RESUMO
The solute specificity profiles (transport and binding) for the nucleobase cation symporter 1 (NCS1) proteins, from the closely related C4 grasses Zea mays and Setaria viridis, differ from that of Arabidopsis thaliana and Chlamydomonas reinhardtii NCS1. Solute specificity profiles for NCS1 from Z. mays (ZmNCS1) and S. viridis (SvNCS1) were determined through heterologous complementation studies in NCS1-deficient Saccharomyces cerevisiae strains. The four Viridiplantae NCS1 proteins transport the purines adenine and guanine, but unlike the dicot and algal NCS1, grass NCS1 proteins fail to transport the pyrimidine uracil. Despite the high level of amino acid sequence similarity, ZmNCS1 and SvNCS1 display distinct solute transport and recognition profiles. SvNCS1 transports adenine, guanine, hypoxanthine, cytosine, and allantoin and competitively binds xanthine and uric acid. ZmNCS1 transports adenine, guanine, and cytosine and competitively binds, 5-fluorocytosine, hypoxanthine, xanthine, and uric acid. The differences in grass NCS1 profiles are due to a limited number of amino acid alterations. These amino acid residues do not correspond to amino acids essential for overall solute and cation binding or solute transport, as previously identified in bacterial and fungal NCS1, but rather may represent residues involved in subtle solute discrimination. The data presented here reveal that within Viridiplantae, NCS1 proteins transport a broad range of nucleobase compounds and that the solute specificity profile varies with species.
Assuntos
Adenina/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Plantas/metabolismo , Setaria (Planta)/genética , Zea mays/genética , Sequência de Aminoácidos , Arabidopsis , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Cloroplastos/metabolismo , Genes de Plantas , Teste de Complementação Genética , Cinética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Transporte Proteico , Saccharomyces cerevisiae , Setaria (Planta)/metabolismo , Especificidade por Substrato , Zea mays/metabolismoRESUMO
The single cell alga Chlamydomonas reinhardtii is capable of importing purines as nitrogen sources. An analysis of the annotated C. reinhardtii genome reveals at least three distinct gene families encoding for known nucleobase transporters. In this study the solute transport and binding properties for the lone C. reinhardtii nucleobase cation symporter 1 (CrNCS1) are determined through heterologous expression in Saccharomyces cerevisiae. CrNCS1 acts as a transporter of adenine, guanine, uracil and allantoin, sharing similar - but not identical - solute recognition specificity with the evolutionary distant NCS1 from Arabidopsis thaliana. The results suggest that the solute specificity for plant NCS1 occurred early in plant evolution and are distinct from solute transport specificities of single cell fungal NCS1 proteins.
Assuntos
Chlamydomonas reinhardtii/metabolismo , Genes de Plantas , Nitrogênio/metabolismo , Proteínas de Transporte de Nucleobases/metabolismo , Proteínas de Plantas/metabolismo , Purinas/metabolismo , Alantoína/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Chlamydomonas reinhardtii/genética , Evolução Molecular , Proteínas Fúngicas/metabolismo , Dados de Sequência Molecular , Proteínas de Transporte de Nucleobases/genética , Proteínas de Plantas/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Locus At5g03555 encodes a nucleobase cation symporter 1 (AtNCS1) in the Arabidopsis genome. Arabidopsis insertion mutants, AtNcs1-1 and AtNcs1-3, were used for in planta toxic nucleobase analog growth studies and radio-labeled nucleobase uptake assays to characterize solute transport specificities. These results correlate with similar growth and uptake studies of AtNCS1 expressed in Saccharomyces cerevisiae. Both in planta and heterologous expression studies in yeast revealed a unique solute transport profile for AtNCS1 in moving adenine, guanine and uracil. This is in stark contrast to the canonical transport profiles determined for the well-characterized S. cerevisiae NCS1 proteins FUR4 (uracil transport) or FCY2 (adenine, guanine, and cytosine transport).
