Functional characterization of Cyberlindnera jadinii carboxylate transporters in Saccharomyces cerevisiae

Abstract

Introduction In Saccharomyces cerevisiae, two permeases are responsible for the uptake of carboxylates (CA) at the plasma membrane, Jen1p a monocarboxylate proton symporter (Major Facilitator Superfamily) and Ady2p an acetate permease (AceTr Family). In Cyberlindnera jadinii, different uptake systems for CAs were functionally characterized however until now the genes encoding these transporters remain unidentified. In this work, CA transporter homolog genes from C. jadinii were identified and expressed in S. cerevisiae. Materials and Methods The S. cerevisiae strain W303-1A jen1Δ ady2Δ, lacking carboxylate uptake capacity, was used to express C. jadinii ScJEN1 and ScADY2 homologs. Genes were identified through sequence alignment and homology prediction and cloned in the p416GPD vector, under the control of a GPD constitutive promoter. GFP-fusions versions were used to determine protein expression and localization. Transport activity was determined through growth on different carbon sources and measurement of the uptake of labelled CAs, namely D,L-[U-14C] lactic acid, [2,3-14C] succinic acid and [1-14C] acetic acid. Results In C. jadinii, 6 genes homolog to ScJEN1 (Cjj23088, Cjj21966, Cjj22358, Cjj21989, Cjj21602, Cjj25129) and 4 genes homolog to ScADY2 (Cja24587, Cja20823, Cja20690, Cja20822) were identified. All proteins were expressed and localized at the plasma membrane. Regarding transporter specificity CJJEN1-6 and CJAD3 encode lactate transporters, CJAD1 and 4, lactate and acetate transporters, and CJAD2 encodes a lactate, acetate and succinate transporter. Conclusions In this work, we identified 6 CjJEN1 and 4 CjADY2 homologs that are functional carboxylate transporters in S. cerevisiae. All the CjJEN1 homologs are lactate transporters and CjADY2 homologs present different specificities. Further studies are underway to fully characterize these ten new plasma membrane transporters from C. jadinii. References Soares-Silva, I., et al. (2007). Mol Membr Biol. 24(5-6), 464-474.