Calcium-Regulated Proteolysis of eEF1A

Abstract

Abstract Eukaryotic elongation factor 1α (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 mmCa2+ 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 μmCaCl2 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.