Molecular Mechanism of Phosphoinositide-specific Phospholipase C
We demonstrated that the C2 domain alone is capable of targeting plant PLC to the membrane in response to a Calcium signal. We have deciphered the mechanism by which plant Phospholipase c (PLC) upon stimulation by calcium bring about the hydrolysis of its substrate and produce second messenger. With the help of fluorescence spectroscopy, we have demonstrated that in presence of calcium the surface hydrophobicity increases in C2 domain, this initiate membrane targeting of PLC. We have also identified two conserved motifs in known plant PLC sequences, which are specific to plant PLC and therefore can be used to annotate plant PLC. We have also provided evidence that plant PLC are soluble and are targeted to the membrane upon stimulation by calcium.
Regulation of Lipid Metabolism by Phosphoinositide-specific Phospholipase C
We provide evidence for the regulation of lipid biosynthesis by phosphatidylinositol-specific phospholipase C (PLC) through UASino and two trans-acting elements. Gene expression analysis and radiolabeling experiments demonstrated that the overexpression of rice PLC in yeast cells altered phospholipid biosynthesis at the levels of transcriptional and enzyme activity. This is the first report, implicating PLC in the direct regulation of lipid biosynthesis. Taken together, these data suggest that regulation by PLC appears to be a mechanism for controlling the carbon flux toward phospholipid and sphingolipid synthesis by regulating either phosphatidic acid synthesis or the expression of genes involved in the utilization of phosphatidic acid.
Bifunctional Oleosin is regulated by phosphorylation, a regulator of lipid metabolic functions.
Triacylglycerol (TAG) is an important neutral
lipid molecule that serves as the primary mechanism of fuel storage
in eukaryotes. In plants, fatty oils are generally stored in spherical
intracellular organelles referred to as oleosomes that are covered by
proteins such as oleosin. We have isolated a catalytically active detergent
resistance, 14 S multiprotein complex capable of acylating monoacylglycerol
(MAG) from the microsomal membranes of developing peanut cotyledons
and oleosin3 as a part of the complex. The recombinant OLE3 microsomes
from Saccharomyces cerevisiae have been shown to have both a
monoacylglycerol acyltransferase (MGAT) and a phospholipase A2 activity.
A serine/threonine/tyrosine protein kinase phosphorylates oleosin. Using
bimolecular fluorescence complementation analysis, we demonstrate that
this kinase interacts with OLE3 and the fluorescence was associated
with chloroplasts. Oleosin3-green fluorescent protein fusion protein
exclusively associated with the chloroplasts. This is the first time
our laboratory had shown that oleosin3 protein targeted to chloroplast.
Phosphorylated OLE3 exhibited reduced monoacylglycerol acyltransferase
and increased phospholipase A2 activities. Phosphorylation levels of
OLE3 during seed germination were determined to be higher than in developing
peanut seeds. Overexpression of the oleosin3 (OLE3) gene in S. cerevisiae
resulted in an increased accumulation of diacylglycerol and triacylglycerols
and decreased phospholipids. These findings provide a direct role for
a structural protein (OLE3) in the biosynthesis and mobilization of
Regulation of lipid biosynthesis by phosphatidylinositol-specific phospholipase C through the transcriptional repression of upstream activating sequence inositol containing genes. Sunny D. Rupwate, Preeti S. Rupwate, Ram Rajasekharan. DOI: 10.1016/j.febslet.2012.04.022 published online 23 April 2012.
Serine/Threonine/Tyrosine protein kinase phosphorylates oleosin, a regulator of lipid metabolic functions. Parthibane V, Iyappan R, Vijayakumar A, Venkateshwari V, Rajasekharan R. Plant Physiol. 2012 May;159(1):95-104. Epub 2012 Mar 20.
Oleosin is bifunctional enzyme that has both monoacylglycerol acyltransferase and phospholipase activities. Parthibane V, Rajakumari S, Venkateshwari V, Iyappan R, Rajasekharan R. J Biol Chem. 2012 Jan 13;287(3):1946-54. Epub 2011 Nov 29.
C2 domain is responsible for targeting rice phosphoinositide specific phospholipase C. Rupwate SD, Rajasekharan R. Plant Mol Biol. 2012 Feb;78(3):247-58. Epub 2011 Nov 29.
Defective in cuticular ridges (DCR) of Arabidopsis thaliana, a gene associated with surface cutin formation, encodes a soluble diacylglycerol acyltransferase. Rani SH, Krishna TH, Saha S, Negi AS, Rajasekharan R. J Biol Chem. 2010 Dec 3;285(49):38337-47. Epub 2010 Oct 4.
Triacylglycerol lipolysis is linked to sphingolipid and phospholipid metabolism of the yeast Saccharomyces cerevisiae. Rajakumari S, Rajasekharan R, Daum G. Biochim Biophys Acta. 2010 Dec;1801(12):1314-22. Epub 2010 Aug 19.
At4g24160, a soluble acyl-coenzyme A-dependent lysophosphatidic acid acyltransferase. Ghosh AK, Chauhan N, Rajakumari S, Daum G, Rajasekharan R. Plant Physiol. 2009 Oct;151(2):869-81. Epub 2009 Aug 21.
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