Extracellular loops matter - subcellular location and function of the lysine transporter Lyp1 from Saccharomycescerevisiae.

Joury S Van‘T Klooster,Frans Bianchi,Mirco Lorenzon,Jarnick H Lusseveld,Bert Poolman,Ruben B Doorn,Christiaan M Punter

doi:10.1111/febs.15262

Abstract

Yeast amino acid transporters of the APC superfamily are responsible for the proton motive force‐driven uptake of amino acids into the cell, which for most secondary transporters is a reversible process. The l‐lysine proton symporter Lyp1 of Saccharomyces cerevisiae is special in that the Michaelis constant from out‐to‐in transport ( Kmout→in ) is much lower than Kmin→out , which allows accumulation of l‐lysine to submolar concentration. It has been proposed that high intracellular lysine is part of the antioxidant mechanism of the cell. The molecular basis for the unique kinetic properties of Lyp1 is unknown. We compared the sequence of Lyp1 with APC para‐ and orthologues and find structural features that set Lyp1 apart, including differences in extracellular loop regions. We screened the extracellular loops by alanine mutagenesis and determined Lyp1 localization and activity and find positions that affect either the localization or activity of Lyp1. Half of the affected mutants are located in the extension of extracellular loop 3 or in a predicted α‐helix in extracellular loop 4. Our data indicate that extracellular loops not only connect the transmembrane helices but also serve functionally important roles.

Highlights

The transport of amino acids in Saccharomyces cerevisiae is facilitated by Yeast Amino acid Transporters (YATs)[1], which are members of the APC-superfamily
Modeling and bioinformatics analysis of loop regions Transmembrane -helices (TMHs) can be predicted with relative high accuracy, using topology prediction programs and multiple sequence alignments of homologous proteins, but loop regions, which often vary a lot in length and structure, are difficult to analyze without proper template
Noticeable is the similarity of both models in predicting the extracellular loops; minor differences are marked by color coding of the amino acid residues

Summary

Introduction

The transport of amino acids in Saccharomyces cerevisiae is facilitated by Yeast Amino acid Transporters (YATs)[1], which are members of the APC-superfamily. Basic amino acids are transported by only a subset of YATs i.e., the proteins encoded by Gap, Hip, Alp, Can and Lyp. At present there is no structure of a YAT available, but models have been built on the basis of bacterial APC structures. Ghaddar et al[12] was able to re-engineer the specificity of Can from Arg to Lys on the basis of the structure of AdiC. Modelling of extracellular loop regions is more challenging as they are often not well resolved in crystal structures and are typically shorter in prokaryotic homologues and unique for eukaryotic membrane proteins[13,14]

Methods

Results

Conclusion