Both ALG-2 and Peflin, Penta-EF-hand (PEF) Proteins, Are Stabilized by Dimerization through Their Fifth EF-Hand Regions

Abstract

ALG-2 (apoptosis-linked gene-2 protein) and peflin are Ca2+-binding proteins and belong to the penta-EF-hand (PEF) protein family, which includes calpain, sorcin, and grancalcin. ALG-2 forms either a homodimer or a heterodimer with peflin like other PEF proteins. In this study, we found that the fifth-EF-hand (EF-5) regions of both ALG-2 and peflin are essential for dimerization and their stabilities. Exogenously expressed EF-5-deletion (ΔEF-5) mutants of ALG-2 and peflin were unstable and were not detected in HEK293 cells by Western blotting. In a pulse–chase experiment, the ΔEF-5 mutants were rapidly degraded, but they were stabilized by treatment with a proteasome inhibitor, MG132. In MG132-treated cells, ΔEF-5 mutants were recovered in the insoluble fractions. Transient coexpression of ALG-2 increased the peflin level. These results indicate that the absence of a fifth EF-hand results in rapid degradation by the proteasome. On the other hand, stable expression of exogenous peflin decreased the amount of endogenous peflin. The amount of peflin that can dimerize with ALG-2 seems to be restricted in mammalian cells.