential future research directions are discussed.     

Identification of a Rhodococcus Gene Cluster Encoding a Homolog of the 17-kDa Antigen of Brucella and a Putative Regulatory Protein of the AsnC-Lrp Family

By sequence analysis, a gene encoding a homolog of the 17-kDa protein antigen of the Gram-negative pathogen Brucella abortus (Hemmen et al., Clin Diagn Lab Immunol 2: 263, 1995) was identified in the nocardioform actinomycete Rhodococcus sp. NI86/21. Database searching also revealed a partial human cDNA sequence for a putative eukaryotic homolog of this presumptive Brucella-specific protein. These proteins display a low but significant level of similarity with lumazine synthases involved in bacterial riboflavin biosynthesis. In the upstream region, a Rhodococcus gene for a putative regulatory protein of the AsnC family is located. Received: 28 October 1995 / Accepted: 14 December 1995     

Two-domain hemoglobin from the blood clam,Barbatia lima. The cDNA-derived amino acid sequence

The blood clam,Barbatia lima, from Kochi, Japan, expresses a tetrameric ( _{2 2}) and a polymeric hemoglobin in erythrocytes. The latter hemoglobin is composed of unusual 34-kDa hemoglobin with a two-domain structure, and its molecular mass (about 430 kDa) is exceptionally large for an intracellular hemoglobin. The 3 and 5 parts of the cDNA ofB. lima two-domain globin have been amplified separately by polymerase chain reaction and the complete nucleotide sequence of 1147 bp was determined. The open reading frame is 930 nucleotides in length and encodes a protein with 309 amino acid residues, of which 73 amino acids were identified directly by protein sequencing. The mature protein begins with the acetylated Ser, and thus the N-terminus Met is cleaved. The molecular mass for the protein was calculated to be 35,244 Da. The cDNA-derived amino acid sequence ofB. lima two-domain globin shows 89% homology with that of two-domain globin fromB. reeveana, a North American species. The sequence homology between the two domains is 75%, suggesting that the two-domain globin resulted from the gene duplication of an ancestral 17-kDa globin.     

The Arabidopsis nuclear DAL gene encodes a chloroplast protein which is required for the maturation of the plastid ribosomal RNAs and is essential for chloroplast differentiation

Altered pigmentation is an easily scored and sensitive monitor of plastid function. We analyzed in detail a yellow colored transposon-tagged mutant (dal1-2) that is allelic to the dal mutant previously identified (Babiychuk et al., 1997). Mesophyll cells of mutant plants possess abnormal nucleoids and more but smaller plastids than wild type cells. Plastid development in dal1-2 is not altered in the dark but is arrested at the early steps of thylakoid assembly. The amino acid sequence of the protein deduced from our cDNA clone is 21 amino acids longer than the previously published DAL sequence (Babiychuk et al., 1997) and allowed us to show that DAL codes for a chloroplast protein. The dal1-2 mutation has a global negative effect on plastid RNA accumulation and on expression of nuclear encoded photosynthetic genes. We show that the plastid RNA polymerases, the nuclear-encoded NEP and the plastid-encoded PEP, are functional in the mutant. Precursor 16S and 23S rRNA species specifically accumulate at a high level in the mutant but the 5-end and the long 3-end trailer are not modified. We suggest that the dal mutation is involved in plastid rRNA processing and consequently in translation and early chloroplast differentiation.     

Purification and characterization of the reconstitutively active tricarboxylate transporter from rat liver mitochondria

The tricarboxylate transporter has been purified in reconstitutively active form from rat liver mitochondria. The transporter was extracted from mitoplasts with Triton X-114 in the presence of cardiolipin and citrate and was then purified by sequential chromatography on hydroxylapatite, Matrex Gel Orange A, Matrex Gel Blue B, and Affi-Gel 501. Analysis of the purified material via sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated the presence of one main protein band with an apparent molecular mass of 32.5 kDa. Upon incorporation into phospholipid vesicles, the purified transporter catalyzed a 1,2,3-benzenetricarboxylate-sensitive citrate/citrate exchange with a specific transport activity of 3240 nmol/4 min/mg of protein. This value was enhanced 831-fold with respect to the starting material. Substrate competition studies indicated that the reconstituted transport could be substantially inhibited by isocitrate, malate, and phosphoenolpyruvate, but not by alpha-ketoglutarate, succinate, malonate, pyruvate, or inorganic phosphate. Moreover, in addition to 1,2,3-benzenetricarboxylate, the reconstituted exchange was sensitive to the anion transport inhibitor n-butylmalonate but was insensitive to phenylsuccinate, alpha-cyano-4-hydroxycinnamate, and carboxyatractyloside. Finally, studies with covalent modifying agents indicated the purified transporter was inhibited by sulfhydryl reagents and by diethyl pyrocarbonate, 2,3-butanedione, phenylglyoxal, and pyridoxal 5-phosphate. In conclusion, these studies describe the first procedure to yield a highly purified tricarboxylate transport protein that both displays a high specific transport activity and can be obtained in quantities that readily enable further structural as well as functional studies. Based on its substrate specificity and inhibitor sensitivity, the purified 32.5-kDa protein appears to represent the complete tricarboxylate transport system found in rat liver mitochondria. Finally, new information is presented concerning the effect of covalent modifying reagents on the function of this transporter.     

Purification and characterization of the reconstitutively active adenine nucleotide carrier from mitochondria of Jerusalem artichoke (Helianthus tuberosus L.) tubers

The adenine nucleotide carrier from Jerusalem artichoke (Helianthus Tuberosus L.) tubers mitochondria was solubilized with Triton X-100 and purified by sequential chromatography on hydroxapatite and Matrex Gel Blue B in the presence of cardiolipin and asolectin. SDS gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent molecular mass of 33 kDa. When reconstituted in liposomes, the adenine nucleotide carrier catalyzed a pyridoxal 5-phosphate-sensitive ATP/ATP exchange. It was purified 75-fold with a recovery of 15% and a protein yield of 0.18% with respect to the mitochondrial extract. Among the various substrates and inhibitors tested, the reconstituted protein transported only ATP, ADP, and GTP and was inhibited by bongkrekate, phenylisothiocyanate, pyridoxal 5-phosphate, mersalyl and p-hydroxymercuribenzoate (but not N-ethylmaleimide). Atractyloside and carboxyatractyloside (at concentrations normally inhibitory in animal and plant mitochondria) were without effect in Jerusalem artichoke tubers mitochondria. V _max of the reconstituted ATP/ATP exchange was determined to be 0.53 mol/min per mg protein at 25°C. The half-saturation constant K _m and the corresponding inhibition constant K _i were 20.4 M for ATP and 45 M for ADP. The activation energy of the ATP/ATP exchange was 28 KJ/mol between 5 and 30°C. The N-terminal amino acid partial sequence of the purified protein showed a partial homology with the ANT protein purified from mitochondria of maize shoots.     

A unique protein-kinase activity is responsible for the phosphorylation of the 26- and 14-kDa proteins but not of the 67-kDa protein in the chloroplast envelope membranes of spinach

Protein-phosphorylation activity has been reported in chloroplast envelope membranes of several species. In spinach (Spinacia oleracea L.), we found three major phosphoproteins after incubation in vitro of envelope membranes in the presence of [-³²P]ATP. A 67-kDa phosphoprotein was associated with both inner and outer envelope membranes whereas 26- and 14-kDa proteins were observed in the inner membrane. Although the phosphorylation of the 67-kDa protein is likely to take place via its phosphoglucomutase activity (Salvucci et al., 1990, Plant Physiol. 93, 105–109), the mechanism by which ³²P is incorporated into the 26- and 14-kDa proteins remains to be elucidated. To this aim, we have compared the conditions under which phosphorylation occurs in these three proteins. The effects of Mg²⁺, Ca²⁺, pH, ATP and H7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine], a specific inhibitor of protein-kinase C, as well as pulse-chase experiments with cold ATP, showed that the phosphorylation mechanism was identical for the 26- and 14-kDa proteins but quite different for the 67-kDa one. The protein kinase involved in the phosphorylation of the 26- and 14-kDa proteins was Ca²⁺-dependent, which was not the case of the 67-kDa protein. In addition, the use of a Triton X-114 phase-separation treatment indicated that both the 26- and 14-kDa proteins exhibited strong hydrophobic properties, in contrast to the hydrophilic character of the 67-kDa phosphoprotein. As indicated by analyses of phosphoamino acids, the three proteins were exclusively phosphorylated on serine residues. Furthermore, a treatment of envelopes by phospholipase C prior to the phosphorylation process inhibited ³²P incorporation into the three phospho-proteins to different extents (61%, 50% and 29% inhibition for the 67-, 14- and 26-kDa proteins, respectively). These results show that phosphatidylcholine and — or phosphatidylglycerol but not phosphatidylinositol were involved in this phosphorylation process.Abbreviations EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - H7 1-(5-isoquinolinesulfonyl)-2-methylpiperazine - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - SDS sodium dodecyl sulfate The authors are grateful to Mrs. Delphine Herrmann and Mr. Daniel Leemann for their skillful technical assistance. This study was supported by the Swiss National Science Foundation (Grant No. 31.26386.89). This work is part of a doctoral program which is carried out by L.B. in the Laboratoire de Physiologie végétale, Université de Neuchâtel.