Quaternary Structure and Lipid Affinity of PLC Figure 4. Cys7 plays critical role in CrPLC dimer stabilization. (A) Chromatogram of different CrPLC mutants indicated in different colors, overlaid with wild type CrPLC (CrPLC WT) shown in black. (B) SDS-PAGE analysis of eluents obtained at 70 ml and 82 ml with preparatory size exclusion chromatography of CrPLC WT and CrPLC C7A mutant under reducing (+DTT) and non-reducing (2DTT) conditions, respectively. (C and D) Immunoblot analysis of CrPLC WT and CrPLC C7A mutant protein respectively, under reducing (+DTT) and non-reducing (2DTT) conditions. Lanes are labeled by elution volume of monomer and dimer peak fractions obtained by preparatory size exclusion chromatography. M denotes the marker. doi:10.1371/journal.pone.0039258.g004 CrPLC Mainly Localizes to the Plasma Membrane in C. reinhardtii Phospholipase has been proposed to function at the plasma membrane for regulation of different signaling cascades [29,30]. Overall, cellular localization of CrPLC in C. reinhardtii cell displayed the preferential localization of CrPLC to the plasma membrane (Figures 6A and B). Labeling of the cells with the membrane tracer FM 4-64 (Figure 6C) suggested the co- localization of CrPLC with the plasma membrane and sub- cellular membrane vesicle compartment. This preferential localization might be due to high lipid affinity of CrPLC. Co- localization of CrPLC with the membrane of sub-cellular compartment does not rule out the possibility of translocation of CrPLC protein via sub-cellular membrane vesicle compart- ments to the plasma membrane. The co-localization signal of CrPLC with the membrane periphery and absence of such signal with preimmune serum or in autofluorescence images, confirmed its association mainly with the plasma membrane (Figures 6D– G). Membrane association of CrPLC could be essential for C. reinhardtii cell as it provides its access and availability to interacting partners. It is required to elucidate the functional importance of cellular localization of CrPLC after which the importance of the localization in the plasma membrane may become more obvious. PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e39258