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Lysosome transporter purification and reconstitution identifies Ypq1 pH-gated lysine transport and regulation

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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Autores principales: Arines, Felichi Mae, Wielenga, Aleksander, Burata, Olive E., Garcia, Francisco Narro, Stockbridge, Randy B., Li, Ming
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10081341/
https://www.ncbi.nlm.nih.gov/pubmed/37034749
http://dx.doi.org/10.1101/2023.03.31.535002
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author Arines, Felichi Mae
Wielenga, Aleksander
Burata, Olive E.
Garcia, Francisco Narro
Stockbridge, Randy B.
Li, Ming
author_facet Arines, Felichi Mae
Wielenga, Aleksander
Burata, Olive E.
Garcia, Francisco Narro
Stockbridge, Randy B.
Li, Ming
author_sort Arines, Felichi Mae
collection PubMed
description 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.
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spelling pubmed-100813412023-04-08 Lysosome transporter purification and reconstitution identifies Ypq1 pH-gated lysine transport and regulation Arines, Felichi Mae Wielenga, Aleksander Burata, Olive E. Garcia, Francisco Narro Stockbridge, Randy B. Li, Ming bioRxiv Article 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. Cold Spring Harbor Laboratory 2023-03-31 /pmc/articles/PMC10081341/ /pubmed/37034749 http://dx.doi.org/10.1101/2023.03.31.535002 Text en https://creativecommons.org/licenses/by-nc-nd/4.0/This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (https://creativecommons.org/licenses/by-nc-nd/4.0/) , which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
spellingShingle Article
Arines, Felichi Mae
Wielenga, Aleksander
Burata, Olive E.
Garcia, Francisco Narro
Stockbridge, Randy B.
Li, Ming
Lysosome transporter purification and reconstitution identifies Ypq1 pH-gated lysine transport and regulation
title Lysosome transporter purification and reconstitution identifies Ypq1 pH-gated lysine transport and regulation
title_full Lysosome transporter purification and reconstitution identifies Ypq1 pH-gated lysine transport and regulation
title_fullStr Lysosome transporter purification and reconstitution identifies Ypq1 pH-gated lysine transport and regulation
title_full_unstemmed Lysosome transporter purification and reconstitution identifies Ypq1 pH-gated lysine transport and regulation
title_short Lysosome transporter purification and reconstitution identifies Ypq1 pH-gated lysine transport and regulation
title_sort lysosome transporter purification and reconstitution identifies ypq1 ph-gated lysine transport and regulation
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10081341/
https://www.ncbi.nlm.nih.gov/pubmed/37034749
http://dx.doi.org/10.1101/2023.03.31.535002
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