Calmodulin activation of an endoplasmic reticulum-located calcium pump involves an interaction with the N-terminal autoinhibitory domain

To investigate how calmodulin regulates a unique subfamily of Ca2+ pumps found in plants, rye examined the kinetic properties of isoform ACA2 identified in Arabidopsis. A recombinant ACA2 was expressed in a yeast K616 mutant deficient in two endogenous Ca2+ pumps. Orthovanadate-sensitive Ca-45(2+) transport into Vesicles isolated from transformants demonstrated that ACA2: is a Ca2+ pump. Ca2+ pumping by the full-length protein (ACA2-1) was 4- to 10-fold lower than that of the N-terminal truncated ACA2-2 (Delta 2-80), indicating that the N-terminal domain normally acts to inhibit the pump. An inhibitory sequence (IC50 = 4 mu M) was localized to a region within valine-20 to leucine-44, because a peptide corresponding to this sequence lowered the V-max and increased the K-m for Ca2+ of the constitutively active ACA2-2 to values comparable to the full-length pump. The peptide also blocked the activity (IC50 = 7 mu M) of a Ca2+ pump (AtECA1) belonging to a second family of Ca2+ pumps. This inhibitory sequence appears to overlap with a calmodulin-binding site in ACA2, previously mapped between asparatate-19 and arginine-36 (J.F. Harper, B. Hong, I. Hwang, H.Q. Guo, R. Stoddard, J.F. Huang, M.G. Palmgren, H. Sze [1998] J Biol Chem 273: 1099-1106). These results support a model in which the pump is kept "unactivated" by an intramolecular interaction between an autoinhibitory sequence located between residues 20 and 44 and a site in the Ca2+ pump core that is highly conserved between different Ca2+ pump families. Results further support a model in which activation occurs as a result of Ca2+ induced binding of calmodulin to a site overlapping or immediately adjacent to the autoinhibitory sequence.