Cellular localization of CrPLC.

<p>(A) CrPLC localizes in the plasma membrane of <i>C. reinhardtii</i> cell as shown by green channel (B) Magnified image of a single cell. (C) Plasma membrane traced by using FM4-64 tracker dye is shown in red. (D) Merged image represent the overlay, where green and red channels are merged together to confirm CrPLC localization in the plasma membrane. (E) Cells visualized in DIC mode of light microscopy. (F and G) Immunolocalization with pre-immune serum and auto-fluorescence served as negative control.</p

Homology analysis of PI-PLC isozymes from different organisms.

<p>Sequences of Phospholipase C from different organisms including <i>Chlamydomonas reinhatdtii</i> (CrPLC), <i>Drosophila melanogaster</i> (DmPLC), <i>Arabidopsis thaliana</i> (AtPLC), <i>Homo sapiens</i> (HsPLC), and <i>Mus musculus</i> (MmPLC) were compared. Identical amino acid residues are represented in white with black background and residues with greater than 85% similarity in sequences are highlighted in black with grey background. Residues present in the X–Y domain and catalytic domain are indicated by black and grey solid bars respectively. Catalytically important and conserved amino acid residues for substrate binding are marked by solid black circles.</p

CrPLC dimerizes both <i>in vitro</i> and <i>in vivo</i>.

<p>(A) Immunoblotting of crosslinked (Glutaraldehyde; GA) dimer and monomer peak fractions. Numbers above each lane are the elution volume in ml with 70 ml (dimer peak fraction) and 82 ml (monomer peak fraction). (B) Cellular detection of CrPLC dimer and monomer species by glutaraldehyde (GA) crosslinking of <i>Chlamydomonas</i> total cell protein (Cr TCL) followed by immunoblotting. (C) Immunoblot analysis of CrPLC dimer and monomer peak fractions of size exclusion chromatography in both presence (reducing) and absence (non-reducing) of dithiothreitol (DTT). (D) Comparative chromatogram of recombinant CrPLC performed under reducing condition using 5 mM DTT (black) and in non-reducing condition (red). (E) Cellular detection of CrPLC in <i>Cr</i> TCL under reducing and non-reducing conditions. Dimer and Monomer in the blots are marked as D and M respectively.</p

Quaternary structures of recombinant CrPLC.

<p>(A) Size exclusion chromatogram of recombinant CrPLC. Straight line depicts the calibration of standard molecular weight marker including, thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B12 (1.35 kDa). (B) Size-exclusion profile of CrPLC of various concentrations. Elution profiles of CrPLC corresponding to various concentrations of recombinant CrPLC (5, 10 and 50 µM) were recorded using analytical grade size exclusion chromatography column and shown in different line patterns as mentioned in the inset of figure. (C and D) Size exclusion chromatogram of reloaded monomer and dimer fractions using preparatory grade chromatography column are shown in black, overlaid with chromatogram of CrPLC (dotted line).</p