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

Summary, in English

The vacuolar calmodulin (CaM)-stimulated Ca(2+)-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 Ca(2+)-dependent manner. A synthetic peptide corresponding to the CaM-binding domain of BCA1p (Ala-19 to Leu-43) strongly inhibited ATP-dependent Ca(2+) 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.

• Författare: Susanna Malmström; Hans-Erik Åkerlund; Per Askerlund
• Avdelning/ar: Biokemi och Strukturbiologi
• Publiceringsår: 2000
• Språk: Engelska
• Sidor: 517-526
• Publikation/Tidskrift/Serie: Plant Physiology
• Volym: 122
• Issue: 2
• Länkar: http://dx.doi.org/10.1104/pp.122.2.517
• Dokumenttyp: Artikel i tidskrift
• Förlag: American Society of Plant Biologists
• Ämne: Biological Sciences
• Status: Published
• ISBN/ISSN/Övrigt: ISSN: 1532-2548