Characterization of two integral membrane proteins located in the protein bodies of pumpkin seeds

Two integral membrane proteins, MP28 and MP23, were found in protein bodies isolated from pumpkin (Cucurbita sp.) seeds. Molecular characterization revealed that both MP28 and MP23 belong to the seed TIP (tonoplast intrinsic protein) subfamily. The predicted 29 kDa precursor to MP23 includes six putative membrane-spanning domains, and the loop between the first and second transmembrane domains is larger than that of MP28. The N-terminal sequence of the mature MP23 starts from residue 66 in the first loop, indicating that an N-terminal 7 kDa fragment that contains one transmembrane domain is post-translationally removed. During maturation of pumpkin seeds, mRNAs for MP28 and MP23 became detectable in cotyledons at the early stage, and their levels increased slightly until a rapid decrease occurred at the late stage. This is consistent with the accumulation of the 29 kDa precursor and MP28 in the cotyledons at the early stage. By contrast, MP23 appeared at the late stage simultaneously with the disappearance of the 29 kDa precursor. Thus, it seems possible that the conversion of the 29 kDa precursor to the mature MP23 might occur in the vacuoles after the middle stage of seed maturation. Both proteins were localized immunocytochemically on the membranes of the vacuoles at the middle stage and the protein bodies at the late stage. These results suggest that both MP28 and the precursor to MP23 accumulate on vacuolar membranes before the deposition of storage proteins, and then the precursor is converted to the mature MP23 at the late stage. These two TIPs might have a specific function during the maturation of pumpkin seeds.     

Apparent processing of a soybean oil body protein accompanies the onset of oil mobilization

The membrane surrounding the oil body contains several different specific polypeptides. To study the biosynthesis and posttranslational modification of these polypeptides we have prepared monoclonal antibodies against purified oil bodies of soybean (Glycine max). Three of the five monoclonals selected recognize a molecular mass 34 kilodalton protein (P34). Epitope mapping of CNBr and proteolytic fragments of P34 indicates that two of the anti-P34 monoclonal antibodies are directed at different epitopes. P34 is accumulated during seed maturation at the same time as the reserve proteins and oil. SDS/PAGE-immunoblots of germinating soybean seed cotyledons indicate that the protein is initially present as a molecular mass 34 kilodalton polypeptide and is processed to molecular mass 32 kilodalton on the fourth through sixth days of seedling growth simultaneously with the onset of oil mobilization. A comparison of reduced and carboxymethylated oil body proteins with nonreduced proteins by SDS/PAGE indicates that P34 exists in vivo as a dimer of molecular mass 58 kilodalton. Comparing the amino terminal sequences of P34 and P32 indicates that their difference is at least in part due to the removal of the amino terminus of P34. The amino terminal sequences of P34 and P32 were aligned to show that the transition of P34 to P32 was accompanied by the removal of a hydrophilic decapeptide (KKMKKEQYSC) at the amino terminus of P34. Hopp-Woods hydrophilicity analysis of the deleted amino terminus of P34 shows that it is more hydrophilic and charged than the sequence of the protein which immediately follows.     

Expression and activation of the vacuolar processing enzyme in Saccharomyces cerevisiae

Vacuolar processing enzymes (VPEs) are cysteine proteinases responsible for maturation of various vacuolar proteins in plants. A larger precursor to VPE synthesized on rough endoplasmic reticulum is converted to an active enzyme in the vacuoles. In this study, a precursor to castor bean VPE was expressed in a pep4 strain of the yeast Saccharomyces cerevisiae to examine the mechanism of activation of VPE. Two VPE proteins of 59 and 46 kDa were detected in the vacuoles of the transformant. They were glycosylated in the yeast cells, although VPE is not glycosylated in plant cells in spite of the presence of two N-linked glycosylation sites. During the growth of the transformant, the level of the 59 kDa VPE increased slightly until a rapid decrease occurred after 9 h. By contrast, the 46 kDa VPE appeared simultaneously with the disappearance of the 59 kDa VPE. Vacuolar processing activity increased with the accumulation of the 46 kDa VPE, but not of the 59 kDa VPE. The specific activity of the 46 kDa VPE was at a similar level to that of VPE in plant cells. The 46 kDa VPE instead of proteinase A mediated the conversion of procarboxypeptidase Y to the mature form. This indicates that proteinase A responsible for maturation of yeast vacuolar proteins can be replaced functionally by plant VPE. These findings suggest that an inactive VPE precursor synthesized on the endoplasmic reticulum is transported to the vacuoles in the yeast cells and then processed to make an active VPE by self-catalytic proteolysis within the vacuoles.     

Synthesis and maturation of the yeast vacuolar enzymes carboxypeptidase Y and aminopeptidase I

We have studied the two vacuolar enzymes carboxypeptidase Y and aminopeptidase I from Saccharomyces cerevisiae with respect to biosynthesis, maturation and transfer from their site of synthesis into the organelle. The levels of translatable mRNA for these two proteins increase more than 10-fold at the end of the exponential growth period on glucose as carbon source and decrease again in the stationary phase. Two precursors of carboxypeptidase Y have been identified by in vivo pulse-labelling with [35S]methionine. These differ in their amount of carbohydrate as shown by inhibition of N-linked glycosylation with tunicamycin. The first is a protein with an apparent molecular weight of 67 kDa, which can be converted into the mature 60-kDa protein via an intermediate of 69 kDa. In the pep4-3 mutant, which is disturbed in the maturation of several vacuolar enzymes (Hemmings, B.A., Zubenko, G.S., Hasilik, A. and Jones, E.W. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 435-439), the 69-kDa precursor accumulates in the vacuole. This suggests that the final proteolytic cleavage of carboxypeptidase Y can occur in the vacuole.     

Role of posttranslational cleavage in glycinin assembly.

Glycinin, like other 11S seed storage proteins, undergoes a complex series of posttranslational events between the time proglycinin precursors are synthesized in endoplasmic reticulum and the mature glycinin subunits are deposited in vacuolar protein bodies. According to the current understanding of this process, proglycinin subunits aggregate into trimers in endoplasmic reticulum, and then the trimers move to the vacuolar protein bodies where a protease cleaves them into acidic and basic polypeptide chains. Stable glycinin hexamers, rather than trimers, are isolated from mature seeds. We used a re-assembly assay in this study to demonstrate that proteolytic cleavage of the proglycinin subunits is required for in vitro assembly of glycinin oligomers beyond the trimer stage. The possibility that the cleavage is a regulatory step and that it triggers the deposition of 11S seed storage proteins as insoluble aggregates in vivo is considered.     

Synthesis of the major oil-body membrane protein in developing rapeseed (Brassica napus) embryos. Integration with storage-lipid and storage-protein synthesis and implications for the mechanism of oil-body formation.

The synthesis of the major protein and lipid storage reserves during embryogenesis in oilseed rape (Brassica napus L., cv. Mikado) has been examined by biochemical, immunological and immunocytochemical techniques. The mature seeds contained about 45% (w/w) storage oil and 25% (w/w) protein. There were three major seed protein components, i.e. about 40-50% total protein was cruciferin, 20% was napin and 20% was a 18 kDa hydrophobic polypeptide associated with the proteinaceous membrane surrounding the storage oil bodies. Embryogenesis was divided into four overlapping stages with regard to the synthesis of these storage components: (1) for the first 3 weeks after flowering, little, if any, synthesis of storage components was observed; (2) storage-oil synthesis began at about week 3, and maximal rates were from weeks 4 to 7; (3) synthesis of the soluble storage proteins cruciferin and napin started at week 6 and rates were maximal between weeks 8 and 11; (4) the final stage was the synthesis of the 19 kDa oil-body polypeptide, which started at weeks 8-10 and was at a maximal rate between weeks 10 and 12. The synthesis of the 19 kDa oil-body protein therefore occurred independently of the synthesis of the soluble seed storage proteins. This former synthesis did not occur until shortly before the insertion of the 19 kDa polypeptide into the oil-body membrane. No evidence was found, either from sucrose-density-gradient-centrifugation experiments or from immunogold-labelling studies, for its prior accumulation in the endoplasmic reticulum. Conventional and immunogold-electron-microscopic studies showed that oil bodies were synthesized in the early to middle stages of seed development without a strongly electron-dense membrane. Such a membrane was only found at later stages of seed development, concomitantly with the synthesis of the 19 kDa protein. It is proposed that, in rapeseed embryos, oil bodies are initially formed with no proteinaceous membrane. Such a membrane is formed later in development after insertion by ribosomes of the hydrophobic 19 kDa polypeptide directly into the oil bodies.     

Stable oil bodies sheltered by a unique oleosin in lily pollen

Stable oil bodies were purified from mature lily (Lilium longiflorum Thunb.) pollen. The integrity of pollen oil bodies was maintained via electronegative repulsion and steric hindrance possibly provided by their surface proteins. Immunodetection revealed that a major protein of 18 kDa was exclusively present in pollen oil bodies and massively accumulated in late stages of pollen maturation. According to mass spectrometric analyses, this oil body protein possessed a tryptic fragment of 13 residues matching that of a theoretical rice oleosin. A complete cDNA fragment encoding this putative oleosin was obtained by PCR cloning with primers derived from its known 13-residue sequence. Sequence analysis as well as immunological non-cross-reactivity suggests that this pollen oleosin represents a distinct class in comparison with oleosins found in seed oil bodies and tapetum. In pollen cells observed by electron microscopy, oil bodies were presumably surrounded by tubular membrane structures, and encapsulated in the vacuoles after germination. It seems that pollen oil bodies are mobilized via a different route from that of glyoxysomal mobilization of seed oil bodies after germination.     

Purification of a p47 phosphoprotein from Xenopus laevis oocytes and identification as an in vivo and in vitro p34cdc2 substrate

This paper describes the purification of a 47 kDa protein from Xenopus laevis oocytes that becomes phosphorylated when the oocytes undergo meiotic maturation. This protein (p47) is part of a high molecular mass complex containing at least two other proteins of molecular mass 30 and 36 kDa. This complex can be isolated from stage VI oocytes before maturation. We obtained a pattern for phosphopeptides in p47 phosphorylated in vivo very similar to that of the purified protein phosphorylated in vitro by p34cdc2 (a H1 kinase which is a component of the M-phase promoting factor) and [gamma-32P]ATP. Therefore, the purified p47, already described as a marker of MPF activity, is the first reported in vivo substrate for the cell division control kinase.     

Cosuppression of the alpha subunits of beta-conglycinin in transgenic soybean seeds induces the formation of endoplasmic reticulum-derived protein bodies

The expression of the alpha and alpha' subunits of beta-conglycinin was suppressed by sequence-mediated gene silencing in transgenic soybean seed. The resulting seeds had similar total oil and protein content and ratio compared with the parent line. The decrease in beta-conglycinin protein was apparently compensated by an increased accumulation of glycinin. In addition, proglycinin, the precursor of glycinin, was detected as a prominent polypeptide band in the protein profile of the transgenic seed extract. Electron microscopic analysis and immunocytochemistry of maturing transgenic soybean seeds indicated that the process of storage protein accumulation was altered in the transgenic line. In normal soybeans, the storage proteins are deposited in pre-existing vacuoles by Golgi-derived vesicles. In contrast, in transgenic seed with reduced beta-conglycinin levels, endoplasmic reticulum (ER)-derived vesicles were observed that resembled precursor accumulating-vesicles of pumpkin seeds and the protein bodies accumulated by cereal seeds. Their ER-derived membrane of the novel vesicles did not contain the protein storage vacuole tonoplast-specific protein alpha-TIP, and the sequestered polypeptides did not contain complex glycans, indicating a preGolgi and nonvacuolar nature. Glycinin was identified as a major component of these novel protein bodies and its diversion from normal storage protein trafficking appears to be related to the proglycinin buildup in the transgenic seed. The stable accumulation of proteins in a protein body compartment instead of vacuolar accumulation of proteins may provide an alternative intracellular site to sequester proteins when soybeans are used as protein factories.     

Aminopeptidase I of Saccharomyces cerevisiae is localized to the vacuole independent of the secretory pathway

The Saccharomyces cerevisiae APE1 gene product, aminopeptidase I (API), is a soluble hydrolase that has been shown to be localized to the vacuole. API lacks a standard signal sequence and contains an unusual amino-terminal propeptide. We have examined the biosynthesis of API in order to elucidate the mechanism of its delivery to the vacuole. API is synthesized as an inactive precursor that is matured in a PEP4-dependent manner. The half-time for processing is approximately 45 min. The API precursor remains in the cytoplasm after synthesis and does not enter the secretory pathway. The precursor does not receive glycosyl modifications, and removal of its propeptide occurs in a sec-independent manner. Neither the precursor nor mature form of API are secreted into the extracellular fraction in vps mutants or upon overproduction, two additional characteristics of soluble vacuolar proteins that transit through the secretory pathway. Overproduction of API results in both an increase in the half-time of processing and the stable accumulation of precursor protein. These results suggest that API enters the vacuole by a posttranslational process not used by most previously studied resident vacuolar proteins and will be a useful model protein to analyze this alternative mechanism of vacuolar localization.     

Co- and posttranslational modification of the alpha(1B)-adrenergic receptor: effects on receptor expression and function

We have characterized the maturation, co- and posttranslational modifications, and functional properties of the alpha(1B)-adrenergic receptor (AR) expressed in different mammalian cells transfected using conventional approaches or the Semliki Forest virus system. We found that the alpha(1B)-AR undergoes N-linked glycosylation as demonstrated by its sensitivity to endoglycosidases and by the effect of tunicamycin on receptor maturation. Pulse-chase labeling experiments in BHK-21 cells demonstrate that the alpha(1B)-AR is synthesized as a 70 kDa core glycosylated precursor that is converted to the 90 kDa mature form of the receptor with a half-time of approximately 2 h. N-Linked glycosylation of the alpha(1B)-AR occurs at four asparagines on the N-terminus of the receptor. Mutations of the N-linked glycosylation sites did not have a significant effect on receptor function or expression. Surprisingly, receptor mutants lacking N-linked glycosylation migrated as heterogeneous bands in SDS-PAGE. Our findings demonstrate that N-linked glycosylation and phosphorylation, but not palmitoylation or O-linked glycosylation, contribute to the structural heterogeneity of the alpha(1B)-AR as it is observed in SDS-PAGE. The modifications found are similar in the different mammalian expression systems explored. Our findings indicate that the Semliki Forest virus system can provide large amounts of functional and fully glycosylated alpha(1B)-AR protein suitable for biochemical and structural studies. The results of this study contribute to elucidate the basic steps involved in the processing of G protein-coupled receptors as well as to optimize strategies for their overexpression.     

Molecular cloning of a protein associated with soybean seed oil bodies that is similar to thiol proteases of the papain family

A 34,000-Da protein (P34) is one of the four major soybean oil body proteins observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of isolated organic solvent-extracted oil bodies from mature seeds. P34 is processed during seedling growth to a 32,000-Da polypeptide (P32) by the removal of an amino-terminal decapeptide (Herman, E.M., Melroy, D.L., and Buckhout, T.J. (1990) Plant Physiol, in press). A soybean lambda ZAP II cDNA library constructed from RNA isolated from midmaturation seeds was screened with monoclonal antibodies directed against two different epitopes of P34. The isolated cDNA clone encoding P34 contains 1,350 base pairs terminating in a poly(A)+ tail and an open reading frame 1,137 base pairs in length. The open reading frame includes a deduced amino acid sequence which matches 23 of 25 amino-terminal amino acids determined by automated Edman degradation of P34 and P32. The cDNA predicts a mature protein of 257 amino acids and of 28,641 Da. The open reading frame extends 5' from the known amino terminus of P34 encoding a possible precursor and signal sequence segments with a combined additional 122 amino acids. Prepro-P34 is deduced to be a polypeptide of 42,714 Da, indicating that the cDNA clone apparently encodes a polypeptide of 379 amino acids. A comparison of the nucleotide and deduced amino acid sequences in the GenBank Data Bank with the sequence of P34 has shown considerable sequence similarity to the thiol proteases of the papain family. Southern blot analysis of genomic DNA indicated that the P34 gene has a low copy number.     

Embryology in Norway spruce (Picea abies). Immunochemical studies on transport of a seed storage protein

One of the main seed storage proteins of Norway spruce ( Picea abies ), is a salt-soluble protein with an average molecular mass of 42 kDa. This protein was localized by immunocytochemical methods in ultrathin sections of megagametophytes active in storage protein synthesis, as analyzed by SDS-PAGE. The megagametophyte in spruce starts accumulating storage materials, proteins and lipids, as the young embryo grows into the gametophytic tissue. It then continues to accumulate these storage products throughout seed development (Hakman 1993). Megagametophytes at an early stage of storage protein accumulation were chosen in this study for analysing the likely transport pathway of the proteins, since only a small amount of lipid had yet accumulated in the cells, and cell organelles were still easy to distinguish. An antibody against the 42 kDa storage protein showed very good reactivity with the 42 kDa protein in immunoblot experiments with total protein extracts from megagametophytes and embryos. In ultrathin sections of the megagametophyte, the antibodies were preferentially localized in the lumen of Golgi cisterna, in Golgi-associated vesicles, protein deposits close to the vacuolar membrane and in protein storage vacuoles (protein bodies). These observations indicate that the transport is mediated by the Golgi apparatus.
Also, proteins present in storage vacuoles in mature zygotic and somatic embryos showed intense labelling with these antibodies in ultrathin sections.     

An in vitro system to examine the effective phospholipids and structural domain for protein targeting to seed oil bodies.

An in vitro system was established to examine the targeting of proteins to maturing seed oil bodies. Oleosin, the most abundant structural protein, and caleosin, a newly identified minor constituent in seed oil bodies, were translated in a reticulocyte lysate system and simultaneously incubated with artificial oil emulsions composed of triacylglycerol and phospholipid. The results suggest that oil body proteins could spontaneously target to artificial oil emulsions in a co-translational mode. Incorporation of oleosin to artificial oil emulsions extensively protected a fragment of approximately 8 kDa from proteinase K digestion. In a competition experiment, in vitro translated caleosin and oleosin preferentially target to artificial oil emulsions instead of microsomal membranes. In oil emulsions with neutral phospholipids, relatively low protein targeting efficiency was observed. The targeting efficiency was substantially elevated when negatively charged phospholipids were supplemented to oil emulsions to mimic the native phospholipid composition of oil bodies. Mutated caleosin lacking various structural domains or subdomains was examined for its in vitro targeting efficiency. The results indicate that the subdomain comprising the proline knot motif is crucial for caleosin targeting to oil bodies. A model of direct targeting of oil-body proteins to maturing oil bodies is proposed.     

Dicyclohexylcarbodiimide and vanadate sensitive ATPase of lung lamellar bodies.

Lung surfactant is synthesized in lung epithelial type II cells and stored in the lamellar bodies prior to its secretion onto the alveolar surface. The lamellar bodies, like other secretory organelles, maintain an ATP-dependent pH gradient that is sensitive to inhibitors of H(+)-ATPase. This report shows that the ATPase activity of lamellar bodies is enriched in a fraction prepared from lamellar bodies that were disrupted after isolation. The apparent Vmax for this enzyme was 150 nmol ATP hydrolyzed per min per mg protein and apparent Km for ATP was approximately 50 microM. The enzyme activity was sensitive to N-ethylmaleimide (NEM), dicyclohexylcarbodiimide (DCCD) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) (all inhibitors of vacuolar-type H(+)-ATPase) and vanadate (inhibitor of phosphoenzyme-type ATPase). Besides, the activity could also be inhibited with diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and Ca2+. Two proteins (of approximately 45 kDa and 17 kDa) of this fraction showed acid-stable phosphorylation with ATP. The labeling of proteins with ATP (-gamma-32P) could be chased with unlabelled ATP, suggesting that phosphorylation and dephosphorylation of these proteins is associated with the ATPase activity. Our results on inhibition characteristics of the enzyme activity suggest that besides a vacuolar type H(+)-ATPase, the lamellar bodies also contain a phosphoenzyme type ATPase that is sensitive to inhibitors of vacuolar type H(+)-ATPase.     

Incorporation of radiolabelled amino acids by adult Schistosoma japonicum: further characterization of a putative eggshell precursor protein.

The synthesis patterns of female-specific proteins of Schistosoma japonicum were further investigated with particular reference to the 34 kDa putative eggshell precursor protein. Adult male and female worms of S. japonicum were metabolically labelled with 14C-tyrosine, 14C-glycine and 35S-methionine in vitro. The rates of amino acid incorporation for female worms were significantly higher than for males in all radiolabelling experiments. Labelled proteins were resolved by two-dimensional gel electrophoresis and visualized by fluorography. By using 14C-tyrosine and 14C-glycine, the 34 kDa female protein band resolved into three major spots with pI 6.0, 5.8 and 5.6. On the other hand, labelling studies using 35S-methionine failed to reveal synthesis of any corresponding spots at Mr 34 kDa. These results, together with the observations that eggshell hydrolysates are very rich in glycine but poor in methionine, suggested that the 34 kDa putative eggshell precursor protein of S. japonicum consists of at least three isoelectric forms. In addition, we have demonstrated several other female-specific polypeptides synthesized by this worm.     

Subtilisin-like autotransporter serves as maturation protease in a bacterial secretion pathway

Proteins of Gram-negative bacteria destined to the extracellular milieu must cross the two cellular membranes and then fold at the appropriate time and place. The synthesis of a precursor may be a strategy to maintain secretion competence while preventing aggregation or premature folding (especially for large proteins). The secretion of 230 kDa filamentous haemagglutinin (FHA) of Bordetella pertussis requires the synthesis and the maturation of a 367 kDa precursor that undergoes the proteolytic removal of its approximately 130 kDa C-terminal intramolecular chaperone domain. We have identified a specific protease, SphB1, responsible for the timely maturation of the precursor FhaB, which allows for extracellular release of FHA. SphB1 is a large exported protein with a subtilisin-like domain and a C-terminal domain typical of bacterial autotransporters. SphB1 is the first described subtilisin-like protein that serves as a specialized maturation protease in a secretion pathway of Gram-negative bacteria. This is reminiscent of pro-protein convertases of eukaryotic cells.     

Bean homologs of the mammalian glucose-regulated proteins: induction by tunicamycin and interaction with newly synthesized seed storage proteins in the endoplasmic reticulum

Treatment of developing bean cotyledons with the inhibitor of N-glycosylation tunicamycin enhanced the synthesis of at least two polypeptides with molecular mass 78 kDa and 97 kDa. Pulse-chase experiments and subcellular fractionation indicated that these are endoplasmic reticulum (ER) residents. The 78 kDa protein is a major component of the ER protein fraction and, by N-terminal sequencing, was identified as a bean homolog of the mammalian 78 kDa glucose-regulated protein (GRP78). This is a molecular chaperone that is probably involved in the folding and oligomerization of several animal and yeast proteins in the ER. When newly synthesized storage glycoproteins phaseolin, phytohemagglutinin or alpha-amylase inhibitor were immunoprecipitated from an ER preparation of tunicamycin-treated tissue, the GRP78 homolog was always co-precipitated. Bound GRP78 homolog could be released by ATP treatment. These results suggest that, at least when glycosylation is inhibited, this protein plays a role in the early stages of the synthesis of vacuolar storage proteins.     

Structural characteristics of hen egg ovalbumin expressed in yeast Pichia pastoris

The recombinant ovalbumin (OVA) produced in yeast Pichia pastoris was purified from the culture medium by anion exchange chromatography, and its structural characteristics were compared with those of hen egg OVA, mainly from the point of view of posttranslational modification. The expressed OVA consisted of two molecular species immmunoreactive with antibody for hen egg OVA. The two molecular species, 45 and 47 kDa in molecular size, were thought to correspond to mono-glycosylated form and di-glycosylated form respectively. The non-glycosylated form was not produced in the system. The other posttranslational modifications (N-terminal acetylation and phosphorylation) observed in hen egg OVA were not detected in either of the molecular species. The two recombinant proteins displayed almost exactly the same circular dichroism and intrinsic tryptophan fluorescence spectra as hen egg OVA. The melting temperature, Tm, which was determined from the thermal unfolding curve, was almost identical in the two recombinant proteins, despite the difference in glycosylation levels, while it decreased by about 2.5 degrees C as compared with that of hen egg OVA (77.3 degrees C). These data indicate that the additional glycosylation to Asn-311 in the recombinant protein does not affect protein conformation or thermostability.     

Temporal association of Ca(2+)-dependent protein kinase with oil bodies during seed development in Santalum album L.: its biochemical characterization and significance

Calcium-dependent protein kinase (CDPK) is expressed in sandalwood (Santalum album L.) seeds under developmental regulation, and it is localized with spherical storage organelles in the endosperm [Anil et al. (2000) Plant Physiol. 122: 1035]. This study identifies these storage organelles as oil bodies. A 55 kDa protein associated with isolated oil bodies, showed Ca(2+)-dependent autophosphorylation and also cross-reacted with anti-soybean CDPK. The CDPK activity detected in the oil body-protein fraction was calmodulin-independent and sensitive to W7 (N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide) inhibition. Differences in Michaelis Menton kinetics, rate of histone phosphorylation and sensitivity to W7 inhibition between a soluble CDPK from embryos and the oil body-associated CDPK of endosperm suggest that these are tissue-specific isozymes. The association of CDPK with oil bodies of endosperm was found to show a temporal pattern during seed development. CDPK protein and activity, and the in vivo phosphorylation of Ser and Thr residues were detected strongly in the oil bodies of endosperm from maturing seed. Since oil body formation occurs during seed maturation, the observations indicate that CDPK and Ca(2+) may have a regulatory role during oil accumulation/oil body biogenesis. The detection of CDPK-protein and activity in oil bodies of groundnut, sesame, cotton, sunflower, soybean and safflower suggests the ubiquity of the association of CDPKs with oil bodies.