Description
Lactic, acetic and propionic acids have been used for many years in industrial and pharmaceutical companies. In Saccharomyces cerevisiae, Jen1p is a major monocarboxylate:H+ symporter specific primarily for lactate, pyruvate and for acetate (TC # 2.A.1.12.2) (Casal et al., 1999). A phylogenetic tree of ScJen1p homologues (Casal et al., 2008) showed the existence of two main clusters: a Jen1 group (monocarboxylate transporters) and a Jen2-like (dicarboxylate transporters).
Structure-function relationships in Jen1p have been approached by using a rational mutational analysis of conserved amino acid residues (Soares-Silva et al., 2007). Analysis of the conserved sequence 379NXX[S/T]HX[S/T]QDXXXT391, located in transmembrane segment seven (TMS-VII), showed that residues N379, H383 or D387 are necessary for function and specificity, while Q386 is important for the kinetics of Jen1p-mediated transport.
In this work, we rationally designed and analyzed novel mutations in conserved regions located in TMS-II, TMS-V and TMS-XI of Jen1p, which we predicted to affect Jen1p specificity (distinction between mono and dicarboxylates) and function. Among the residues studied, F270 (TMS-V) and Q498 (TMS-XI) are specificity determinants for the distinction of mono- from dicarboxylates, and N501 (TMS-XI) is critical for function.
Using a model based on Jen1p similarity with the GlpT permease, we show that all polar residues critical for function within TMS-VII and TMS-XI are aligned along the protein pore and substrate docking studies reveal a potential substrate translocation trajectory consisting mostly of the polar residues genetically identified as important for function.
Overall, our results constitute a first step towards the genetic manipulation of substrate specificity in the lactate/pyruvate:H+ symporter subfamily and a tool for the in silico prediction of the function of Jen1p homologues in other fungi (Soares-Silva et al., 2011).
; Silva, Isabel Soares 
