Evolutionary proteomics uncovers ciliary signaling components

Monika Abedin Sigg; Tabea Menchen; Jeffery Johnson; Chanjae Lee; Semil P. Choksi; Galo Garcia; Henriette Busengdal; Gerard Dougherty; Petra Pennekamp; Claudius Werner; Fabian Rentzsch; Nevan Krogan; John B. Wallingford; Heymut Omran; Jeremy F. Reiter

To obtain a sufficient amount of material for mass spectrometry of choanoflagellate cilia, 169 we performed two separate cilia preparations and pooled the samples before analysis. The 170 sample was fractionated by high resolution reverse phase before mass spectrometry. To identify 171 proteins selectively enriched in choanoflagellate flagella, we analyzed fractions containing cell 172 bodies (fraction 1) and microvilli (fraction 10) alongside the ciliary fractions (fractions 4 and 5) 173 ( Figure 1G ). We defined proteins with a ciliary fraction peptide count higher than that of the cell 174 body and microvilli fraction as candidate members of the choanoflagellate ciliary proteome, or 175 'ciliome '. 176 Metazoan developmental signaling pathways are proposed to have emerged with the 177 advent of multicellularity or with major transitions in animal evolution (Pincus et al., 2008; 178 Pires-daSilva and Sommer, 2003). Many signal transduction components are membrane-179 associated, suggesting that the membrane plus matrix fraction of sea urchin cilia would be 180 enriched for signal transduction components relative to the axonemal fraction. Therefore, we 181 calculated the degree of membrane plus matrix enrichment for each sea urchin protein by 182 comparing its peptide count in the membrane plus matrix fraction to that of the axonemal 183 fraction ( Figure 2C and Table S2 ). 184