US EPA

1532607 

Journal Article 

Proteomic Analysis of Extracellular ATP-Regulated Proteins Identifies ATP Synthase beta-Subunit as a Novel Plant Cell Death Regulator 

Chivasa, S; Tome, DFA; Hamilton, JM; Slabas, AR 

2011 

1 

Molecular and Cellular Proteomics
ISSN: 1535-9476
EISSN: 1535-9484 

10 

3 

21156838 

10.1074/mcp.M110.003905 

WOS:000287847200013 

Extracellular ATP is an important signal molecule required
to cue plant growth and developmental programs, interactions with other organisms, and responses
to environmental stimuli. The molecular targets mediating the physiological effects of
extracellular ATP in plants have not yet been identified. We developed a well characterized
experimental system that depletes Arabidopsis cell suspension culture extracellular ATP via
treatment with the cell death-inducing mycotoxin fumonisin B1. This provided a platform for
protein profile comparison between extracellular ATP-depleted cells and fumonisin B1-treated
cells replenished with exogenous ATP, thus enabling the identification of proteins regulated by
extracellular ATP signaling. Using two-dimensional difference in-gel electrophoresis and matrix-
assisted laser desorption-time of flight MS analysis of microsomal membrane and total soluble
protein fractions, we identified 26 distinct proteins whose gene expression is controlled by the
level of extracellular ATP. An additional 48 proteins that responded to fumonisin B1 were
unaffected by extracellular ATP levels, confirming that this mycotoxin has physiological effects
on Arabidopsis that are independent of its ability to trigger extracellular ATP depletion.
Molecular chaperones, cellular redox control enzymes, glycolytic enzymes, and components of the
cellular protein degradation machinery were among the extracellular ATP-responsive proteins. A
major category of proteins highly regulated by extracellular ATP were components of ATP
metabolism enzymes. We selected one of these, the mitochondrial ATP synthase beta-subunit, for
further analysis using reverse genetics. Plants in which the gene for this protein was knocked
out by insertion of a transfer-DNA sequence became resistant to fumonisin B1-induced cell death.
Therefore, in addition to its function in mitochondrial oxidative phosphorylation, our study
defines a new role for ATP synthase beta-subunit as a pro-cell death protein. More significantly,
this protein is a novel target for extracellular ATP in its function as a key negative regulator
of plant cell death. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.003905, 1-13, 2011.