Nucleic acid (cDNA) and amino acid sequences of isocitrate lyase from castor bean

A cDNA library was constructed to mRNA enriched for isocitrate-lyase mRNA from castor-bean (Ricinus communis var. zanzibarensis) endosperms. Nine clones for isocitrate lyase (EC 4.1.3.1) were identified. The insert of 2.2 kb from clone ICL4 was sequenced and proved to contain the entire coding region, 1731 bp, for isocitrate lyase. The amino acid sequence of isocitrate lyase was deduced from the nucleic acid sequence. By analogy with muscle aldolase a lysine residue that possibly takes part in the binding of the substrate was identified. The 3 untranslated region contained three putative polyadenylation addition signals and two direct repeats.     

Cloning and expression of amino acid transporters from broad bean

This work describes the isolation of a full-length (VfAAP2) and three partial amino acid transporter genes (VfAAPa, VfAAPb, VfAAPc) from broad bean (Vicia faba L.). The function of VfAAP2 was tested by heterologous expression in a yeast mutant deficient in proline uptake. VfAAP2 mediates proton-dependent proline uptake with an apparent K_m of about 1 mM. Analysis of substrate specificity by competition experiments showed that aromatic amino acids, neutral aliphatic acids and L-citrulline are the best competitors, whereas basic amino acids do not compete with proline. Northern analysis indicates that all VfAAPs exhibit different patterns of expression. VfAAP2 is most strongly expressed in the stem and at a lower level in sink leaves and pods. VfAAPa, VfAAPb and VfAAPc are most strongly expressed in the flowers, but their expression in the other organs varies.     

Isolation and characterization of a general amino acid permease from the ectomycorrhizal fungus Amanita muscaria

A cDNA coding for a fungal amino acid transporter ( AmAAP1 ) was identified from Amanita muscaria ectomycorrhizas. The transporter gene was expressed at a basal level under all conditions investigated, but its expression was enhanced 10-fold in the absence of a N source utilized by the fungus. Nitrate was not a suitable N source for A. muscaria and resulted in maximal AmAAP1 expression. The expression of AmAAP1 in a yeast mutant revealed its function as a high-affinity amino acid transporter with a broad substrate spectrum. AmAAP1 takes up all investigated amino acids with K _m values between 22 μM for histidine and up to 100 μM for proline. Gene expression and amino acid uptake data together indicate two main functions for AmAAP1: uptake of amino acids from soil for fungal nutrition, and prevention of an amino acid loss by hyphal leakage in the absence of a suitable N source.     

Genome-wide identification,characterization and expression analysis of the amino acid permease gene family in tea plants (Camellia sinensis)

Amino acid permeases (AAPs) are involved in transporting a broad spectrum of amino acids and regulating physiological processes in plants. In this study, 19 AAP genes were identified from the tea plants genome database and named CsAAP1–19. Based on phylogenetic analysis, the CsAAP genes were classified into three groups, having significantly different structures and conserved motifs. In addition, an expression analysis revealed that most of CsAAP genes were specifically expressed in different tissues, especially CsAAP19 was expressed only in root. These genes also were significantly expressed in the Baiye 1 and Huangjinya cultivars. Nitrogen treatments indicated that the CsAAPs were obviously expressed in root. CsAAP2, −6, −12, −13 and − 16 were significantly expressed at 6 d after the glutamate treatment, while the expression trend at 24 h after contained the ammonium. These results improve our understanding of the CsAAP genes and their functions in nitrogen utilization in tea plants.     

Functional estimation of loop-helix boundaries in the lactose permease of Escherichia coli by single amino acid deletion analysis

Mutants with single amino acid deletions in the loops of lactose permease retain activity, while mutants with single deletions in transmembrane helices are inactive, and the loop--helix boundaries of helices IV, V, VII, VIII, and IX have been approximated functionally by the systematic deletion of single residues [Wolin, C. D., and Kaback, H. R. (1999) Biochemistry 38, 8590-8597]. The experimental approach is applied here to the remainder of the permease. Periplasmic and cytoplasmic loop-helix boundaries for helices I, II, X, XI, and XII and the cytoplasmic boundary of helix III are in reasonable agreement with structural predictions. In contrast, the periplasmic end of helix III appears to be five to eight residues further into the transmembrane domain than predicted. Taken together with the previous findings, the analysis estimates that 11 of the 12 transmembrane helices have an average length of 21 residues. Surprisingly, deletion analysis of loop V/VI, helix VI, and loop VI/VII does not yield an activity profile typical of the rest of the protein, as individual deletion of only three residues in this region abolishes activity. Thus, transmembrane domain VI which is probably on the periphery of the 12-helix bundle may make few functionally important contacts.     

Cloning and characterization of the acid lipase from castor beans

Castor bean endosperm contains a well known acid lipase activity that is associated with the oil body membrane. In order to identify this enzyme, proteomic analysis was performed on purified oil bodies. A approximately 60-kDa protein was identified (RcOBL1), which shares homology with a lipase from the filamentous fungus Rhizomucor miehei. RcOBL1 contains features that are characteristic of an alpha/beta-hydrolase, such as a putative catalytic triad (SDH) and a conserved pentapeptide (GXSXG) surrounding the nucleophilic serine residue. RcOBL1 was expressed heterologously in Escherichia coli and shown to hydrolyze triolein at an acid pH (optima approximately 4.5). RcOBL1 can hydrolyze a range of triacylglycerols but is not active on phospholipids. The activity is sensitive to the serine reagent diethyl p-nitrophenyl phosphate, indicating that RcOBL1 is a serine esterase. Antibodies raised against RcOBL1 were used to show that the protein is restricted to the endosperm where it is associated with the surface of oil bodies. This is the first evidence for the molecular identity of an oil body-associated lipase from plants. Sequence comparisons reveal that families of OBL1-like proteins are present in many species, and it is likely that they play an important role in regulating lipolysis.     

Novel motifs in amino acid permease genes from Leishmania

Eight amino acid permease genes from the protozoan parasite Leishmania donovani (AAPLDs) were cloned, sequenced, and shown to be expressed in promastigotes. Seven of these belong to the amino acid transporter-1 and one to the amino acid polyamino-choline superfamilies. Using these sequences as well as known and characterized amino acid permease genes from all kingdoms, a training set was established and used to search for motifs, using the MEME motif discovery tool. This study revealed two motifs that are specific to the genus Leishmania, four to the family trypanosomatidae, and a single motif that is common between trypanosomatidae and mammalian systems A1 and N. Interestingly, most of these motifs are clustered in two regions of 50-60 amino acids. Blast search analyses indicated a close relationship between the L. donovani and Trypanosoma brucei amino acid permeases. The results of this work describe the cloning of the first amino acid permease genes in parasitic protozoa and contribute to the understanding of amino acid permease evolution in these organisms. Furthermore, the identification of genus-specific motifs in these proteins might be useful to better understand parasite physiology within its hosts.     

Partial purification and characterization of CTP:cholinephosphate cytidylyltransferase from castor bean endosperm

CTP: cholinephosphate cytidylyltransferase (EC 2.7.7.15) has been purified approximately 600-fold from postgermination endosperm of castor bean. The enzyme was solubilized with n-octyl beta-D-glucopyranoside and then subjected to ion exchange and gel filtration chromatography. The Km's of the purified enzymatic activity were 0.37 and 1.1 mM for CTP and choline phosphate, respectively. Magnesium was required for activity. The purified cytidylyltransferase activity was inhibited by both phosphate and ATP. The extent of ATP inhibition was dependent on preincubation time, temperature, and Mg2+ and Ca2+ concentrations. The possible regulation of cytidylyltransferase in castor bean endosperm by protein phosphorylation is discussed.     

Isolation and characterization of a mutant of Neurospora crassa deficient in general amino acid permease activity.

A mutant of Neurospora crassa (pm-nbg27) was isolated on the basis of its resistance of p-fluoro-phenylalanine on ammonium-deficient Vogel's medium. This mutant was found to be devoid of both conidial and post-conidial (after 180 min of preincubation) transport activity of all amino acids. Genetic analysis of pm-nbg27 by crossing it to wild-type (74A) resulted in the predicted segregants exhibiting transport characteristics of pm-n, pm-b, pm-g, pm-nb, pm-ng, pm-bg and parental types. The above observations confirm the postulated general amino acid permease system as well as a single genetic locus control of that activity.     

Control of the citric acid cycle in mitochondria from germinating castor bean endosperm

Only a part of the citric acid cycle seems to be functional in the endosperm of germinating castor bean seeds. Mitochondria isolated from the endosperm can oxidize all of the citric acid cycle substrates. This was investigated further by studying the enzymic activities of isolated mitochondria during germination. Whilst all enzymic activities increase during germination there is an imbalance in the absolute levels of activities, with very low activities of those enzymes involved in converting pyruvate to succinate. It is suggested that the enzymic activity represents a coarse control of the cycle in this tissue.     

Isolation and characterization of NAD(P)H-dehydrogenases from seeds of the castor bean

Two pyridine nucleotide dehydrogenases have been isolated from castor bean seed extracts by a combination of ion exchange chromatography on DEAE-Sepharose and gel permeation chromatography on Sephadex G-200. The enzymes were designated D-I and D-II according to their elution position on DEAE-Sepharose. Both enzymes D-I and D-II are globular proteins which have MWs of 66 000 and 60 000, respectively. Dehydrogenation is observed with both NADH and NADPH as electron donors, while the electron acceptor specificity demonstrates that the enzymes are probably NAD(P)H: quinone oxidoreductases. Successful coupling of dehydrogenase activity with that of peroxidase indicates a possible role of the enzymes in seed germination.     

Heterologous expression and characterization of recombinant purple acid phosphatase from red kidney bean

Purple acid phosphatases (PAPs) are dinuclear metallohydrolases of widespread occurrence. In a first step to understand structure-function relationship of PAP from red kidney bean (kbPAP), we cloned its cDNA and functionally expressed the enzyme in insect cells. kbPAP cDNA encodes a protein of 459 amino acids with 99% identity to the published primary structure (T. Klabunde et al., Eur. J. Biochem. 226 (1994) 369-375). N-terminally the cDNA encodes 27 amino acids with characteristics for a signal directing the nascent protein to the endoplasmic reticulum. A baculovirus vector was constructed containing cDNAs of kbPAP and green fluorescent protein, the latter to serve as transfection and infection marker. Heterologous expression in High Five insect cells afforded a dimeric, disulfide-linked phosphatase of 110 kDa, identical to the mass of native kbPAP. Purification in three steps yielded 1.5 mg recombinant protein per liter of culture medium with a specific activity of 266 units/mg, slightly exceeding that of native kbPAP. The recombinant protein was functionally indistinguishable from native kbPAP, despite differences in glycosylation and sensitivity to redox reagents.     

Amino acid sequences of two nonspecific lipid-transfer proteins from germinated castor bean

The amino acid sequence of two nonspecific lipid-transfer proteins (nsLTP) B and C from germinated castor bean seeds have been determined. Both the proteins consist of 92 residues, as for nsLTP previously reported, and their calculated Mr values are 9847 and 9593 for nsLTP-B and nsLTP-C, respectively. The sequences of nsLTP-B and nsLTP-C, compared to the known sequence of nsLTP-A from the same source, are 68% and 35% similar, respectively. No variation was found at the positions of the cysteine residues, indicating that they might be involved in disulfide bridges.     

Activity-dependent reversible inactivation of the general amino acid permease

The general amino acid permease, Gap1p, of Saccharomyces cerevisiae transports all naturally occurring amino acids into yeast cells for use as a nitrogen source. Previous studies have shown that a nonubiquitinateable form of the permease, Gap1p(K9R,K16R), is constitutively localized to the plasma membrane. Here, we report that amino acid transport activity of Gap1p(K9R,K16R) can be rapidly and reversibly inactivated at the plasma membrane by the presence of amino acid mixtures. Surprisingly, we also find that addition of most single amino acids is lethal to Gap1p(K9R,K16R)-expressing cells, whereas mixtures of amino acids are less toxic. This toxicity appears to be the consequence of uptake of unusually large quantities of a single amino acid. Exploiting this toxicity, we isolated gap1 alleles deficient in transport of a subset of amino acids. Using these mutations, we show that Gap1p inactivation at the plasma membrane does not depend on the presence of either extracellular or intracellular amino acids, but does require active amino acid transport by Gap1p. Together, our findings uncover a new mechanism for inhibition of permease activity in response to elevated amino acid levels and provide a physiological explanation for the stringent regulation of Gap1p activity in response to amino acids.     

Characterization of glyoxysomes from castor bean endosperm

Electron micrographs are presented which establish the identity of the components of the 3 major bands observed after sucrose density centrifugation of the crude particulate fraction from the endosperm of germinating castor bean seedlings. These are: mitochondria (density 1.19 g/cc), proplastids (density 1.23 g/cc) and glyoxysomes (density 1.25 g/cc). Further evidence is provided on the enzymatic composition of the glyoxysomes. Essentially all of the particulate malate synthetase, isocitrate lyase, catalase, and glycolic oxidase is present in these organelles. The distribution of glyoxysomal enzymes on sucrose density gradients is contrasted with that of the strictly mitochondrial enzymes fumarase, NADH oxidase, and succinoxidase. Malate dehydrogenase and citrate synthetase are present in both organelles. The functional role of glyoxysomes and their relationship to cytosomes from other tissues is discussed.