Figure 2. CDPK4 activity facilitates the assembly of the pre-replicative complex during microgametogenesis.

(A) GO term enrichment analysis of proteins co-immunoprecipitated with CDPK4-3xHA in non-activated gametocytes reveals the kinase is interacting with proteins of the pre-replicative complex. Bonferroni corrected p-values are indicated. (B) 178 proteins are immunoprecipitated with both CDPK4-3xHA and MCM5-3xHA including all components of the ORC and the MCM complex. The relative abundance of proteins was determined as the number of spectral counts for each protein divided by the number of total spectral counts in the respective immunoprecipitate. (C) The interaction between CDPK4-3xHA and MCM2-7/Cdt1 proteins is stable during the first 15 s following XA stimulation while interaction with ORC1-5/Cdc6 proteins is increased when the kinase is activated. Data are representative of two independent biological replicates. (D) MCM5-3xHA is enriched in chromatin-enriched NaCl fractions at 15 s post-activation in gametocytes but not when 1294 is added; α-tubulin was used as a soluble control. Error bars show standard deviations, n = 3, ** Student's T-test, p≤0.01.

DOI: http://dx.doi.org/10.7554/eLife.26524.005

Figure 2—figure supplement 1. CDPK4 activity is required to assemble the pre-replicative complex.

(1) Complementation of a CDPK4-KO line with a CDPK4-2xmyc allele. (2) A transgenic line expressing CDPK4-2xmyc does not show exflagellation defects. Error bars are standard deviations from two biological replicates. (3) Western blot and silver staining analysis of CDPK4-3xHA immunoprecipitation. Eluates show specific isolation of the 3xHA fusion protein. Molecular weights are indicated in kDa. hc = heavy chain, lc = light chain. (4) Generation and genotyping of a non-clonal line expressing MCM5-3xHA. (5) Western blot analysis of MCM5-3xHA lysates produced from non-activated gametocytes. (6) CDPK4-3xHA is incorporated in the exflagellated male gamete. Scale bar is 1 μm. (7) Relative abundance of proteins co-immunoprecipitated with CDPK4-3xHA in non-activated gametocytes compared with 15 second-activated gametocytes in presence or absence of 1 µM 1294. The six components of the ORC/cdc6 complex show a significant increase in abundance when CDPK4 is active. (8) Relative abundance of proteins co-immunoprecipitated with CDPK4-2xmyc in non-activated gametocytes compared with 15 second-activated gametocytes in presence or absence of 1 µM 1294.

DOI: http://dx.doi.org/10.7554/eLife.26524.006