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 Ca(2+) 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 Ca(2+) pumps. Orthovanadate-sensitive (45)Ca(2+) transport into vesicles isolated from transformants demonstrated that ACA2 is a Ca(2+) pump. Ca(2+) pumping by the full-length protein (ACA2-1) was 4- to 10-fold lower than that of the N-terminal truncated ACA2-2 (Delta2-80), indicating that the N-terminal domain normally acts to inhibit the pump. An inhibitory sequence (IC(50) = 4 microM) 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 Ca(2+) of the constitutively active ACA2-2 to values comparable to the full-length pump. The peptide also blocked the activity (IC(50) = 7 microM) of a Ca(2+) pump (AtECA1) belonging to a second family of Ca(2+) pumps. This inhibitory sequence appears to overlap with a calmodulin-binding site in ACA2, previously mapped between aspartate-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 Ca(2+) pump core that is highly conserved between different Ca(2+) pump families. Results further support a model in which activation occurs as a result of Ca(2+)-induced binding of calmodulin to a site overlapping or immediately adjacent to the autoinhibitory sequence.

Figure 1

Expression of an N-terminal truncated ACA2 enhanced growth of yeast mutant K616 on medium containing submicromolar Ca²⁺. A, Time-course of yeast growth on medium containing 0.16 μm Ca²⁺ and 10 mm EGTA. K616 strains were transformed with control vector alone (▴), vector containing ACA2-1 (○), and ACA2-2 (●). Transformants were suspended in SC-URA medium containing 10 mm EGTA to an initial A₆₀₀ of 0.1. B, Submicromolar levels of extracellular Ca²⁺ support growth of wild-type yeast and transformants harboring the truncated ACA2-2. Transformants were suspended in SC-URA medium containing varying levels EGTA to give free [Ca²⁺] ranging from 0.16 to 55 μm. Cells were then incubated for 24 h. Wild-type yeast transformed with vector control (Δ), and K616 mutant transformed with vector alone (▴), ACA2-1 (○), or ACA2-2 (●).

Figure 2

N-terminal-truncated ACA2 is an active Ca²⁺ pump. Membranes were isolated from mutants transformed with ACA2-1 (○), ACA2-2 (●), or vector alone (▴). ⁴⁵Ca²⁺ uptake into vesicles was determined in the presence of bafilomycin and CCCP with or without ATP. EGTA was used to give a free [Ca²⁺] of 0.1 μm. Activity is expressed as ATP-dependent Ca²⁺ uptake. A23187 was added (arrow) to a final concentration of 2.5 μg/mL.

Figure 3

Calmodulin stimulated the full-length ACA2 pump but not the N-terminal truncated ACA2. Membranes were isolated from mutants transformed with ACA2-1 (○), ACA2-2 (●), or vector alone (▴). Net Ca²⁺ uptake (10 min) into vesicles was measured with or without 200 μm Na orthovanadate in the presence of CCCP and bafilomycin. The mixture contained 100 μm Ca²⁺ and 100 μm EGTA to give a final [Ca²⁺] of 2.6 μm, and bovine calmodulin ranged from 0 to 1 μm. Activity is expressed as vanadate-sensitive Ca²⁺ transport. The average of two independent experiments representative of five experiments is shown.

Figure 4

Calmodulin enhanced the V_max and the affinity for Ca²⁺ of the full-length ACA2 protein. Membranes were isolated from ACA2-1 transformants. Initial rate of vanadate-sensitive Ca²⁺ transport was determined at 30 s with (●) or without (○) 0.5 μm calmodulin in the presence of bafilomycin and CCCP. EGTA (100 μm) was added to reaction mixtures containing 1-100 μm Ca²⁺ to give free Ca²⁺ concentrations from 1 nm to 2.6 μm. The K_m for Ca²⁺ was 0.67 or 0.39 μm without or with calmodulin, respectively. The average of duplicates from one of three similar experiments is shown.

Figure 5

N-terminal truncated ACA2 pumps Ca²⁺ with high velocity and affinity. Membranes were isolated from yeast expressing ACA2-2. The initial rate of vanadate-sensitive Ca²⁺ uptake (30 s) was determined as a function of external [Ca²⁺] in the presence of bafilomycin and CCCP. The K_m for Ca²⁺ of 0.2 μm was estimated using a Lineweaver-Burk plot. The average of duplicates from one of three experiments is shown.

Figure 6

Sensitivity of the truncated ACA2 pump to various inhibitors: A, Orthovanadate; B, erythrosin B; C, thapsigargin; and D, cyclopiazonic acid. Vesicles isolated from ACA2-2 transformants were incubated with inhibitors for 10 min at room temperature in the reaction mixtures. Then net ATP-dependent Ca²⁺ uptake was measured in the presence of bafilomycin and CCCP. Activity (5.2 nmol mg⁻¹ protein⁻¹) without the inhibitors used in A through C was set to 100%. To test cyclopiazonic acid in D, ATP was lowered to 0.6 mm, which gave a control activity of 1.5 nmol/mg.

Figure 7

Peptide 20-44 inhibited Ca²⁺ pumping of the truncated ACA2 (left panel) and ECA1 (right panel). Membranes isolated from either ACA2-2 or ECA1 transformants were incubated with varying concentrations of peptides 12-36 (▴), 20-44 (●), or 30-54 (○). Peptides correspond to overlapping sequences between residues 12 through 54 of ACA2 shown at the top. Charged residues are marked with + or −; asterisk (*) indicates potential phosphorylation sites. Vanadate-inhibited Ca²⁺ transport (10 min) was measured at 50 nm Ca²⁺. The results are the mean of duplicates. Activity in the absence of peptide was set to 100%, which corresponds to 1.25 and 0.89 nmol/mg for ACA2 and ECA1, respectively.

Figure 8

Peptide 20-44 antagonized the stimulation of the full-length pump by calmodulin. Membranes isolated from ACA2-1 transformants were incubated with 0, 0.25, or 1.0 μm calmodulin alone, or with peptide 20-44 for 10 min. ATP was added to initiate transport at saturating Ca²⁺ (2.6 μm). Orthovanadate-inhibited uptake at 10 min is plotted. In the absence of peptide, the activity (100%) stimulated by 0.25 or 1.0 μm calmodulin was 2.85 or 4.0 nmol/mg, respectively. The data are from two experiments.

Figure 9

Working model of the regulation of ACA2 pump by an N-terminal domain. In an unstimulated cell, the full-length pump (ACA2-1) is inhibited by an intramolecular interaction of an N-terminal region with part(s) of the catalytic and hydrophilic loop(s) (top). After a stimulus, an increase in [Ca²⁺] leads to the binding of Ca²⁺-calmodulin complex to a region near the autoinhibitory sequence. The resulting conformational change displaces the autoinhibitory domain from its interaction with the hydrophilic loop(s), thus activating the pump (left). An N-terminal truncated pump (ΔN2-80) is therefore constitutively active and unresponsive to calmodulin (right). Numbers refer to putative transmembrane domains. N and C refer to the amino and carboxyl termini, respectively.