Characterization of putative acetate transporters in Chlamydomonas reinhardtii

The unicellular green alga C. reinhardtii can grown either phototrophically with CO2 as the sole carbon source, heterotrophically by consuming acetate in the dark and mixotrophically by using both carbon sources in the light. Despite significant knowledge gained on acetate metabolism, the genes coding for acetate transporter/permease are still unknown in this alga. However, recent analyses1,2 have shown five functionally uncharacterized members of the GPR1/FUN34/yaaH (GFY), a protein family which includes genes involved in carboxylic organic acid uptake/sensing already described in bacteria, yeasts and filamentous fungi. Thus, the five genes identified in C. reinhardtii as Cre17.g700450 (GFY1), Cre17.g.700650 (GFY2), Cre17.g.700750 (GFY3), Cre17.g.702900 (GFY4) and Cre17.g.702950 (GFY5) encode for putative acetate transporter proteins given that they are structured in 6 hydrophobic transmembrane helices. They are characterized by a close gene structure (Fig. 1) and very high similarity in their coding sequence (CDS) except for a clear distinction at the N-terminus amino acid sequences (Fig. 2).
A reverse functional genomics approach by using artificial micro RNA (amiRNA) gene silencing was adopted to target the five genes one-by-one. Until now, ~160 transformants were generated for each amiRNA construct and their characterization is ongoing. A further characterization of the mutants will follow to have an understanding of the gene function in the acetate metabolism.