Regulatory role of the N terminus of the vacuolar calcium-ATPase in cauliflower

The vacuolar calmodulin (CaM)-stimulated Ca2+-ATPase, BCA1p, in cauliflower (Brassica oleracea) has an extended N terminus, which was suggested to con tain a CaM-binding domain (S. Malmstrom, P. Askerlund, M.G. Palmgren [1997] FEES Lett 400: 324-328). The goal of the present study was to determine th e 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 tr ypsin 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 ha s an autoinhibitory function. The expressed N terminus of BCA1p and a synth etic peptide (Ala-19 to Met-39) were good substrates for phosphorylation by protein kinase C. Sequencing of the phosphorylated fusion protein and pept ide suggested serine-16 and/or serine-28 as likely targets for phosphorylat ion. Phosphorylation of serine-28 had no effect on CaM binding to the alani ne-19 to methionine-39 peptide. Our results demonstrate the regulatory impo rtance of the N terminus of BCA1p as a target for CaM binding, trypsin clea vage, and phosphorylation, as well as its importance as an autoinhibitory d omain.