Calmodulin Activation of an Endoplasmic Reticulum-Located Calcium Pump Involves an Interaction with the N-Terminal Autoinhibitory Domain

Abstract

To investigate how calmodulin regulates a unique subfamily of Ca²⁺ pumps found in plants, we 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-sensitive⁴⁵Ca²⁺ 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 (Δ2–80), indicating that the N-terminal domain normally acts to inhibit the pump. An inhibitory sequence (IC₅₀ = 4 μm) was localized to a region within valine-20 to leucine-44, because a peptide corresponding to this sequence lowered the V_max and increased theK_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 μm) 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.