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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...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Cold Spring Harbor Laboratory
2023
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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. |
format | Online Article Text |
id | pubmed-10081341 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Cold Spring Harbor Laboratory |
record_format | MEDLINE/PubMed |
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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