Previous mutational analysis of Jen1p, a Saccharomyces cerevisiae monocarboxylate/H⁺ symporter of the Major Facilitator Superfamily, has suggested that the consensus sequence ³⁷⁹NXX[S/T]HX[S/T]QD³⁸⁷ in transmembrane segment VII (TMS-VII) is part of the substrate translocation pathway. Here, we rationally design, analyse and show that several novel mutations in TMS-V and TMS-XI directly modify Jen1p function. Among the residues studied, F270 (TMS-V) and Q498 (TMS-XI) are critical specificity determinants for the distinction of mono- from dicarboxylates, and N501 (TMS-XI) is a critical residue for function. Using a model created on the basis of Jen1p similarity with the GlpT permease, we show that all polar residues critical for function within TMS-VII and TMS-XI (N379, H383, D387, Q498, N501) are perfectly aligned in an imaginary axis that lies parallel to the protein pore. This model and subsequent mutational analysis further reveal that an additional polar residue facing the pore, R188 (TMS-II), is irreplaceable for function. Our model also justifies the role of F270 and Q498 in substrate specificity. Finally, docking calculations reveal a 'trajectory-like' substrate displacement within the Jen1p pore, where R188 plays a major dynamic role mediating the orderly relocation of the substrate by subsequent H-bond interactions involving itself and residues H383, N501 and Q498.