Ca2+ binding protein calreticulin in Chlamydomonas reinhardtii (Chlorophyta): Biochemical characterization, differential expression during sexual reproduction and phylogenetic analysis.

The occurrence of calreticulin, the main Ca2+ binding protein in the endoplasmic reticulum of eukaryotic cells, was investigated in the unicellular green alga Chlamydomonas reinhardtii Dangeard. The biochemical characterization of a diethylaminoethyl purified extract highlighted the presence, on SDS-PAGE, of a 55-kDa protein that stained blue with the Stains All dye, a diagnostic feature of acidic Ca2+ binding proteins. Immunoblot analyses revealed a strong cross-reaction of the Chlamydomonas reinhardtii protein with antibodies to plant calreticulins and the endoplasmic reticulum retention signal HDEL. Furthermore, the 55-kDa protein bound [Ca-45(2+)] and had an acidic isoelectric point (pI = 4.9) but was neither glycosylated nor phosphorylated. N-terminal sequencing revealed strong amino acid sequence similarity to calreticulin from other sources. The presence of calreticulin in Chlamydomonas reinhardtii suggested that an endoplasmic reticulum Ca2+ buffering mechanism was present in this unicellular chlorophyte. The data suggest an early origin and high conservation of endoplasmic-reticulum-mediated Ca2+ functions in eukaryotes, whereby specific posttranslational modifications of the protein have been specifically acquired in different Lineages of photosynthetic eukaryotes. Moreover, northern and western blot analysis experiments showed a regulation of calreticulin expression during Chlamydomonas sexual reproduction with a high abundance of calreticulin mRNA and protein in reproductive cells.