Figure 5. Functional characterisation of three identified substrates of CDPK4.
(A) A SOC1-KO line shows a strong reduction in exflagellation (n = 3). SOC1 is required only for the 1N/2N transition as parasites reaching the 2N level were able to further progress to the octoploid state (n = 2). Error bars show standard deviations, *** Student's T-test, p≤0.001. (B) SOC1-3xHA shows a diffuse cytoplasmic distribution (scale bar = 2 µm) but interacts with CDPK4 and proteins of the MCM complex. (C) SOC2-KO line is strongly impaired for exflagellation (n = 3). SOC2 is not required for the 1N/2N transition but for each following transition indicating it is not involved in the initiation of DNA replication nor for DNA replication per se but most likely during the three successive endomitoses (n = 2). Consistently, a strong reduction in parasites exhibiting mitotic spindles at 1 min post-activation is observed in the SOC2-KO line (n = 2). However no defect in axoneme assembly could be detected (n = 2). Error bars show standard deviations, *** Student's T-test, p≤0.001. (D) SOC2 is predicted to be a 645 kDa polytopic protein. Immunoprecipitation of SOC2-3xHA recovers peptides covering the last 1707 a.a. only; corresponding unique spectral counts are indicated in pink. Similarly co-immunoprecipitation of CDPK4 only recovered peptides covering the last 1137 a.a. of SOC2; corresponding unique spectral counts are indicated in blue. The C-terminal fragment colocalises with mitotic spindle α-tubulin but not with peripheral α-tubulin. Scale bar = 2 µm. (E) SOC3 is not required for DNA replication (n = 2) and axoneme assembly (n = 2) but is essential for exflagellation (n = 3). Error bars show standard deviations, *** Student's T-test, p≤0.001. (F) SOC3 colocalises with axonemal α-tubulin in exflagellating gametes. Scale bar = 2 µm.
Figure 5—figure supplement 1. Characterisation of three identified substrates of CDPK4.
