| Autor: |
Arines FM; Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109., Wielenga A; Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109., Henn D; Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109., Burata OE; Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109., Garcia FN; Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109., Stockbridge RB; Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109., Li M; Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109. |
| Abstrakt: |
Lysosomes achieve their function through numerous transporters that import or export nutrients across their membrane. However, technical challenges in membrane protein overexpression, purification, and reconstitution hinder our understanding of lysosome transporter function. Here, we developed a platform to overexpress and purify the putative lysine transporter Ypq1 using a constitutive overexpression system in protease- and ubiquitination-deficient yeast vacuoles. Using this method, we purified and reconstituted Ypq1 into proteoliposomes and showed lysine transport function, supporting its role as a basic amino acid transporter on the vacuole membrane. We also found that the absence of lysine destabilizes purified Ypq1 and causes it to aggregate, consistent with its propensity to be downregulated in vivo upon lysine starvation. Our approach may be useful for the biochemical characterization of many transporters and membrane proteins to understand organellar transport and regulation. |
