Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.
FEBS J. 2020 Oct;287(20):4401-4414. doi: 10.1111/febs.15262. Epub 2020 Mar 11.
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 ( ) is much lower than , 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.
酵母 APC 超家族氨基酸转运蛋白负责质子动力驱动的氨基酸进入细胞，对于大多数次级转运蛋白来说，这是一个可逆的过程。酿酒酵母的 l-赖氨酸质子协同转运蛋白 Lyp1 很特殊，因为其外向到内向转运的米氏常数（ ）远低于 ，这使得 l-赖氨酸可以积累到亚摩尔浓度。有人提出，细胞内高赖氨酸是细胞抗氧化机制的一部分。Lyp1 独特动力学特性的分子基础尚不清楚。我们比较了 Lyp1 与 APC 旁系和直系同源物的序列，发现了使其与众不同的结构特征，包括细胞外环区的差异。我们通过丙氨酸诱变筛选细胞外环，确定了 Lyp1 的定位和活性，并找到了影响 Lyp1 定位或活性的位置。一半受影响的突变体位于细胞外环 3 的延伸部分或细胞外环 4 中的预测α螺旋中。我们的数据表明，细胞外环不仅连接跨膜螺旋，而且还具有重要的功能作用。
