Abstract
Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the first step of both the glycerolipid and the triacylglycerol (TAG) biosynthetic pathways. In plants, there are different isozymes for GPAT in different organelles, including the endoplasmic reticulum (ER) membrane-bound GPAT and the soluble chloroplast (plastid) GPAT. In microalgae, studies on GPAT have been limited; only the microsomal TpGPAT from the diatom Thalassiosira pseudonana has been characterized by enzymatic assay using heterologous expression in Saccharomyces cerevisiae. In the present study, we report the cloning and sequence analysis of two genes encoding GPAT isozymes from the green microalga Chlamydomonas reinhardtii, CrGPATer and CrGPATcl. CrGPATer is a homolog to Arabidopsis thaliana AtGPAT9, which encodes an ER-located GPAT, and CrGPATcl is a homolog to A. thaliana ATS1, which encodes a chloroplast-located GPAT. We mapped the 3 ' UTRs of both CrGPATer and CrGPATcl and identified three alternative splicings in CrGPATer mRNA and two in CrGPATcl mRNA. Interestingly, one of these splicings results from a trans-splicing event in CrGPATer mRNA. The heterologous expression of the cDNAs from each gene in the S. cerevisiae gat1 Delta mutant demonstrated, for the first time, that both CrGPATer and CrGPATcl show GPAT activity. Moreover, GPAT activity for CrGPATer was detected in the membrane extract, while that for CrGPATcl was detected in both the soluble and membrane extracts. Overall, these findings represent an important contribution to the better understanding of lipid metabolism in C. reinhardtii and in green microalgae in general.