Glutaraldehyde Crosslinking Protein crosslinking was performed by using 2.5% (v/v) freshly prepared glutaraldehyde (Sigma). 25 mM of heterologously expressed CrPLC or total cell protein of C. reinhardtii dissolved in 60 ml of PBS was incubated with 5 ml of 2.5% glutaraldehyde at 37uC. Reactions were stopped after 5 min by adding 1 M Tris-Cl, pH 8. Crosslinked protein samples were resolved on 10% SDS- PAGE and detected by immunoblotting. Immunolocalization of CrPLC in Chlamydomonas Cells in early log phase were harvested and resuspended in PBS. Aliquots of approximately 200 ml of cell suspension were seeded on acid washed coverslips and fixed with 3.7% paraformaldehyde in PBS. Algal cells on the slide were permeabilized by submerging them in cold 100% ethanol at 220uC for 10 min. Cells were then washed with PBS containing 0.25 M NaCl at room temperature for 10 min, followed by PBS for 5 min. After brief washings with PBS containing 0.5% triton X-100 (PBST), incubation with freshly diluted rabbit CrPLC antiserum (1:125 dilutions) or preimmune serum was carried out overnight at 4uC. Samples were incubated with FITC conjugated anti-rabbit IgG secondary antibody. Followed by brief washings, coverslips were mounted on slides applying antifade reagent (Slow Fade Gold, Molecular Probes). Slides were visualized by Leica TCS SP5 confocal microscope at central instrument facility, University of Delhi South Campus. Plasma membrane was labeled by tracker dye FM 4-64 (Invitrogen) at concentration 5 mg/ml. Lipid Overlay Assay Lipid overlay assay was conducted using commercial PIP strips membranes (Molecular probes) [15] with spotted blots of synthetic phospholipids. Membrane was blocked with 1% skimmed milk in PBS buffer supplemented with 0.1% tween-20 for 1 hour at room temperature. The membrane was then incubated overnight on rocker with 2 mg/ml of recombinant CrPLC protein dissolved in PBST buffer, washed three times with PBST buffer for 10 min. Bound protein was then detected by standard immunoblotting as discussed earlier. Densitometry of blots was performed by Image J software (http://www.macbiophotonics.ca/imagej). CD Spectroscopy Affinity purified CrPLC WT and C7A mutant were further purified by size exclusion chromatography against 10 mM Sodium Phosphate buffer pH 7.4. All the Far UV-CD measurements were performed in Jasco J815 spectrophotometer using quartz cuvette with the path length of 0.1 cm at room temperature. Spectra of each protein for protein concentration 0.2 mg/ml were recorded between 190–260 nm with the 1 nm band width and scan speed of 50 nm/min. Each spectrum was averaged over three scans and corrected by subtracting buffer baseline. The fractional a-helical, b-sheet, turns and random coil content of CrPLC WT and mutant protein was calculated using Rausseans et al method [31]. References 1. Blazer-Yost BL, Nofziger C (2005) Phosphoinositide lipid second messengers: new paradigms for transepithelial signal transduction. Pflugers Arch 450: 75–82. 2. Miao B, Skidan I, Yang J, Lugovskoy A, Reibarkh M, et al. (2010) Small molecule inhibition of phosphatidylinositol-3,4,5-triphosphate (PIP3) binding to pleckstrin homology domains. Proc Natl Acad Sci U S A 107: 20126–20131. 3. Berridge MJ (1987) Inositol trisphosphate and diacylglycerol: two interacting second messengers. Annu Rev Biochem 56: 159–193. 4. Hokin MR, Hokin LE (1953) Enzyme secretion and the incorporation of P32 into phospholipides of pancreas slices. J Biol Chem 203: 967–977. 5. Kaftan EJ, Ehrlich BE, Watras J (1997) Inositol 1,4,5-trisphosphate (InsP3) and calcium interact to increase the dynamic range of InsP3 receptor-dependent calcium signaling. J Gen Physiol 110: 529–538. Quaternary Structure and Lipid Affinity of PLC Supporting Information Figure S1 Phylogenetic analysis of CrPLC. Phylogenetic analysis comparing C. reinhardtii phospholipase C (CrPLC) with other PLC isoforms including sequences (accession numbers are given in brackets): Homo sapiens (AAH10668.2), Mus Musculus (CAM22088.1), Catfish (AAA87954.1), Zea Mays (ACG25330.1), Oryza sativa (ABA98951.2), Nicotiana tabacum (ABP57375.1), Dro- sophila melanogaster (ACZ95198.1), Arabidopsis thaliana (AAN75042.1), Ostreococcus lucimarinus (XP_003080695.1). The number at each branch point represents the bootstrap probability. (TIF) Figure S2 Predicted tertiary structure of wild type CrPLC. (A) Cartoon representation of the 3D structure of the CrPLC modeled using automated Swiss model server. (B) Position of aromatic amino acid and cysteine residues present on the surface of the molecule are shown in blue and red respectively. (TIF) Figure S3 SDS-PAGE analysis of recombinant CrPLC. Purification profile of the recombinant CrPLC purified by immobilized metal affinity chromatography and resolved on 10% SDS-PAGE. (TIF) Figure S4 Immunoblot analysis of dimer and monomer fractions of CrPLC. Separated monomer and dimer fractions purified by size exclusion chromatography corresponding to elution volume 67–76 ml and 79–88 ml respectively immuno- blotted with CrPLC specific antibody. (TIF) Figure S5 Immunoblot analysis of monomer and dimer fractions of wild type and various mutants of CrPLC under reducing and non-reducing conditions. (A–E) Immunoblotting of both dimer (right panel) and monomer elution fractions (left panel) of CrPLC wild type and mutants under reducing (+DTT) and non-reducing (2DTT) conditions with CrPLC specific antibody. (TIF) Acknowledgments We would like to thank Dr. Sindhu K.V for her scientific advice and critical discussion. All lab members are also acknowledged for the critical reading of manuscript. We thank CIF UDSC staff especially for sequencing, CD spectroscopy and confocal microscopy studies. Author Contributions Conceived and designed the experiments: SK. Performed the experiments: MA JB. Analyzed the data: SK MA JB. Contributed reagents/materials/ analysis tools: MA JB. Wrote the paper: SK MA JB. 6. Dabdoub A, Payne R (1999) Protein kinase C activators inhibit the visual cascade in Limulus ventral photoreceptors at an early stage. J Neurosci 19: 10262–10269. 7. Eichwald C, Kaiser F (1993) Model for receptor-controlled cytosolic calcium oscillations and for external influences on the signal pathway. Biophys J 65: 2047–2058. 8. Berridge MJ, Bootman MD, Roderick HL (2003) Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol 4: 517–529. 9. Rhee SG, Bae YS (1997) Regulation of phosphoinositide-specific phospholipase C isozymes. J Biol Chem 272: 15045–15048. PLoS ONE | www.plosone.org 10 June 2012 | Volume 7 | Issue 6 | e39258