Calcium-dependent phosphorylation of symbiosome membrane proteins from nitrogen-fixing soybean nodules : evidence for phosphorylation of nodulin-26

By using a peptide (CK-15) based on the COOH-terminal sequence of nodulin-26, we have demonstrated the presence of a Ca²⁺-dependent protein kinase in soluble as well as particulate fractions of nitrogen-fixing soybean (Glycine max) root nodules. Substantial enzyme activity was found in symbiosome membranes. The soluble enzyme was purified 1570-fold. The enzyme was fractionated from endogenous calmodulin and yet was fully activated by Ca²⁺ (K_0.5 = 0.4 micromolar) in the absence of exogenous calmodulin, phosphatidylserine and 1,2-dioleylglycerol, oleic acid, and platelet activating factor. CK-15 was used to generate a site-specific antibody to nodulin-26. The antibody reacted with a protein in the symbiosome membrane with an apparent molecular mass of 27,000 daltons, consistent with the molecular mass predicted for nodulin-26 from the deduced amino acid sequence. A symbiosome membrane protein with an identical electrophoretic mobility was phosphorylated in vitro in a Ca²⁺-dependent manner. Additionally, this symbiosome membrane protein was phosphorylated when nodules were incubated with ³²P-phosphate. Overall, the results show the existence of a Ca²⁺-dependent and calmodulin/lipid-independent enzyme in nitrogen-fixing soybean root nodules and suggest that nodulin-26 is a substrate for Ca²⁺-dependent phosphorylation.     

Protein phosphorylation stimulates the rate of malate uptake across the peribacteroid membrane of soybean nodules

Incubation of intact isolated symbiosomes with [gamma-32P]ATP, followed by isolation of the peribacteroid membrane and polypeptide analysis, showed that a single major polypeptide at 26 kDa was labelled. Antibodies raised against nodulin 26 reacted with a similar sized polypeptide. Incubation of the symbiosomes with alkaline phosphatase removed the label from this polypeptide. Pre-incubation with ATP stimulated malate accumulation by isolated symbiosomes, but only slightly (10-30%). Pre-treatment of symbiosomes with alkaline phosphatase inhibited malate uptake substantially and this inhibition was completely relieved by addition of ATP. The ATP stimulation of malate uptake was not affected by ATPase inhibitors. It is suggested that the rate of malate uptake across the peribacteroid membrane is controlled by phosphorylation of nodulin 26.     

Nodulin-26, a peribacteroid membrane nodulin is expressed independently of the development of the peribacteroid compartment.

The peribacteroid membrane (pbm) of root nodules is derived from the plant cell plasma membrane but contains in addition several nodule-specific host proteins (nodulins). Antibodies raised against purified pbm of soybean were used to immunoprecipitate polysomes to isolate an RNA fraction that served as a template for the synthesis of a cDNA probe for screening a nodule-specific cDNA library. Clone p1B1 was found to encode a 26.5 kDa polypeptide (nodulin-26) which is immunoprecipitable specifically with the anti-pbm serum. Nodulin-26 has features of a transmembrane protein and its structure differs from that of nodulin-24 which appears to be a surface protein of pbm. The expression of these two pbm nodulins was examined in nodules induced by Bradyrhizobium japonicum Tn5 mutants that arrest nodule development at different stages of pbm biosynthesis. Nodules that do not show release of bacteria from the infection thread express nodulin-24 at a very low level. In contrast, the expression of nodulin-26 occurs fully in nodules that form infection threads only and is not affected by the release of bacteria from the threads.     

Ferrous iron is transported across the peribacteroid membrane of soybean nodules

Symbiosomes and bacteroids isolated from soybean (Glycine max Merr.) nodules are able to take up ferrous iron. This uptake activity was completely abolished in the presence of ferrous-iron chelators. The kinetics of uptake were characterized by initially high rates of iron internalization, but no saturation was observed with increasing iron concentration. This process does not appear to involve the ferric reductase of the peribacteroid membrane. The transport of ferrous iron was inhibited by other transition metals, particularly copper. Ferrous iron was taken up by symbiosomes more efficiently than the ferric form. This indicates that the iron transport from the plant host cell to the microsymbiont in vivo may occur mainly as the ferrous form. Received: 11 February 1998 / Accepted: 29 May 1998     

Specificity and regulation of the dicarboxylate carrier on the peribacteroid membrane of soybean nodules

Malate and succinate were taken up rapidly by isolated, intact peribacteroid units (PBUs) from soybean (Glycine max (L.) Merr.) root nodules and inhibited each other in a competitive manner. Malonate uptake was slower and was severely inhibited by equimolar malate in the reaction medium. The apparent K_m for malonate uptake was higher than that for malate and succinate uptake. Malate uptake by PBUs was inhibited by (in diminishing order of severity) oxaloacetate, fumarate, succinate, phthalonate and oxoglutarate. Malonate and butylmalonate inhibited only slightly and pyruvate,isocitrate and glutamate not at all. Of these compounds, only oxaloacetate, fumarate and succinate inhibited malate uptake by free bacteroids. Malate uptake by PBUs was inhibited severely by the uncoupler carbonylcyanidem-chlorophenyl hydrazone and the respiratory poison KCN, and was stimulated by ATP. We conclude that the peribacteroid membrane contains a dicarboxylate transport system which is distinct from that on the bacteroid membrane and other plant membranes. This system can catalyse the rapid uptake of a range of dicarboxylates into PBUs, with malate and succinate preferred substrates, and is likely to play an important role in symbiotic nitrogen fixation. Energization of both the bacteroid and peribacteroid membranes controls the rate of dicarboxylate transport into peribacteroid units.     

ATPase activity and anion transport across the peribacteroid membrane of isolated soybean symbiosomes

Addition of ATP to intact symbiosomes isolated from soybean nodules, resulted in generation of a membrane potential (positive inside) across the peribacteroid membrane (PBM). This energisation was monitored as oxonol fluorescence quenching. The rate of fluorescence quenching was inhibited by the inclusion of permeant anions in the reaction medium. Using this inhibition as a measure of anion uptake across the PBM, the presence of a phthalonate-sensitive dicarboxylate carrier on the PBM was confirmed. Following dissipation of the membrane potential by a permeant anion, a pH gradient, measured using [¹⁴C]methylamine uptake, was slowly established across the PBM. This pH was abolished by addition of an uncoupler but was insensitive to inhibitors of bacteroid respiration. The difference in pH between the external medium and the symbiosome interior was estimated to be in the range of 1–1.6 pH units. The magnitude in planta will depend on the concentrations of ATP and permeant anions in the cytosol of the host cell.Abbreviations PBM peribacteroid membrane - electrical membrane potential - MA methylamine The term symbiosome refers to the peribacteroid unit consisting of bacteroids enclosed in the host-derived peribacteroid membrane     

Channel-mediated permeation of ammonia gas through the peribacteroid membrane of soybean nodules

Ammonia permeability of the peribacteroid membrane (PBM) from N(2)-fixing soybean nodules was measured (8x10(-5) m/s) using isolated PBM in a stopped-flow spectrofluorimeter. Ammonia (NH(3)) uptake into PBM vesicles was inhibited by up to 42% by HgCl(2) (EC(50)=2.9 microM, mercaptoethanol-reversible) and reduced by ATP pre-incubation. The activation energy of NH(3) uptake (52 kJ/mol) increased (118 kJ/mol) with HgCl(2). Water transport was also HgCl(2)-sensitive (EC(50)=52.6 microM), but increased by ATP pre-incubation. NH(3) and H(2)O may permeate via different pathways through Nodulin 26 or there is another protein on the PBM that is permeable to NH(3).     

The galactolipid digalactosyldiacylglycerol accumulates in the peribacteroid membrane of nitrogen-fixing nodules of soybean and Lotus

The peribacteroid membrane (PBM) surrounding nitrogen fixing rhizobia in the nodules of legumes is crucial for the exchange of ammonium and nutrients between the bacteria and the host cell. Digalactosyldiacylglycerol (DGDG), a galactolipid abundant in chloroplasts, was detected in the PBM of soybean (Glycine max) and Lotus japonicus. Analyses of membrane marker proteins and of fatty acid composition confirmed that DGDG represents an authentic PBM lipid of plant origin and is not derived from the bacteria or from plastid contamination. In Arabidopsis, DGDG is known to accumulate in extraplastidic membranes during phosphate deprivation. However, the presence of DGDG in soybean PBM was not restricted to phosphate limiting conditions. Complementary DNA sequences corresponding to the two DGDG synthases, DGD1 and DGD2 from Arabidopsis, were isolated from soybean and Lotus. The two genes were expressed during later stages of nodule development in infected cells and in cortical tissue. Because nodule development depends on the presence of high amounts of phosphate in the growth medium, the accumulation of the non-phosphorus galactolipid DGDG in the PBM might be important to save phosphate for other essential processes, i.e. nucleic acid synthesis in bacteroids and host cells.     

Identification with proteomics of novel proteins associated with the peribacteroid membrane of soybean root nodules

Soybean peribacteroid membrane (PBM) proteins were isolated from nitrogen-fixing root nodules and subjected to N-terminal sequencing. Sequence data from 17 putative PBM proteins were obtained. Six of these proteins are homologous to proteins of known function. These include three chaperones (HSP60, BiP [HSP70], and PDI) and two proteases (a serine and a thiol protease), all of which are involved in some aspect of protein processing in plants. The PBM homologs of these proteins may play roles in protein translocation, folding, maturation, or degradation in symbiosomes. Two proteins are homologous to known, nodule-specific proteins from soybean, nodulin 53b and nodulin 26B. Although the function of these nodulins is unknown, nodulin 53b has independently been shown to be associated with the PBM. All of the eight proteins with identifiable homologs are likely to be peripheral rather than integral membrane proteins. Possible reasons for this apparent bias are discussed. The identification of homologs of HSP70 and HSP60 associated with the PBM is the first evidence that the molecular machinery for co- or post-translational import of cytoplasmic proteins is present in symbiosomes. This has important implications for the biogenesis of this unique, nitrogen-fixing organelle.     

Particle density and protein composition of the peribacteroid membrane from soybean root nodules is affected by mutation in the microsymbiont Bradyrhizobium japonicum

Particle frequency of the peribacteroid membrane (PBM) from nodules of Glycine max (L.) Merr. cv. Maple Arrow infected with Bradyrhizobium japonicum 61-A-101 (wild-type strain) was determined by freeze-fracturing to be about 2200·m^-2 in the protoplasmic fracture face and 700·m^-2 in the exoplasmic fracture face. In membranes isolated from nodules infected with the mutant RH 31-Marburg of B. japonicum, the particle frequency was similar in both fracture faces with 1200–1300 particles·m^-2. Analysis of particlesize distribution on peribacteroid membranes showed a loss, especially of particle sizes larger than 11 nm, in the mutant-infected nodules. Two-dimensional gel electrophoresis (isoelectric focussing and sodium dodecyl sulfate-polyacrylamide) showed 27 different polypeptides in the PBM from nodules infected with the wild-type strain, four of which were absent from the PBM of nodules infected with the mutant RH 31-Marburg, which also exhibited one extra small-molecular-weight polypeptide. At least 14 of the 27 polypeptides in the PBM from the wild-type-infected nodule were glycoproteins. In three of these glycoproteins, post-translational modifications were either lacking or different when the membrane was derived from mutant-infected nodules.Abbreviations EF exoplasmatic fracture face - HRPO horse radish peroxidase - IEF Isoelectric focussing - PBM peribacteroid membrane - PF protoplasmatic fracture face - PNA peanut agglutinin - PSA Pisum sativum agglutinin - SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Topology analysis of the SecY protein, an integral membrane protein involved in protein export in Escherichia coli.

The secY (prlA) gene product is an essential component of the Escherichia coli cytoplasmic membrane, and its function is required for the translocation of exocytoplasmic proteins across the membrane. We have analyzed the orientation of the SecY protein in the membrane by examining the hydropathic character of its amino acid sequence, by testing its susceptibility to proteases added to each side of the membrane, and by characterizing SecY-PhoA (alkaline phosphatase) hybrid proteins constructed by TnphoA transpositions. The orientation of the PhoA portion of the hybrid protein with respect to the membrane was inferred from its enzymatic activity as well as sensitivity to external proteases. The results suggest that SecY contains 10 transmembrane segments, five periplasmically exposed parts, and six cytoplasmic regions including the amino- and carboxyterminal regions.     

Erythropoietin rapidly alters phosphorylation of pp43, an erythroid membrane protein

The phosphorylation of a prominent 43-kDa phosphoprotein (pp43) in the membranes of normal murine erythroid cells was reduced markedly by exposure of the membranes to highly purified erythropoietin. A virtually identical reduction of pp43 phosphorylation was seen when erythropoietin-responsive Rauscher murine erythroleukemia cell membranes were exposed to the hormone. This effect was both time-dependent, occurring within 30 min after erythropoietin exposure, and concentration-dependent. Phosphoamino acid analysis revealed that pp43 is phosphorylated on serine residues. The results provide the first evidence that rapid alterations in membrane protein phosphorylation may serve as a trans-membrane signal for erythropoietin.     

Determination of the site of phosphorylation of nodulin 26 by the calcium-dependent protein kinase from soybean nodules.

Nodulin 26 is a nodule-specific protein that is associated with the symbiosome membrane of soybean root nodules. Nodulin 26 is an endogenous substrate for a novel calcium-dependent protein kinase (CDPK) of soybean root nodules. By phosphopeptide mapping of endoproteinase Lys-C-digested nodulin 26 and automated and manual peptide sequence analyses, we have identified the site on nodulin 26 phosphorylated by CDPK. We have also established that the phosphorylation site of nodulin 26 is identical to the phosphorylation site of CK-15, a synthetic peptide with the carboxyl-terminal sequence of nodulin 26. The phosphorylation of nodulin 26 occurs at position Ser262, and the phosphorylation of CK-15 occurs at the analogous position, Ser,6 in vitro. Thus, the CK-15 sequence apparently contains sufficient structural features of the phosphorylation site of nodulin 26 to be recognized by CDPK. On the basis of peptide mapping analysis of nodulin 26 from nodules that are metabolically labeled with [32P]phosphate, it appears that the site of nodulin 26 that is phosphorylated in vitro is also labeled in vivo. The data indicate that the carboxyl terminus of nodulin 26 is phosphorylated by CDPK and provide initial sequence data for the phosphorylation site of an endogenous substrate for a plant CDPK.     

Seryl-phosphorylation of soybean nodule sucrose synthase (nodulin-100) by a Ca-dependent protein kinase

Sucrose synthase (SS; EC 2.4.1.13) was radiolabeled in situ by incubating detached soybean nodules with ³²Pi. Phosphoamino acid analysis indicated that SS was phosphorylated on a serine residue(s). In-vitro phosphorylation of purified nodule SS by desalted nodule extracts was Ca²⁺-dependent. This SS-kinase was partially purified (2200-fold) from nodules harvested from illuminated plants. The molecular mass of the SS-kinase was about 55 000 on a Superdex 75 size-exclusion column or in a denaturing autophosphorylation gel. With either purified nodule SS or Syntide 2 as substrate, exogenous calmodulin and phosphatidylserine showed little or no effect on the in-vitro activity of this partially purified protein kinase. However, its activity was inhibited by W-7. The purified nodule SS-kinase (or CDPK) phosphorylated nodule PEP carboxylase (PEPC; EC 4.1.1.31) in the presence of Ca²⁺. In contrast, a partially purified nodule PEPC-kinase preparation was incapable of phosphorylating nodule SS. Unlike nodule PEPC [Zhang et al. (1995) Plant Physiol. 108, 1561–1568], the phosphorylation state of SS is not likely modulated in planta by photosynthate supply from the shoots.     

Influence of the membrane potential on the free energy of an intrinsic protein

A modified Poisson-Boltzmann equation is developed from statistical mechanical considerations to describe the influence of the transmembrane potential on macromolecular systems. Using a Green's function formalism, the electrostatic free energy of a protein associated with the membrane is expressed as the sum of three terms: a contribution from the energy required to charge the system's capacitance, a contribution corresponding to the interaction of the protein charges with the membrane potential, and a contribution corresponding to a voltage-independent reaction field free energy. The membrane potential, which is due to the polarization interface, is calculated in the absence of the protein charges, whereas the reaction field is calculated in the absence of transmembrane potential. Variations in the capacitive energy associated with typical molecular processes are negligible under physiological conditions. The formulation of the theory is closely related to standard algorithms used to solve the Poisson-Boltzmann equation and only small modifications to current source codes are required for its implementation. The theory is illustrated by examining the voltage-dependent membrane insertion of a simple polyalanine alpha-helix and by computing the electrostatic potential across a 60-A-diameter sphere meant to represent a large intrinsic protein.     

Involvement of protein kinase C in the phosphorylation of an insulin-granule membrane protein.

The involvement of protein kinase C in the Ca2+-dependent phosphorylation of a 29 000-Mr insulin-granule membrane protein prepared from a rat insulinoma was investigated. Protein kinase C activity towards exogenous lysine-rich histone was detected in a cytosolic fraction prepared from an insulinoma homogenate in the presence of EGTA. This activity bound reversibly to insulin granules in a Ca2+-dependent manner. Phosphatidylserine liposomes removed both protein kinase C activity and the 29 000-Mr protein-phosphorylating activity from the cytosolic fraction in a Ca2+-dependent fashion. Protein kinase C activity and the enzymic activity responsible for the phosphorylation of the 29 000-Mr granule protein behaved identically on sucrose-density-gradient centrifugation, ion-exchange chromatography, (NH4)2SO4 fractionation and gel filtration of the cytosolic fraction. These results are consistent with protein kinase C being the enzyme responsible for the phosphorylation of the 29 000-Mr insulin-granule membrane protein.     

The importance of intrinsic disorder for protein phosphorylation

Reversible protein phosphorylation provides a major regulatory mechanism in eukaryotic cells. Due to the high variability of amino acid residues flanking a relatively limited number of experimentally identified phosphorylation sites, reliable prediction of such sites still remains an important issue. Here we report the development of a new web-based tool for the prediction of protein phosphorylation sites, DISPHOS (DISorder-enhanced PHOSphorylation predictor, http://www.ist.temple. edu/DISPHOS). We observed that amino acid compositions, sequence complexity, hydrophobicity, charge and other sequence attributes of regions adjacent to phosphorylation sites are very similar to those of intrinsically disordered protein regions. Thus, DISPHOS uses position-specific amino acid frequencies and disorder information to improve the discrimination between phosphorylation and non-phosphorylation sites. Based on the estimates of phosphorylation rates in various protein categories, the outputs of DISPHOS are adjusted in order to reduce the total number of misclassified residues. When tested on an equal number of phosphorylated and non-phosphorylated residues, the accuracy of DISPHOS reaches 76% for serine, 81% for threonine and 83% for tyrosine. The significant enrichment in disorder-promoting residues surrounding phosphorylation sites together with the results obtained by applying DISPHOS to various protein functional classes and proteomes, provide strong support for the hypothesis that protein phosphorylation predominantly occurs within intrinsically disordered protein regions.     

TIP,an integral membrane protein of the protein-storage vacuoles of the soybean cotyledon undergoes developmentally regulated membrane accumulation and removal

Protein storage vacuoles (PSVs) in soybean (Glycine max (L.) Merr.) cotyledon cells are formed by subdivision of the central vacuole early in seed maturation. They persist until the fifth or sixth day after germination when the central vacuole re-forms. The major integral membrane protein of PSVs, called Tonoplast Integral Protein or TIP, is highly conserved in the seeds of higher plants (K.D. Johnson et al. 1989, Plant Physiol. 91, 1006–1013). The primary sequence of TIP indicates that it may be a pore protein, although of unknown function (K.D. Johnson et al. 1990, Plant Cell 2, 525–532). TIP is apparently seed-specific and is localized in the protein-storage-vacuole membrane of the storageparenchyma cells and the tonoplast of provascular cells. Using correlated immunoblot and electron microscopicimmunocytochemical assays, we have studied TIP accumulation during seed maturation and its disappearance during seed germination. We have determined that the accumulation of TIP in the protein-storage-vacuole membrane is not correlated with the presence or concentration of stored protein in the organelle. Accumulation of TIP occurs primarily after the division of the central vacuole into protein-storage vacuoles is complete and most of the stored protein has been deposited. Transport of TIP to the PSV membrane is apparently mediated by the Golgi apparatus. Quantitative SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis)-immunoblots indicate that, after germination is initiated, TIP abundance is unchanged for the first 4d, but that between days 5 and 7 of growth its abundance decreases drastically. TIP is removed from the PSV membrane prior to the completion of storageprotein mobilization and concurrently with re-formation of the central vacuole. The mechanism of TIP removal appears to involve autophagic sequestering of membrane inside the PSV. The developmental regulation of TIP insertion and removal indicates a physiological function of TIP during late seed maturation or early seedling growth.Abbreviations PSV protein storage vacuoles - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TIP Tonoplast Integral Protein The mention of vendor or product does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over vendors of similar products not mentionedWe are grateful to Drs. Ken D. Johnson and Maarten J. Chrispeels (University of California/San Diego, La Jolla, USA) for the gift of anti-TIP antiserum and for their continuing interest in this project. We are also grateful to Dr. Robert Yaklich (Plant Germplasm and Quality Enhancement Laboratory, U.S. Department of Agriculture, Beltsville, Md.) for the soybeans used in this study. We thank Dr. Maria L. Ghirardi for her assistance with the laser densitometry.