Abstract
Malaria parasites are protists of the genus Plasmodium, whose transmission to mosquitoes is initiated by the production of gametes. Male gametogenesis is an extremely rapid process that is tightly controlled to produce eight flagellated microgametes from a single haploid gametocyte within 10 minutes after ingestion by a mosquito. Regulation of the cell cycle is poorly understood in divergent eukaryotes like Plasmodium, where the highly synchronous response of gametocytes to defined chemical and physical stimuli from the mosquito has proved to be a powerful model to identify specific phosphorylation events critical for cell-cycle progression. To reveal the wider network of phosphorylation signalling in a systematic and unbiased manner, we have measured a high-resolution time course of the phosphoproteome of P. berghei gametocytes during the first minute of gametogenesis. The data show an extremely broad response in which distinct cell-cycle events such as initiation of DNA replication and mitosis are rapidly induced and simultaneously regulated. We identify several protein kinases and phosphatases that are likely central in the gametogenesis signalling pathway and validate our analysis by investigating the phosphoproteomes of mutants in two of them, CDPK4 and SRPK1. We show these protein kinases to have distinct influences over the phosphorylation of similar downstream targets that are consistent with their distinct cellular functions, which is revealed by a detailed phenotypic analysis of an SRPK1 mutant. Together, the results show that key cell-cycle systems in Plasmodium undergo simultaneous and rapid phosphoregulation. We demonstrate how a highly resolved time-course of dynamic phosphorylation events can generate deep insights into the unusual cell biology of a divergent eukaryote, which serves as a model for an important group of human pathogens.
