Lysosomal membrane transporter purification and reconstitution for functional studies

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 pla...

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Bibliographic Details
Published in:	Molecular biology of the cell Vol. 35; no. 3; p. ar28
Main Authors:	Arines, Felichi Mae, Wielenga, Aleksander, Henn, Danielle, Burata, Olive E, Garcia, Francisco Narro, Stockbridge, Randy B, Li, Ming
Format:	Journal Article
Language:	English
Published:	United States American Society for Cell Biology 01-03-2024
Subjects:	Analysis Cell membranes Chemical processes Identification and classification Lysine - metabolism Lysosomes Lysosomes - metabolism Membrane Proteins - metabolism Membrane Transport Proteins - metabolism Methods Saccharomyces cerevisiae - metabolism Saccharomyces cerevisiae Proteins - metabolism Ubiquitin-proteasome system United States
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Summary:	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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1059-1524 1939-4586
DOI:	10.1091/mbc.E23-06-0259