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Plant Physiol, January 2000, Vol. 122, pp. 157-168
Calmodulin Activation of an Endoplasmic Reticulum-Located Calcium
Pump Involves an Interaction with the N-Terminal Autoinhibitory
Domain1
Ildoo
Hwang,
Jeffrey F.
Harper,
Feng
Liang,2 and
Heven
Sze*
Department of Cell Biology and Molecular Genetics, and Maryland
Agricultural Experiment Station, University of Maryland, College
Park, Maryland 20742 (I.H., F.L., H.S.); and Department of Cell
Biology, The Scripps Research Institute, La Jolla, California
92037 (J.F.H.).
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
45Ca2+ 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 ( 2-80),
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.
1
This work was supported in part by the
Department of Energy (grant no. DE-FG02-95ER20200 to H.S. and grant
no. DE-FG03-94ER20152 to J.F.H.), and a joint grant from the National
Aeronautics and Space Administration and National Science Foundation
(grant no. IBN-9416038) for the Plant Sensory Systems Collaborative
Research Network.
2
Present address: Department of Bioinformatics,
The Institute for Genomic Research, 9712 Medical Center Drive,
Rockville, MD 20850.
*
Corresponding author; e-mail hs29{at}umail.umd.edu; fax
301-314-9082.
© 2000 American Society of Plant Physiologists
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