Tomato annexins p34 and p35 bind to F-actin and display nucleotide phosphodiesterase activity inhibited by phospholipid binding.
Annexins are a family of proteins found in a range of eukaryotic cell types. They share a characteristic amino acid sequence and a Ca(2+)-dependent affinity for specific phospholipids. In plants, proteins with common properties and significant homology with annexins have been identified in a number of species and implicated in diverse cellular functions known to be modulated by Ca2+. This study describes several novel biochemical properties of the tomato annexins p34 and p35 that are relevant to our understanding of their functions in the plant. First, the annexins were found to bind to actin in a calcium- and pH-dependent interaction that was specific for F-actin and not G-actin. Second, an enzyme activity defined as a nucleotide phosphodiesterase activity was found associated with the purified annexin preparation. Selective immunoprecipitation of p34 and p35 strongly suggests that the enzyme activity is a property of the annexins and constitutes 60% of the total soluble activity found in root extracts capable of hydrolyzing free ATP. The substrate specificity of the enzyme within in vitro assays is broad. ATP is the preferred substrate, but nearly identical rates of hydrolysis of GTP and substantial hydrolysis of other nucleotide tri- and diphosphates are observed. The enzyme activity was found to be a property of both p34 and p35, although the specific activity was routinely higher for p34. Third, the enzyme activity of the annexins was not affected by F-actin binding but could be abolished by the specific Ca(2+)-dependent interaction of the annexins with phospholipids. Our results showed that p34 and p35 account for substantial enzyme activity in tomato root cells. This activity was exhibited when the proteins were either in soluble form or attached to actin filaments. Enzyme activity was not exhibited when the annexins were bound to phospholipids. These properties suggest a role for the proteins in mediating Ca(2+)-dependent events involving interactions of the cytoskeleton and cellular membranes.
Calvert
C M
CM
Department of Biology, University of York, United Kingdom.
Gant
S J
SJ
Bowles
D J
DJ
D000199:Actins / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D017302:Annexins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002846:Chromatography, Affinity; D004533:Egtazic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D018551:Solanum lycopersicum / Q000378:metabolism*; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000378:metabolism; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010743:Phospholipids / Q000378:metabolism* / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits
D000199:Actins; D017302:Annexins; D010726:Phosphodiesterase Inhibitors; D010743:Phospholipids; D004533:Egtazic Acid; D010727:Phosphoric Diester Hydrolases; D000251:Adenosine Triphosphatases; D002118:Calcium