Molecular Cloning and Characterization of the pyrB1 and pyrB2 Genes Encoding Aspartate Transcarbamoylase in Pea (Pisum sativum L.)

Williamson, Cynthia L.; Slocum, Robert D.

doi:10.1104/pp.105.1.377

Cited by 32 publications

(21 citation statements)

References 34 publications

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“…Sequence alignments revealed that identical or similar sequence motifs are present in the transit peptides of ferredoxin-2 and Asp carbamoyltransferase (ATCase; Fig. 8; Williamson and Slocum, 1994). In particular, the FP motif in T4 and the KKFET motif in T7 are present in the transit peptide of ATCase.…”

Section: Sequence Motifs Found In the Transit Peptide Of Rbcs Are Alsmentioning

confidence: 99%

Functional Characterization of Sequence Motifs in the Transit Peptide of Arabidopsis Small Subunit of Rubisco

et al. 2005

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The transit peptides of nuclear-encoded chloroplast proteins are necessary and sufficient for targeting and import of proteins into chloroplasts. However, the sequence information encoded by transit peptides is not fully understood. In this study, we investigated sequence motifs in the transit peptide of the small subunit of the Rubisco complex by examining the ability of various mutant transit peptides to target green fluorescent protein reporter proteins to chloroplasts in Arabidopsis (Arabidopsis thaliana) leaf protoplasts. We divided the transit peptide into eight blocks (T1 through T8), each consisting of eight or 10 amino acids, and generated mutants that had alanine (Ala) substitutions or deletions, of one or two T blocks in the transit peptide. In addition, we generated mutants that had the original sequence partially restored in single-or double-T-block Ala (A) substitution mutants. Analysis of chloroplast import of these mutants revealed several interesting observations. Single-T-block mutations did not noticeably affect targeting efficiency, except in T1 and T4 mutations. However, double-T mutants, T2A/T4A, T3A/T6A, T3A/ T7A, T4A/T6A, and T4A/T7A, caused a 50% to 100% loss in targeting ability. T3A/T6A and T4A/T6A mutants produced only precursor proteins, whereas T2A/T4A and T4A/T7A mutants produced only a 37-kD protein. Detailed analyses revealed that sequence motifs ML in T1, LKSSA in T3, FP and RK in T4, CMQVW in T6, and KKFET in T7 play important roles in chloroplast targeting. In T1, the hydrophobicity of ML is important for targeting. LKSSA in T3 is functionally equivalent to CMQVW in T6 and KKFET in T7. Furthermore, subcellular fractionation revealed that Ala substitution in T1, T3, and T6 produced soluble precursors, whereas Ala substitution in T4 and T7 produced intermediates that were tightly associated with membranes. These results demonstrate that the transit peptide contains multiple motifs and that some of them act in concert or synergistically.

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Section: Sequence Motifs Found In the Transit Peptide Of Rbcs Are Alsmentioning

confidence: 99%

Functional Characterization of Sequence Motifs in the Transit Peptide of Arabidopsis Small Subunit of Rubisco

et al. 2005

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“…Thus, inhibition of ATC by UMP seems to be important in partitioning CP between pyrimidine and arginine synthesis. Williamson and Slocum (1994) cloned cDNAs from pea that encode two different isoforms of ATC (pyrB1 and pyrB2), by complementation of an E. coli delta pyrB mutant. The two cDNAs encode polypeptides of 386 and 385 amino acid residues, respectively that contain typical chloroplast transit peptide sequences.…”

Section: Aspartate Transcarbamoylases (Atc Ec 2132)mentioning

confidence: 99%

Purine and Pyrimidine Nucleotide Synthesis and Metabolism

Moffatt

Ashihara

2002

The Arabidopsis Book

263

249

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“…There are a few exceptions, e.g. the finding of three aspartate transcarbamoylase (ATCase) genes from Pisum sativum (Williamson and Slocum, 1994) and Glycine max or two different cDNAs for uridine monophosphate synthase (UMPSase) from Oryza sativa. From these findings, we conclude that pyrimidine de novo synthesis is an essential process and that complete loss of any one function leads to death of the plant.…”

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Functional Analysis of the Pyrimidine de Novo Synthesis Pathway in Solanaceous Species

2005

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Pyrimidines are particularly important in dividing tissues as building blocks for nucleic acids, but they are equally important for many biochemical processes, including sucrose and cell wall polysaccharide metabolism. In recent years, the molecular organization of nucleotide biosynthesis in plants has been analyzed. Here, we present a functional analysis of the pyrimidine de novo synthesis pathway. Each step in the pathway was investigated using transgenic plants with reduced expression of the corresponding gene to identify controlling steps and gain insights into the phenotypic and metabolic consequences. Inhibition of expression of 80% based on steady-state mRNA level did not lead to visible phenotypes. Stepwise reduction of protein abundance of Asp transcarbamoylase or dihydro orotase resulted in a corresponding inhibition of growth. This was not accompanied by pleiotropic effects or by changes in the developmental program. A more detailed metabolite analysis revealed slightly different responses in roots and shoots of plants with decreased abundance of proteins involved in pyrimidine de novo synthesis. Whereas in leaves the nucleotide and amino acid levels were changed only in the very strong inhibited plants, the roots show a transient increase of these metabolites in intermediate plants followed by a decrease in the strong inhibited plants. Growth analysis revealed that elongation rates and number of organs per plant were reduced, without large changes in the average cell size. It is concluded that reduced pyrimidine de novo synthesis is compensated for by reduction in growth rates, and the remaining nucleotide pools are sufficient for running basic metabolic processes.Pyrimidine nucleotides are abundant molecules with essential functions in a multitude of biochemical processes. They are of particular importance in dividing and elongating tissues as building blocks for nucleic acid biosynthesis. In addition, as an energy source or precursors for the synthesis of primary and secondary products, they are participants in various metabolic processes. In particular, the pyrimidine nucleotides are directly involved in plant carbohydrate metabolism providing the energy-rich precursor UDP-Glc for many synthetic reactions, such as Suc and cell wall biosyntheses.In recent years, the basic processes of plant nucleotide de novo synthesis have been analyzed in some detail (Giermann et al., 2002;Moffatt and Ashihara, 2002;Boldt and Zrenner, 2003;Stasolla et al., 2003;Kafer et al., 2004). Plants utilize the same reactions as those found in other organisms. The so-called orotate pathway of pyrimidine de novo synthesis is defined as the formation of UMP from carbamoyl phosphate, Asp, and phosphoribosyl pyrophosphate. The pathway consists of the six enzymatic reactions as shown in Figure 1. The first reaction is conducted by the Gln hydrolyzing carbamoyl phosphate synthase. This enzyme is not unique to the pyrimidine pathway but is also involved in Arg biosynthesis. It is composed of two different subunits (carbamoyl phosp...

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Molecular Cloning and Characterization of the pyrB1 and pyrB2 Genes Encoding Aspartate Transcarbamoylase in Pea (Pisum sativum L.)

Cited by 32 publications

References 34 publications

Functional Characterization of Sequence Motifs in the Transit Peptide of Arabidopsis Small Subunit of Rubisco

Functional Characterization of Sequence Motifs in the Transit Peptide of Arabidopsis Small Subunit of Rubisco

Purine and Pyrimidine Nucleotide Synthesis and Metabolism

Functional Analysis of the Pyrimidine de Novo Synthesis Pathway in Solanaceous Species

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