A putative plastidic glucose translocator is expressed in heterotrophic tissues that do not contain starch, during olive (Olea europea L.) fruit ripening

Metabolite-specific transporters are present in the inner membrane of the plastid envelope allowing transport between the plastid and other cellular compartments. A plastidic glucose translocator (pGlcT) in leaf mesophyll cells transports glucose from chloroplast stroma to the cytosol after amylolytic starch degradation at night. Here we report the cloning of a pGlcT expressed in olive fruits (Olea europea L.). Our results showed high expression of pGlcT in non-green heterotrophic fruit tissues. Expression of pGlcT in olive fruits was somewhat higher compared to leaves, and continued until the black, mature fruit stage. We cloned part of tomato pGlcT and found that it is also expressed throughout fruit development implying a role for pGlcT in heterotrophic tissues. Light and electron microscopic characterization of plastid structural changes during olive fruit ripening revealed the transition of chloroplast-like plastids into starchless, non-green plastids; in mature olive fruits only chromoplasts were present. Together, these findings suggest that olive pGlcT is abundant in chromoplasts during structural changes, and provide evidence that pGlcT may play different physiological roles in ripening fruits and possibly in other non-photosynthetic organs.     

Identification, purification, and molecular cloning of N-1-naphthylphthalmic acid-binding plasma membrane-associated aminopeptidases from Arabidopsis

Polar transport of the plant hormone auxin is regulated at the cellular level by inhibition of efflux from a plasma membrane (PM) carrier. Binding of the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) to a regulatory site associated with the carrier has been characterized, but the NPA-binding protein(s) have not been identified. Experimental disparities between levels of high-affinity NPA binding and auxin transport inhibition can be explained by the presence of a low-affinity binding site and in vivo hydrolysis of NPA. In Arabidopsis, colocalization of NPA amidase and aminopeptidase (AP) activities, inhibition of auxin transport by artificial beta-naphthylamide substrates, and saturable displacement of NPA by the AP inhibitor bestatin suggest that PM APs may be involved in both low-affinity NPA binding and hydrolysis. We report the purification and molecular cloning of NPA-binding PM APs and associated proteins from Arabidopsis. This is the first report of PM APs in plants. PM proteins were purified by gel permeation, anion exchange, and NPA affinity chromatography monitored for tyrosine-AP activity. Lower affinity fractions contained two orthologs of mammalian APs involved in signal transduction and cell surface-extracellular matrix interactions. AtAPM1 and ATAPP1 have substrate specificities and inhibitor sensitivities similar to their mammalian orthologs, and have temporal and spatial expression patterns consistent with previous in planta histochemical data. Copurifying proteins suggest that the APs interact with secreted cell surface and cell wall proline-rich proteins. AtAPM1 and AtAPP1 are encoded by single genes. In vitro translation products of ATAPM1 and AtAPP1 have enzymatic activities similar to those of native proteins.     

The plastidic pentose phosphate translocator represents a link between the cytosolic and the plastidic pentose phosphate pathways in plants

Plastids are the site of the reductive and the oxidative pentose phosphate pathways, which both generate pentose phosphates as intermediates. A plastidic transporter from Arabidopsis has been identified that is able to transport, in exchange with inorganic phosphate or triose phosphates, xylulose 5-phosphate (Xul-5-P) and, to a lesser extent, also ribulose 5-phosphate, but does not accept ribose 5-phosphate or hexose phosphates as substrates. Under physiological conditions, Xul-5-P would be the preferred substrate. Therefore, the translocator was named Xul-5-P/phosphate translocator (XPT). The XPT shares only approximately 35% to 40% sequence identity with members of both the triose phosphate translocator and the phosphoenolpyruvate/phosphate translocator classes, but a higher identity of approximately 50% to glucose 6-phosphate/phosphate translocators. Therefore, it represents a fourth group of plastidic phosphate translocators. Database analysis revealed that plant cells contain, in addition to enzymes of the oxidative branch of the oxidative pentose phosphate pathway, ribose 5-phosphate isomerase and ribulose 5-phosphate epimerase in both the cytosol and the plastids, whereas the transketolase and transaldolase converting the produced pentose phosphates to triose phosphates and hexose phosphates are probably solely confined to plastids. It is assumed that the XPT function is to provide the plastidic pentose phosphate pathways with cytosolic carbon skeletons in the form of Xul-5-P, especially under conditions of a high demand for intermediates of the cycles.     

Analysis of the plastidic phosphate translocator gene family in Arabidopsis and identification of new phosphate translocator-homologous transporters,classified by their putative substrate-binding site

Analysis of the Arabidopsis genome revealed the complete set of plastidic phosphate translocator (pPT) genes. The Arabidopsis genome contains 16 pPT genes: single copies of genes coding for the triose phosphate/phosphate translocator and the xylulose phosphate/phosphate translocator, and two genes coding for each the phosphoenolpyruvate/phosphate translocator and the glucose-6-phosphate/phosphate translocator. A relatively high number of truncated phosphoenolpyruvate/phosphate translocator genes (six) and glucose-6-phosphate/phosphate translocator genes (four) could be detected with almost conserved intron/exon structures as compared with the functional genes. In addition, a variety of PT-homologous (PTh) genes could be identified in Arabidopsis and other organisms. They all belong to the drug/metabolite transporter superfamily showing significant similarities to nucleotide sugar transporters (NSTs). The pPT, PTh, and NST proteins all possess six to eight transmembrane helices. According to the analysis of conserved motifs in these proteins, the PTh proteins can be divided into (a) the lysine (Lys)/arginine group comprising only non-plant proteins, (b) the Lys-valine/alanine/glycine group of Arabidopsis proteins, (c) the Lys/asparagine group of Arabidopsis proteins, and (d) the Lys/threonine group of plant and non-plant proteins. None of these proteins have been characterized so far. The analysis of the putative substrate-binding sites of the pPT, PTh, and NST proteins led to the suggestion that all these proteins share common substrate-binding sites on either side of the membrane each of which contain a conserved Lys residue.     

Identification and cloning of xp95, a putative signal transduction protein in Xenopus oocytes

A 95-kDa protein in Xenopus oocytes, Xp95, was shown to be phosphorylated from the first through the second meiotic divisions during progesterone-induced oocyte maturation. Xp95 was purified and cloned. The Xp95 protein sequence exhibited homology to mouse Rhophilin, budding yeast Bro1, and Aspergillus PalA, all of which are implicated in signal transduction. It also contained three conserved features including seven conserved tyrosines, a phosphorylation consensus sequence for the Src family of tyrosine kinases, and a proline-rich domain near the C terminus that contains multiple SH3 domain-binding motifs. We showed the following: 1) that both Xp95 isolated from Xenopus oocytes and a synthetic peptide containing the Src phosphorylation consensus sequence of Xp95 were phosphorylated in vitro by Src kinase and to a lesser extent by Fyn kinase; 2) Xp95 from Xenopus oocytes or eggs was recognized by an anti-phosphotyrosine antibody, and the relative abundance of tyrosine-phosphorylated Xp95 increased during oocyte maturation; and 3) microinjection of deregulated Src mRNA into Xenopus oocytes increased the abundance of tyrosine-phosphorylated Xp95. These results suggest that Xp95 is an element in a tyrosine kinase signaling pathway that may be involved in progesterone-induced Xenopus oocyte maturation.     

Endo-beta-mannosidase, a plant enzyme acting on N-glycan: purification, molecular cloning, and characterization

Endo-beta-mannosidase is a novel endoglycosidase that hydrolyzes the Manbeta1-4GlcNAc linkage in the trimannosyl core structure of N-glycans. This enzyme was partially purified and characterized in a previous report (Sasaki, A., Yamagishi, M., Mega, T., Norioka, S., Natsuka, S., and Hase, S. (1999) J. Biochem. 125, 363-367). Here we report the purification and molecular cloning of endo-beta-mannosidase. The enzyme purified from lily flowers gave a single band on native-PAGE and three bands on SDS-PAGE with molecular masses of 42, 31, and 28 kDa. Amino acid sequence information from these three polypeptides allowed the cloning of a homologous gene, AtEBM, from Arabidopsis thaliana. AtEBM was engineered for expression in Escherichia coli, and the recombinant protein comprised a single polypeptide chain with a molecular mass of 112 kDa corresponding to the sum of molecular masses of three polypeptides of the lily enzyme. The recombinant protein hydrolyzed pyridylamino derivatives (PA) of Manalpha1-6Manbeta1-4Glc-NAcbeta1-4GlcNAc into Manalpha1-6Man and GlcNAcbeta1-4Glc-NAc-PA, showing that AtEBM is an endo-beta-mannosidase. AtEBM hydrolyzed Man(n)Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc-PA (n = 0-2) but not PA-sugar chains containing Manalpha1-3Manbeta or Xylosebeta1-2Manbeta as for the lily endo-beta-mannosidase. AtEBM belonged to the clan GH-A of glycosyl hydrolases. Site-directed mutagenesis experiments revealed that two glutamic acid residues (Glu-464 and Glu-549) conserved in this clan were critical for enzyme activity. The amino acid sequence of AtEBM has distinct differences from those of the bacterial, fungal, and animal exo-type beta-mannosidases. Indeed, AtEBM-like genes are only found in plants, indicating that endo-beta-mannosidase is a plant-specific enzyme. The role of this enzyme in the processing and/or degradation of N-glycan will be discussed.     

Characterization and molecular cloning of a putative binding protein for heparin-binding growth factors

A novel Mr 17,000 heparin-binding protein was purified from culture medium conditioned by A431 human epidermoid carcinoma cells. This protein, designated HBp17, was found to bind the heparin-binding peptide growth factors HBGF-1 and HBGF-2 in a noncovalent, reversible manner. In addition HBp17 was found to inhibit the biological activities of both HBGF-1 and HBGF-2. Both the binding and inactivation of HBGF-1 and HBGF-2 by HBp17 were abolished by heparin. Full-length 1163-base pair HBp17 cDNA was cloned and sequenced by using the polymerase chain reaction technique. The deduced primary structure of HBp17 consisted of 234 amino acids including each of five partial peptide sequences obtained from proteolytic fragments of purified HBp17. The encoded protein included a 33-residue N-terminal signal sequence for secretion and a single potential N-linked glycosylation site. No homology with any known protein was found for the deduced primary structure of HBp17. The expression of HBp17 mRNA was found to occur preferentially in normal human keratinocytes and in squamous cell carcinomas. This pattern of HBp17 gene expression suggests that this binding protein for HBGFs 1 and 2 has a physiological role in squamous epithelia.     

Identification, molecular cloning, and phylogenetic analysis of a non-respiratory pseudo-hemocyanin of Homarus americanus

Copper-containing hemocyanins serve to transport oxygen in many arthropod species. Here I describe the identification and cDNA cloning of a structurally closely related non-respiratory pseudo-hemocyanin (PHc) of the American lobster, Homarus americanus. This protein has lost the ability to bind copper and, therefore, oxygen because a histidine residue in copper-binding site A is replaced by tyrosine. Like many arthropod hemocyanins, PHc forms a hexamer. It consists of two different subunit types of 660 and 661 amino acids, respectively, that share a 94.4% sequence identity. Whereas Homarus hemocyanin is produced in the hepatopancreas, PHc is synthesized by the ovaries and the heart tissue. Because different levels of PHc were observed in distinct individuals, I propose an association of the synthesis of this protein with the molting or reproduction cycle, similar to the hexamerins, insect storage proteins that are also related to the hemocyanins. However, phylogenetic analyses show that PHc derived independently from crustacean hemocyanins. Therefore, Homarus PHc is a member of a new class within the growing hemocyanin protein superfamily.     

Identification and purification of a liver microsomal glucose 6-phosphatase.

1. Hepatic glucose 6-phosphatase activity was purified 65-fold in good yield over that in cholate-solubilized microsomal fractions. 2. This preparation still contained five major polypeptides and numerous minor contaminants. 3. The smallest of the five major polypeptides (Mr approx. 18 500) could be purified from heat-treated microsomal fractions. 4. Antisera raised against the heat-stable protein doublet was used to immunoprecipitate specifically glucose 6-phosphatase activity from cholate-solubilized microsomal fractions. 5. This work indicates that hepatic microsomal glucose 6-phosphatase appears to be one or both of the low-molecular-weight heat-stable polypeptides.     

Trypanosoma cruzi: molecular cloning of a gene coding for a putative vacuolar protein

We describe the characterization of Tc38, a Trypanosoma cruzi gene coding for a 337-amino-acid protein with a predicted molecular mass of 38 kDa. Tc38 presents similarities to the plant storage vacuolar protein gamma-3-hordein involved in the transport and targeting of prolamins to the vacuole of developing barley endosperm. Western blot analysis using a polyclonal antiserum against recombinant Tc38 revealed that the protein is differentially expressed in the different life stages of the parasite, showing a higher expression in the epimastigote and tripomastigote stages. Immunofluorescence studies suggest that the protein is located in putative vacuolar structures in epimastigotes. The functionality of this protein in T. cruzi remains to be elucidated.     

Sarcolectin: complete purification for molecular cloning.

A great variety of vertebrate cells contain detectable amounts of lectins, able to stimulate the initiation of cellular DNA synthesis. One of them, sarcolectin (SCL) can block interferon (IFN) action, by inhibiting the synthesis and the expression of the IFN dependent secondary proteins. As a result, the IFN-induced antiviral state is abolished in the cells, which likely facilitates their replication. We identified a major 65 kDa and a minor 55 kDa protein, which could carry these cellular functions. Their purification, especially that of the 65 kDa, was difficult, because of the proximity of albumin. We devised therefore a two-step primary separation, followed by a four-step final purification, which are reported here. The purification was controlled by high pressure liquid chromatography (HPLC), SDS-PAGE electrophoresis and identified by Western blots. We found that only the minor 55 kDa protein can be considered as being sarcolectin, while the major 65 kDa band results from the binding of some SCL molecules to albumin. The major biological functions, namely, stimulation of DNA synthesis and cell agglutination were preserved to the end of the last purification step. This work is requisite for establishing the molecular structure of SCL by recombinant DNA technology.     

The purification and molecular characterisation of a putative nicotinic-muscarinic acetylcholine receptor from housefly heads

The purification and characterisation of a (³H)-decamethonium binding component which is a putative acetylcholine receptor with a mixed nicotinic-muscarinic pharmacology is described. The protein fraction obtained after differential centrifugation and gel permeation chromatography bound up to 1.8 μmoles of decamethonium/g protein and was shown to be low in acetylcholine esterase and other contaminating proteins. Preparative isoelectrofocusing of the purified putative receptor fraction produced two protein bands, with pI values of 4.7 and 4.9, which bound 0.65 and 0.1 μmoles of decamethonium/g protein respectively. SDS-polyacrylamide gel elctrophoresis of the purified putative receptor revealed the presence of two glycopeptides with molecular weights of 83,000 and 91,000. Polyacrylamide gel electrophoresis in the absence of SDS also revealed two glycopeptide bands. At least one of the polypeptides was shown to covalently bind the muscarinic antagonist (³H)-propylbenzilylcholine mustard. Cross-linkage of the solubilised protein with various cross-linking reagents produced a protein molecule with a molecular weight between 320,000 and 380,000. The putative acetylcholine receptor was shown to contain 3% by weight of neutral sugars (galactose, mannose and glucose) and between 1.9 and 2.4% by weight of glucosamine. Amino acid analysis of the purified protein indicates that it contains a high proportion of hydrophilic and polar residues and may therefore be a peripheral membrane protein.     

Serine acetyltransferase involved in cysteine biosynthesis from spinach: molecular cloning, characterization and expression analysis of cDNA encoding a plastidic isoform.

A cDNA clone that encodes a chloroplast-localizing isoform of serine acetyltransferase (SATase) (EC 2.3.1.30) was isolated from spinach (Spinacia oleracea L.). The cDNA encodes a polypeptide of 347 amino acids containing a putative transit peptide of ca. 60-70 amino acids at the N-terminal. Deduced amino acid sequence of SATase from spinach exhibited homology with other SATases from plants. DNA blot hybridization analysis showed the presence of 2-3 copies of Sat gene in the genome of spinach. RNA blot hybridization analysis indicated the constitutive expression of Sat gene in green and etiolated seedlings of spinach. Bacterial expression of the cDNA could directly rescue the cysteine auxotrophy of Escherchia coli caused by a lack of SATase locus (cysE). Catalytically active SATase protein was produced in E. coli cells. L-Cysteine, an end product of the cysteine biosynthetic pathway, inhibited the activity of recombinant spinach SATase, indicating the regulatory function of SATase in this metabolic pathway. A chloroplastic localization of this spinach SATase was revealed by the analyses of transgenic plant expressing transit peptide of SATase-beta-glucuronidase (GUS) fusion protein, and transient expression using the transit peptide-green fluorescent protein (GFP) fusion protein. The result from in vitro translation analysis suggests that this cDNA may encode both plastidic and cytosolic SATases.     

Identification and molecular cloning of a functional GDP-fucose transporter in Drosophila melanogaster

Nucleotide sugar transporters play a central role in the process of glycosylation. They are responsible for the translocation of nucleotide sugars from the cytosol, their site of synthesis, into the Golgi apparatus where the activated sugars serve as substrates for a variety of glycosyltransferases. We and others have recently identified and cloned the first GDP-fucose transporters of H. sapiens and C. elegans. Based on sequence similarity, we could identify a putative homolog in Drosophila melanogaster showing about 45% identity on protein level. The gene (CG9620) encodes a highly hydrophobic, multi-transmembrane spanning protein of 38.1 kDa that is localized in the Golgi apparatus. In order to test whether this protein serves as a GDP-fucose transporter, we performed complementation studies with fibroblasts from a patient with LADII (leukocyte adhesion deficiency II) which exhibit a strong reduction of fucosylation due to a point mutation in the human GDP-fucose transporter gene. We show that transient transfection of these cells with the Drosophila CG9620 cDNA corrects the GDP-fucose transport defect and reestablishes fucosylation. This study gives experimental proof that the product of the in silico identified Drosophila gene CG9620 serves as a functional GDP-fucose transporter.