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

• Published in: Plant physiology (Bethesda) Vol. 122; no. 1; pp. 157 - 167
• Main Authors: Hwang, Ildoo, Harper, Jeffrey F., Liang, Feng, Sze, Heven
• Format: Journal Article
• Language: English
• Published: Legacy CDMS American Society of Plant Physiologists 01.01.2000
• Subjects: Adenosine triphosphatases; Biological and medical sciences; Calcium; Calcium - metabolism; Calcium-Transporting ATPases - metabolism; Calmodulin - metabolism; Cell Biology and Signal Transduction; Cell membranes; Cell membranes. Ionic channels. Membrane pores; Cell physiology; Cell structures and functions; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - metabolism; Endoplasmic Reticulum - metabolism; Fundamental and applied biological sciences. Psychology; Fungal Proteins - chemistry; Fungal Proteins - metabolism; Kinetics; Life Sciences (General); Molecular and cellular biology; Physiological regulation; Plant physiology and development; Plants; Plasma membrane and permeation; Protein Binding; Protein Structure, Tertiary; Proteins; Pumps; Saccharomyces cerevisiae - growth & development; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins; Space life sciences; Transcription Factors - chemistry; Transcription Factors - metabolism; Water pumps; Yeasts; Nasa Discipline Plant Biology; Non-Nasa Center; Nasa Program Fundamental Space Biology; Calcium; Cell permeability; Interaction; Binding site; Fungi; Arabidopsis thaliana; Regulation(control); Membrane pump; Cruciferae; Dicotyledones; Angiospermae; Self regulation; Ascomycetes; Spermatophyta; Experimental plant; Molecular domain; Endoplasmic reticulum; Saccharomyces cerevisiae; Calmodulin; Thallophyta; NASA Program Fundamental Space Biology; NASA Discipline Plant Biology; Non-NASA Center
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.122.1.157

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-sensitive ^{45}\text{Ca}^{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 (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 Vmax and increased the Km 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.