Plant Physiol, March 2000, Vol. 122, pp. 957-966

Calcium-Regulated Proteolysis of eEF1A¹

Botany Department, North Carolina State University, Raleigh, North Carolina 27695-7612 (W.D.R.-H., I.B., W.F.B.); and Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506 (X.W.)

Eukaryotic elongation factor 1 (eEF1A) can be post-translationally modified by the addition of phosphorylglycerylethanolamine (PGE). [¹⁴C]Ethanolamine was incorporated into the PGE modification, and with carrot (Daucus carota L.) suspension culture cells, eEF1A was the only protein that incorporated detectable quantities of [¹⁴C]ethanolamine (Ransom et al., 1998). When 1 mM CaCl₂ was added to microsomes containing [¹⁴C]ethanolamine-labeled eEF1A ([¹⁴C]et-eEF1A), there was a 60% decrease in the amount of [¹⁴C]et-eEF1A recovered after 10 min. The loss of endogenous [¹⁴C]et-eEF1A was prevented by adding EGTA. Recombinant eEF1A, which did not contain the PGE modification, also was degraded by microsomes in a Ca²⁺-regulated manner, indicating that PGE modification was not necessary for proteolysis; however, it enabled us to quantify enodgenous eEF1A. By monitoring [¹⁴C]et-eEF1A, we found that treatment with phospholipase D or C, but not phospholipase A₂, resulted in a decrease in [¹⁴C]et-eEF1A from carrot microsomes. The fact that there was no loss of [¹⁴C]et-eEF1A with phospholipase A₂ treatment even in the presence of 1 mM Ca²⁺ suggested that the loss of membrane lipids was not essential for eEF1A proteolysis and that lysolipids or fatty acids decreased proteolysis. At micromolar Ca²⁺ concentrations, proteolysis of eEF1A was pH sensitive. When 1 µM CaCl₂ was added at pH 7.2, 35% of [¹⁴C]et-eEF1A was lost; while at pH 6.8, 10 µM CaCl₂ was required to give a similar loss of protein. These data suggest that eEF1A may be an important downstream target for Ca²⁺ and lipid-mediated signal transduction cascades.