A coccolithophorid calcifying vesicle with a vacuolar-type ATPase proton pump: Cloning and immunolocalization of the V-0 subunit c(1)

Coccolithophorid calcification is subcellular. It relies on a single Golgi apparatus to produce coccoliths consisting of an organic baseplate and calc ite. The calcification reaction is initiated in a calcifying vesicle derive d from the trans-most Golgi, We have cloned a subunit of a V (vacuolar)-ATP ase (EC 3.6.1.3., ATP phosphohydrolase) from a Pleurochrysis (Haptophyceae) cDNA library of transcripts expressed during calcification. Degenerate PCR primers were developed after alignment of the higher plant V-ATPase subuni t c genes to identify conserved consensus sequences. The library was screen ed with a homologous probe obtained by PCR, The cloned gene is found as a s ingle copy on the P. cartarae (strain 136) genome and includes a 495-base p air open reading frame encoding a 164 amino acid polypeptide and deduced mo lecular mass of 16.2 kDa, Its deduced amino acid sequence shows a close rel ationship to subunit c of the V, domain of the vacuolar proton-pumping ATPa se of higher plants. An in vitro-synthesized oligopeptide corresponding to the L2 extramembrane domain was used for rabbit immunization. Affinity-puri fied antiserum detected a polypeptide band with an apparent molecular mass of 24 kDa in immunoblots of highly enriched coccolith vesicle membranes. Im munofluoresence microscopy showed antibody specificity for the membranes of isolated coccolith vesicles. This work provides support for the existence of an authentic, vacuolar-type, proton-pumping ATPase on coccolith vesicle membranes in a calcifying coccolithophorid.