Disulphide Bridges of Phospholipase C of Chlamydomonas reinhardtii Modulates Lipid Interaction and Dimer Stability Mayanka Awasthi, Jyoti Batra, Suneel Kateriya* Department of Biochemistry, University of Delhi, South Campus, New Delhi, India Abstract Background: Phospholipase C (PLC) is an enzyme that plays pivotal role in a number of signaling cascades. These are active in the plasma membrane and triggers cellular responses by catalyzing the hydrolysis of membrane phospholipids and thereby generating the secondary messengers. Phosphatidylinositol-PLC (PI-PLC) specifically interacts with phosphoino- sitide and/or phosphoinositol and catalyzes specific cleavage of sn-3- phosphodiester bond. Several isoforms of PLC are known to form and function as dimer but very little is known about the molecular basis of the dimerization and its importance in the lipid interaction. Principal Findings: We herein report that, the disruption of disulphide bond of a novel PI-specific PLC of C. reinhardtii (CrPLC) can modulate its interaction affinity with a set of phospholipids and also the stability of its dimer. CrPLC was found to form a mixture of higher oligomeric states with monomer and dimer as major species. Dimer adduct of CrPLC disappeared in the presence of DTT, which suggested the involvement of disulphide bond(s) in CrPLC oligomerization. Dimer-monomer equilibrium studies with the isolated fractions of CrPLC monomer and dimer supported the involvement of covalent forces in the dimerization of CrPLC. A disulphide bridge was found to be responsible for the dimerization and Cys7 seems to be involved in the formation of the disulphide bond. This crucial disulphide bond also modulated the lipid affinity of CrPLC. Oligomers of CrPLC were also captured in in vivo condition. CrPLC was mainly found to be localized in the plasma membrane of the cell. The cell surface localization of CrPLC may have significant implication in the downstream regulatory function of CrPLC. Significance: This study helps in establishing the role of CrPLC (or similar proteins) in the quaternary structure of the molecule its affinities during lipid interactions. Citation: Awasthi M, Batra J, Kateriya S (2012) Disulphide Bridges of Phospholipase C of Chlamydomonas reinhardtii Modulates Lipid Interaction and Dimer Stability. PLoS ONE 7(6): e39258. doi:10.1371/journal.pone.0039258 Editor: Eric Cascales, Centre National de la Recherche Scientifique, France Received February 21, 2012; Accepted May 22, 2012; Published June 21, 2012 Copyright: 2012 Awasthi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was carried out with the financial support of the DBT (http://dbtindia.nic.in/index.asp), Govt. of India to SK (BT/PR9090/BRB/10/540/2007) for carrying basic research. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: skateriya@south.du.ac.in Introduction Phosphoinositide (PIP3) has been shown to affect a great variety of cellular responses, including exocytosis, cytoskeleton remodel- ing, chemotaxis and regulation of ion channels [1], and its metabolism inside the cell is stringently controlled [2]. Phospho- inositide-specific phospholipase C (PI-PLC) is the class of enzymes that hydrolyzes the highly phosphorylated phosphatidylinositol 4, 5-bisphosphate [PI(4,5)P2], generating two intracellular products inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG) [3]. This event serves as one of the earliest key steps to trigger further phosphoinositide-mediated intracellular signal transduction path- ways [4]. InsP3 is a universal calcium-mobilizing secondary messenger [5] and DAG is an activator of protein kinase C [6,7]. Both signaling molecules modulate intracellular Ca2+ level. Activated protein kinase C regulates a wide variety of downstream effectors [8]. PLCs are soluble and membrane associated multi- domain proteins that are classified into different isoforms like b, c, d, f, g and e, on the basis of their primary structure and mechanism of their activation [9]. PLCs harboring single domain also exist in nature [10]. Different domains of PLC constitutes catalytic a/b barrel including X and Y region, which represents the incomplete TIM (triose phosphate isomerase) barrel fold, hydrophobic rim region, X/Y-spanning sequence (Z Region), pleckstrin homology (PH) domain, EF-hands, C2 domain and C- terminus extension [11]. These modular domains of PLCs are known to regulate lipid interaction and/or enzyme activity [12,13,14,15]. Dimerization is one of the known molecular behaviours of phospholipases, which also plays a role in regulation of PLC activity. Some isoforms of PLC are known to exist and function as dimers, such as purified PLC from human platelets [16]. In 1994, Banno et. al. suggested that size exclusion chromatography purified PLC-b-dimers are catalytically active enzymes [17]. Crystal structure of avian PLC-b reveals that C-tail is composed of three long helices, forming a coiled-coil structure, which controls dimerization of the enzyme in an antiparallel orientation [18]. PLoS ONE | www.plosone.org 1 June 2012 | Volume 7 | Issue 6 | e39258