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Virulence of apicomplexan parasites is based on their ability to divide rapidly to produce significant biomass. The regulation of their cell cycle is therefore key to their pathogenesis. Phosphorylation is a crucial posttranslational modification that regulates many aspects of the eukaryotic cell cycle. The phosphatase PP1 is known to play a major role in the phosphorylation balance in eukaryotes. We explored the role of TgPP1 during the cell cycle of the tachyzoite form of the apicomplexan parasite Toxoplasma gondii. Using a conditional mutant strain, we show that TgPP1 regulates many aspects of the cell cycle including the proper assembly of the daughter cells' inner membrane complex (IMC), the segregation of organelles, and nuclear division. Unexpectedly, depletion of TgPP1 also results in the accumulation of amylopectin, a storage polysaccharide that is usually found in the latent bradyzoite form of the parasite. Using transcriptomics and phospho-proteomics, we show that TgPP1 mainly acts through posttranslational mechanisms by dephosphorylating target proteins including IMC proteins. TgPP1 also dephosphorylates a protein bearing a starch-binding domain. Mutagenesis analysis reveals that the targeted phospho-sites are linked to the ability of the parasite to regulate amylopectin steady-state levels. Therefore, we show that TgPP1 has pleiotropic roles during the tachyzoite cell cycle regulation, but also regulates amylopectin accumulation. Rapid division and biomass production are important for apicomplexan parasite virulence. This study shows that the phosphatase TgPP1 in Toxoplasma gondii regulates the cell cycle and amylopectin accumulation by dephosphorylating inner membrane complex and starch-binding domain proteins. [ABSTRACT FROM AUTHOR]

The endosymbiosis event resulting in the plastid of photosynthetic eukaryotes was accompanied by the appearance of a novel form of storage polysaccharide in Rhodophyceae, Glaucophyta, and Chloroplastida. Previous analyses indicated that starch synthesis resulted from the merging of the cyanobacterial and the eukaryofic storage polysaccharide metabolism pathways. We performed a comparative bioinformatic analysis of six algal genome sequences to investigate this merger. Specifically, we analyzed two Chlorophyceae, Chlamydomonas reinhardtii and Volvox carterii, and four Prasinophytae, two Ostreococcus strains and two Micromonas pusilla strains. Our analyses revealed a complex metabolic pathway whose intricacies and function seem conserved throughout the green lineage. Comparison of this pathway to that recently proposed for the Rhodophyceae suggests that the complexity that we observed is unique to the green lineage and was generated when the latter diverged from the red algae. This finding corresponds well with the plastidial location of starch metabolism in Chloroplastidae. In contrast, Rhodophyceae and Glaucophyta produce and store starch in the cytoplasm and have a lower complexity pathway. Cytoplasmic starch synthesis is currently hypothesized to represent the ancestral state of storage polysaccharide metabolism in Archaeplastida. The retargeting of components of the cytoplasmic pathway to plastids likely required a complex stepwise process involving several rounds of gene duplications. We propose that this relocation of glucan synthesis to the plastid facilitated evolution of chlorophyll containing light-harvesting complex antennae by playing a protective role within the chloroplast.

To understand the biological function of bacterial glycogen phosphorylase (GIgP), we have produced and characterized Escherichia coli cells with null or altered glgP expression, glgP deletion mutants ([DELTA]glgP) totally lacked glycogen phosphorylase activity, indicating that all the enzymatic activity is dependent upon the glgP product. Moderate increases of glycogen phosphorylase activity were accompanied by marked reductions of the intracellular glycogen levels in cells cultured in the presence of glucose. In turn, both glycogen content and rates of glycogen accumulation in [DELTA]glgP cells were severalfold higher than those of wild-type cells. These defects correlated with the presence of longer external chains in the polysaccharide accumulated by [DELTA]glgP cells. The overall results thus show that GIgP catalyzes glycogen breakdown and affects glycogen structure by removing glucose units from the polysaccharide outer chains in E. coli.

A role for the Escherichia coil glgX gene in bacterial glycogen synthesis and/or degradation has been inferred from the sequence homology between the glgX gene and the genes encoding isoamylase-type debranching enzymes; however, experimental evidence or definition of the role of the gene has been lacking. Construction of E. coil strains with defined deletions in the glgX gene is reported here. The results show that the GlgX gene encodes an isoamylase-type debranching enzyme with high specificity for hydrolysis of chains consisting of three or four glucose residues. This specificity ensures that GlgX does not generate an extensive futile cycle during glycogen synthesis in which chains with more than four glucose residues are transferred by the branching enzyme. Disruption of glgX leads to overproduction of glycogen containing short external chains. These results suggest that the GlgX protein is predominantly involved in glycogen catabolism by selectively debranching the polysaccharide outer chains that were previously recessed by glycogen phosphorylase.