RESUMO
ADP-glucose synthesis through ADP-glucose pyrophosphorylase defines the major rate-controlling step of storage polysaccharide synthesis in both bacteria and plants. We have isolated mutant strains defective in the STA6 locus of the monocellular green alga Chlamydomonas reinhardtii that fail to accumulate starch and lack ADP-glucose pyrophosphorylase activity. We show that this locus encodes a 514-amino-acid polypeptide corresponding to a mature 50-kDa protein with homology to vascular plant ADP-glucose pyrophosphorylase small-subunit sequences. This gene segregates independently from the previously characterized STA1 locus that encodes the large 53-kDa subunit of the same heterotetramer enzyme. Because STA1 locus mutants have retained an AGPase but exhibit lower sensitivity to 3-phosphoglyceric acid activation, we suggest that the small and large subunits of the enzyme define, respectively, the catalytic and regulatory subunits of AGPase in unicellular green algae. We provide preliminary evidence that both the small-subunit mRNA abundance and enzyme activity, and therefore also starch metabolism, may be controlled by the circadian clock.
Assuntos
Chlamydomonas reinhardtii/genética , Mutação , Nucleotidiltransferases/genética , Amido/biossíntese , Sequência de Aminoácidos , Animais , Relógios Biológicos , Fusão Celular , Chlamydomonas reinhardtii/enzimologia , Ritmo Circadiano , Clonagem Molecular , Glucose-1-Fosfato Adenililtransferase , Dados de Sequência Molecular , Nucleotidiltransferases/classificação , Filogenia , Poliploidia , Estrutura Quaternária de Proteína , Protoplastos , RNA Mensageiro/análise , RNA de Plantas/análise , Seleção Genética , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
In Chlamydomonas, as in higher plants, synthesis of ADP glucose catalyzed by ADP-glucose pyrophosphorylase is rate-limiting for the building of starch in the chloroplast. We have isolated disruptions of the STA1 ADP-glucose pyrophosphorylase structural gene that rendered the enzyme less responsive to the allosteric activator 3-phosphoglycerate. The structure and composition of the residual starch synthesized by all mutants of the STA1 locus is dramatically altered. The residual polysaccharide is shown to be devoid of amylose despite the presence of granule-bound starch synthase, the amylose biosynthetic enzyme. In addition, the fine structure of the mutant amylopectin revealed the presence of an altered chain-length distribution. This distribution mimicks that which is observed during growth and photosynthesis and differs markedly from that observed during storage. We therefore propose that low nucleotide sugar concentrations are either directly or indirectly responsible for the major differences observed in the composition or structure of starch during storage and photosynthesis.
