1 Identification of Protein Interactomes from the green chlorophyte Chlamydomonas reinhardtii# *Jacinta S. D’Souza, Venkatramanan G. Rao, Dolly K. Khona, Sirisha V. L., Kanak Gawade and Marilyn P. Sequeira UM-DAE Centre for Excellence in Basic Sciences, Kalina campus, Santacruz (E), Mumbai *Author for Correspondence: (# Work funded by DAE, India) Introduction: The whole set of molecular interactions in cells is called Interactome and its study is called interactomics. It can occur between molecules belonging to different biochemical families (proteins, nucleic acids, lipids, carbohydrates) and also within a given family. The size of an organism's interactome correlates better than genome size with the biological complexity of the organism (Stumpf et al., 2008). Because the interactome considers the whole organism, there is a need to collect a massive amount of information. Thus far, two types of protein interactomes have been observed; Stable and Transient. Stable interactomes are already present in the cell in its active or inactive form. On the other hand, transient interactomes are those that are made freshly when the need arises. Among these pool, proteins that interact with each other at a given point in time and circumstances is called the interactome. We have been using the easy to grow, unicellular, biflagellated, green alga, Chlamydomonas reinhardtii as a model organism for the identification of both the stable and transient interactomes. The ‘9+2’ cilia seems to harbour several stable interactomes and one such interactome has been explored using a protein (FAP174) as a bait. G Identification of a stable interactome from cilia: Using the ciliary proteome map (Pazour et al, 2005) as a guide, a MYCBP-1 homolog (FAP174) from Chlamydomonas reinhardtii was identified, isolated, cloned, His-tagged protein expressed, purified and antibodies raised. A purified polyclonal antibody was used to localize FAP174 protein to the cilia. MYCBP-1 has been shown to be an AKAP interactor. The cilia harboring two AKAPs; one in the radial spokes and the other in the central pair, an AKAP interaction with RSP3 (Radial Spoke AKAP) full-length protein and the RIIBD domain was established (A-D) using modified pull-down assays. In order to establish its interaction with the CP AKAP, a far-western technique using the RII domain from the regulatory subunit of PKA (courtesy of Susan Taylor, HHMI, UCSD) and FAP174 was carried out on ciliary axonemes (E-F). In order to identify other interactors, an immunoprepcipitation (G) was carried out using anti-FAP174 antibody and the polypeptides analyzed using MALDI-TOF. IP No. Protein MW (kDa) Domain IP1 AKAP240 265 Hydin like and MYCBPAP like IP2 A/G kinase 260 Adenylate Guanylate kinase domains IP3 FAP75 139 Nucleotide kinase family IP4 CPC1 Adenylate kinase domain/EF hands and calponin superfamily IP5 FAP70 124 Tertratricopeptide repeats IP6 FAP147 100 MYCBPAP IP7 NS 89 No significant match IP8 HSP70 70 HSP domain The alongside Table lists the proteins that were immunoprecipitated using an anti-FAP174 antibody from the partially purified preparation of central pair apparatus. Since FAP174 is localized in the C2b projection, an interactome of ~1.5 mDa has been projected in the cartoon. In search of a Transient Interactome: Chlamydomonas reinhardtii cells when exposed to various stress agents, responded differentially. When exposed independently to H2O2, menadione or KCl, it underwent PCD; while to high concentrations of NaCl (200 to 800 mM), it underwent necrosis. On the other hand, when exposed to low NaCl concentrations (100 and 150 mM), it exhibited dormant physiology termed as palmelloids. The hallmarks of PCD, necrosis and palmelloidy have been established vis-à-vis these stress agents and these paradigms are being investigated for the presence of transient interactomes. The evidences for these responses, Palmelloidy (Panels A-I) and PCD (Panels J-K) are depicted alongside. D E F G H I A B C D Refs: Pazour GJ, Agrin N, Leszyk J, Witman GB. JCB (2005) 170: Stumpf MP, Thorne T, de Silva E, Stewart R, An HJ, Lappe M, Wiuf C. PNAS (2008) 105: Cells exposed to 100 or 150 mM NaCl showed increased clustering with time (A, arrows). Increased vacuolation, remnants of flagella (C-D), accumulation of starch (D-E), lipid (F-G) and exopolysaccharide (H-I) were observed features of palmelloidy. When exposed to menadione, H2O2 or KCl, characteristic hallmarks of apoptosis were observed – one of which is depicted here (J-L), the others being cell death, TUNEL positivity, disruption of MMP and caspase-3-like enzyme activation (data not shown). Since these responses are induced by an external stimuli, it is expected that transient interactomes are assembled/disassembled in these situations. Current experiments are designed towards identifying these transient interactomes specific to each response.