CA²⁺ BINDING PROTEIN CALRETICULIN IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA): BIOCHEMICAL CHARACTERIZATION, DIFFERENTIAL EXPRESSION DURING SEXUAL REPRODUCTION, AND PHYLOGENETIC ANALYSIS

Abstract

The occurrence of calreticulin, the main Ca 2؉ 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 Ca 2؉ 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 [ 45 Ca 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 Ca 2؉ buffering mechanism was present in this unicellular chlorophyte. The data suggest an early origin and high conservation of endoplasmic-reticulummediated Ca 2؉ functions in eukaryotes, whereby specific posttranslational modifications of the protein 1 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.

Development of a species-specific transformation system using the novel endogenous promoter calreticulin from oleaginous microalgae Ettlia sp.

“…CRT is one of the predominant proteins acting as a molecular calcium-binding chaperone that promotes protein folding by glycoprotein maturation, acts as a buffer, acts as a sensor of intraluminal-free Ca 2+ levels in the ER, and is related to transcriptional regulation and signal transduction in the nucleus 22 . In Chlamydomonas, CRT has shown high expression during sexual reproduction in reproductive cells 23 .…”

“…This lectin can interact transiently with almost all the monoglucosylated glycoproteins that are synthesized in the ER. Moreover, CRT is also found in the nucleus, suggesting that it may play a role in transcription regulation 22 , resulting in high expression during cell proliferation. The LHCSR was also excluded because despite exhibiting high differential gene expression, its expression was lower than that of CRT .…”

Chlamydomonas reinhardtii cellular compartments and their contribution to intracellular calcium signalling

“…In the lumen of the ER of many eukaryotes, Ca 2+ ions are also buffered efficiently by Ca 2+ -binding proteins, among which calreticulin (CR) is one of the most predominant forms. Chlamydomonas reinhardtii cells express a CR homologue (CRT2) that putatively localizes to the ER ( Table 1 ), providing first evidence for the presence of an intraluminal Ca 2+ storage machinery in the C. reinhardtii ER ( Zuppini et al , 1999 ). CR, as an effective Ca 2+ buffer, may in fact allow the transient storage of the ion and its prompt mobilization when Ca 2+ release is triggered, being characterized by high capacity and low affinity ( Zuppini et al , 1999 ).…”

“…Chlamydomonas reinhardtii cells express a CR homologue (CRT2) that putatively localizes to the ER ( Table 1 ), providing first evidence for the presence of an intraluminal Ca 2+ storage machinery in the C. reinhardtii ER ( Zuppini et al , 1999 ). CR, as an effective Ca 2+ buffer, may in fact allow the transient storage of the ion and its prompt mobilization when Ca 2+ release is triggered, being characterized by high capacity and low affinity ( Zuppini et al , 1999 ). Interestingly, CR expression in C. reinhardtii is developmentally regulated during gamete formation, with a progressive increase of the level of the protein in both pre-gametes and gametes.…”