Calcium-dependent protein kinase in the green alga Chara

Abstract

Cytoplasmic streaming in the characean algae is inhibited by micromolar rises in the level of cytosolic free Ca²⁺, but both the mechanism of action and the molecular components involved in this process are unknown. We have used monoclonal antibodies against soybean Ca²⁺-dependent protein kinase (CDPK), a kinase that is activated by micromolar Ca²⁺ and co-localizes with actin filaments in higher-plant cells (Putnam-Evans et al., 1989, Cell Motil. Cytoskel. 12, 12–22) to identify and localize its characean homologue. Immunoblot analysis revealed that CDPK in Chara corralina Klein ex. Wild shares the same relative molecular mass (51–55 kDa) as the kinase purified from soybean, and after electrophoresis in denaturing gels is capable of phosphorylating histone III-S in a Ca²⁺-dependent manner. Immunofluorescence microscopy localized CDPK in Chara to the subcortical actin bundles and the surface of small organelles and other membrane components of the streaming endoplasm. The endoplasmic sites carrying CDPK were extracted from internodal cells by vacuolar perfusion with 1 mM ATP or 10⁻⁴ M Ca²⁺. Both the localization of CDPK and its extraction from internodal cells by perfusion with ATP or high Ca²⁺ are properties similar to that reported for the heavy chain of myosin in Chara (Grolig et al., 1988, Eur. J. Cell Biol. 47, 22–31). Based on its endoplasmic location and inferred enzymatic properties, we suggest that CDPK may be a putative element of the signal-transduction pathway that mediates the rapid Ca²⁺-induced inhibition of streaming that occurs in the characean algae.