Premium
Molecular cloning and functional characterization of two glycerol‐3‐phosphate acyltransferases from the green microalga Chlamydomonas reinhardtii
Author(s) -
DuarteCoello María E.,
HerreraValencia Virginia A.,
EchevarríaMachado Ileana,
CasaisMolina Melissa L.,
PerazaEcheverria Santy
Publication year - 2019
Publication title -
phycological research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.438
H-Index - 44
eISSN - 1440-1835
pISSN - 1322-0829
DOI - 10.1111/pre.12350
Subject(s) - chlamydomonas reinhardtii , biology , acyltransferases , arabidopsis thaliana , biochemistry , heterologous expression , chloroplast , plastid , acyltransferase , chlamydomonas , arabidopsis , endoplasmic reticulum , gene , microbiology and biotechnology , mutant , biosynthesis , recombinant dna
SUMMARY 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 Δ 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.