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Plant Physiol, February 2000, Vol. 122, pp. 517-526
Regulatory Role of the N Terminus of the Vacuolar
Calcium-ATPase in Cauliflower1
Susanna
Malmström,*
Hans-Erik
Åkerlund, and
Per
Askerlund
Department of Plant Biochemistry, Lund University, P.O. Box 117, SE-221 00 Lund, Sweden.
The vacuolar calmodulin
(CaM)-stimulated Ca2+-ATPase, BCA1p, in cauliflower
(Brassica oleracea) has an extended N terminus, which
was suggested to contain a CaM-binding domain (S. Malmström, P. Askerlund, M.G. Palmgren [1997] FEBS Lett 400: 324-328). The goal of
the present study was to determine the role of the N terminus in
regulating BCA1p. Western analysis using three different antisera showed that the N terminus of BCA1p is cleaved off by trypsin and that
the N terminus contains the CaM-binding domain. Furthermore, the
expressed N terminus binds CaM in a Ca2+-dependent manner.
A synthetic peptide corresponding to the CaM-binding domain of BCA1p
(Ala-19 to Leu-43) strongly inhibited ATP-dependent Ca2+
pumping by BCA1p in cauliflower low-density membranes, indicating that
the CaM-binding region of BCA1p also has an autoinhibitory function.
The expressed N terminus of BCA1p and a synthetic peptide (Ala-19 to
Met-39) were good substrates for phosphorylation by protein kinase C. Sequencing of the phosphorylated fusion protein and peptide suggested
serine-16 and/or serine-28 as likely targets for phosphorylation.
Phosphorylation of serine-28 had no effect on CaM binding to the
alanine-19 to methionine-39 peptide. Our results demonstrate the
regulatory importance of the N terminus of BCA1p as a target for CaM
binding, trypsin cleavage, and phosphorylation, as well as its
importance as an autoinhibitory domain.
1
This work was supported by the Swedish
Natural Science Research Council and the European Union
Biotechnology Program.
*
Corresponding author; e-mail susanna.malmstrom{at}plantbio.lu.se;
fax 46-46-2224116.
© 2000 American Society of Plant Physiologists
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