Get access

A COCCOLITHOPHORID CALCIFYING VESICLE WITH A VACUOLAR-TYPE ATPASE PROTON PUMP: CLONING AND IMMUNOLOCALIZATION OF THE V0 SUBUNIT c

Authors

  • Paul L. A. M. Corstjens,

    1. Department of Organismic Biology, Ecology and Evolution, University of California, Los Angeles, Los Angeles, California 90095-1606
    Search for more papers by this author
    • Present address: Department of Molecular Cell Biology, Laboratory for Cytochemistry and Cytometry, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands.

  • Yoko Araki,

    1. Department of Organismic Biology, Ecology and Evolution, University of California, Los Angeles, Los Angeles, California 90095-1606
    Search for more papers by this author
  • Elma L. González

    1. Department of Organismic Biology, Ecology and Evolution, University of California, Los Angeles, Los Angeles, California 90095-1606
    Search for more papers by this author

Abstract

Coccolithophorid calcification is subcellular. It relies on a single Golgi apparatus to produce coccoliths consisting of an organic baseplate and calcite. The calcification reaction is initiated in a calcifying vesicle derived from the trans-most Golgi. We have cloned a subunit of a V (vacuolar)-ATPase (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 subunit c genes to identify conserved consensus sequences. The library was screened with a homologous probe obtained by PCR. The cloned gene is found as a single copy on the P. cartarae (strain 136) genome and includes a 495-base pair open reading frame encoding a 164 amino acid polypeptide and deduced molecular mass of 16.2 kDa. Its deduced amino acid sequence shows a close relationship to subunit c of the Vo domain of the vacuolar proton-pumping ATPase of higher plants. An in vitro-synthesized oligopeptide corresponding to the L2 extramembrane domain was used for rabbit immunization. Affinity-purified antiserum detected a polypeptide band with an apparent molecular mass of 24 kDa in immunoblots of highly enriched coccolith vesicle membranes. Immunofluoresence 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.

Ancillary