Calcium-regulated proteolysis of eEF1A.
Eukaryotic elongation factor 1alpha (eEF1A) can be post-translationally modified by the addition of phosphorylglycerylethanolamine (PGE). [(14)C]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 [(14)C]ethanolamine (Ransom et al., 1998). When 1 mM CaCl(2) was added to microsomes containing [(14)C]ethanolamine-labeled eEF1A ([(14)C]et-eEF1A), there was a 60% decrease in the amount of [(14)C]et-eEF1A recovered after 10 min. The loss of endogenous [(14)C]et-eEF1A was prevented by adding EGTA. Recombinant eEF1A, which did not contain the PGE modification, also was degraded by microsomes in a Ca(2+)-regulated manner, indicating that PGE modification was not necessary for proteolysis; however, it enabled us to quantify enodgenous eEF1A. By monitoring [(14)C]et-eEF1A, we found that treatment with phospholipase D or C, but not phospholipase A(2), resulted in a decrease in [(14)C]et-eEF1A from carrot microsomes. The fact that there was no loss of [(14)C]et-eEF1A with phospholipase A(2) treatment even in the presence of 1 mM Ca(2+) suggested that the loss of membrane lipids was not essential for eEF1A proteolysis and that lysolipids or fatty acids decreased proteolysis. At micromolar Ca(2+) concentrations, proteolysis of eEF1A was pH sensitive. When 1 microM CaCl(2) was added at pH 7.2, 35% of [(14)C]et-eEF1A was lost; while at pH 6.8, 10 microM CaCl(2) was required to give a similar loss of protein. These data suggest that eEF1A may be an important downstream target for Ca(2+) and lipid-mediated signal transduction cascades.
Ransom-Hodgkins
W D
WD
Botany Department, North Carolina State University, Raleigh, North Carolina 27695-7612, USA.
Brglez
I
I
Wang
X
X
Boss
W F
WF
D001550:Benzamidines / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D018552:Daucus carota / Q000187:drug effects / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism; D019856:Ethanolamine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D020648:Peptide Elongation Factor 1 / Q000378:metabolism*; D010739:Phospholipase D / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D010738:Type C Phospholipases / Q000494:pharmacology
D001550:Benzamidines; D020648:Peptide Elongation Factor 1; D011994:Recombinant Proteins; D004533:Egtazic Acid; D019856:Ethanolamine; D010738:Type C Phospholipases; D010739:Phospholipase D; D010450:Endopeptidases; C032157:benzamidine; D002118:Calcium