The pentatricopeptide repeat (PPR) family represents one of the largest gene families in plants, with >440 members annotated in Arabidopsis thaliana. PPR proteins are thought to have a major role in the regulation of posttranscriptional processes in organelles. Recent studies have shown that Arabidopsis PPR proteins play an essential, nonredundant role during embryogenesis. Here, we demonstrate that mutations in empty pericarp4 (emp4), a maize (Zea mays) PPR-encoding gene, confer a seed-lethal phenotype. Mutant endosperms are severely impaired, with highly irregular differentiation of transfer cells in the nutrient-importing basal endosperm. Analysis of homozygous mutant plants generated from embryo-rescue experiments indicated that emp4 also affects general plant growth. The emp4-1 mutation was identified in an active Mutator (Mu) population, and cosegregation analysis revealed that it arose from a Mu3 element insertion. Evidence of emp4 molecular cloning was provided by the isolation of four additional emp4 alleles obtained by a reverse genetics approach. emp4 encodes a novel type of PPR protein of 614 amino acids. EMP4 contains nine 35-amino acid PPR motifs and an N-terminal mitochondrion-targeted sequence peptide, which was confirmed by a translational EMP4-green fluorescent protein fusion that localized to mitochondria. Molecular analyses further suggest that EMP4 is necessary to regulate the correct expression of a small subset of mitochondrial transcripts in the endosperm.

Transfer cells are highly modified plant cells specialized in the transport of solutes. They differentiate at many plant exchange surfaces, including phloem loading and unloading zones such as those present in the sink organs and seeds. In maize (Zea mays) seeds, transfer cells are located at the base of the endosperm. It is currently unknown how apical-basal polarity is established or why the peripheral cells at the base of the endosperm differentiate into transfer instead of aleurone cells. Here, we show that in epidermal cells committed to develop into aleurone cells, the ectopic expression of the transfer cell-specific transcriptional activator Myb-Related Protein-1 (MRP-1) is sufficient to temporarily transform them into transfer cells. These transformed cells acquire distinct transfer cell features, such as cell wall ingrowths and an elongated shape. In addition, they express a number of MRP-1 target genes presumably involved in defense. We also show that the expression of MRP-1 is needed to maintain the transfer cell phenotype. Later in development, an observed reduction in the ectopic expression of MRP-1 was followed by the reversion of the transformed cells, which then acquire aleurone cell features.

The pre-steady-state reaction of Dictyostelium nucleoside diphosphate (NDP) kinase with dideoxynucleotide triphosphates (ddNTP) and AZT triphosphate was studied by quenching of protein fluorescence after manual mixing or by stopped flow. The fluorescence signal, which is correlated with the phosphorylation state of the catalytic histidine in the enzyme active site, decreases upon ddNTP addition according to a monoexponential time course. The pseudo-first order rate constant was determined for different concentrations of the various ddNTPs and was found to be saturable. The data are compatible with a two-step reaction scheme, where fast association of the enzyme with the dideoxynucleotide is followed by a rate-limiting phosphorylation step. The rate constants and dissociation equilibrium constants determined for each dideoxynucleotide were correlated with the steady-state kinetic parameters measured in the enzymatic assay in the presence of the two substrates. It is shown that ddNTPs and AZT triphosphate are poor substrates for NDP kinase with a rate of phosphate transfer of 0.02 to 3.5 s ؊1 and a K S of 1-5 mM. The equilibrium dissociation constants for ADP, GDP, ddADP, and ddGDP were also determined by fluorescence titration of a mutant F64W NDP kinase, where the introduction of a tryptophan at the nucleotide binding site provides a direct spectroscopic probe. The lack of the 3-OH in ddNTP causes a 10-fold increase in K D . Contrary to "natural" NTPs, NDP kinase discriminates between various ddNTPs, with ddGTP the more efficient and ddCTP the least efficient substrate within a range of 100 in k cat values.Nucleoside analogues like 3Ј-deoxy-3Ј-azidothymidine (AZT) 1 and dideoxynucleosides (ddN) are widely used as antiviral drugs, particularly in the multitherapy protocols now used in the treatment of AIDS. These drugs are targeted at the HIV reverse transcriptase as the lack of the 3Ј-OH required for the 3Ј-5Ј phosphodiester bond formation during DNA elongation blocks viral DNA synthesis. To exert their antiviral activity, the nucleoside analogues must be phosphorylated into triphosphates derivatives by cellular kinases. Although AZT, ddT, and ddC are structurally related, they show different patterns of intracellular phosphorylation (1). The synthesis of mono-and diphospho-derivatives involves kinases specific for either purines (for example deoxyguanosine kinase) or pyrimidines (for example deoxycytidine kinase or thymidine kinase). In all cases, the last step in the pathway leading to the triphospho-derivative is catalyzed by nucleoside diphosphate (NDP) kinase (EC 2.7.4.6), which has little specificity toward the nucleobase (2).NDP kinase phosphorylates all nucleoside diphosphates into triphosphates using ATP as the major phosphate donor. The reaction involves the formation of a phosphorylated intermediate according to a ping-pong bi-bi mechanism following the scheme below, where E-P is the phosphorylated intermediate on the catalytic histidine (3).EϳP ϩ N 2 DP 7 N 2 TP ϩ E (reaction B) SCHEME IIn recent yea...

Nucleoside diphosphate (NDP) kinase is the enzyme responsible in the cell for the phosphorylation of nucleoside or deoxynucleoside diphosphates into the corresponding triphosphates at the expense of ATP. Transfer of the gamma-phosphate is very fast (turnover number above 1000 s-1) and involves the phosphorylation of a histidine residue at the active site of the enzyme. We have used intrinsic protein fluorescence of the single tryptophan of Dictyostelium discoideum NDP kinase as a sensitive probe for monitoring the interaction of the enzyme with its substrates. We demonstrate that the 20% quenching of steady-state fluorescence observed upon addition of ATP is due to formation of the phosphorylated intermediate. Time-resolved fluorescence indicates that the Trp-137 side chain is rigidly bound to the protein core with a unique lifetime of 4.5 ns for the free enzyme at 20 degrees C and that it remains tightly immobilized during the time course of the reaction. Phosphorylation of this catalytic residue (His-122) in the presence of ATP induces a similar decrease in mean lifetime, due to the splitting of the signal and the appearance of a shorter decay. This splitting is discussed in terms of a slow conformational equilibrium. We demonstrate that, in the wild-type enzyme, the conserved His-55 quenches the fluorescence of Trp-137 as the H55A mutant protein fluorescence displays an increase in quantum yield. Even though H55A mutant enzyme is active, the absence of the imidazole ring prevents the detection of the phosphorylated state of His-122 by Trp-137. We conclude that His-55 serves as a relay between His-122 and Trp-137.