将液泡膜转运蛋白Ypq1纯化并重组到蛋白脂质体中的实验方案。

Protocol for purifying and reconstituting a vacuole membrane transporter Ypq1 into proteoliposomes.

作者信息

Arines Felichi Mae, Wielenga Aleksander, Stockbridge Randy B, Li Ming

机构信息

Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA.

出版信息

STAR Protoc. 2024 Dec 20;5(4):103483. doi: 10.1016/j.xpro.2024.103483. Epub 2024 Dec 10.

DOI:10.1016/j.xpro.2024.103483

PMID:39661504

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11697543/

Abstract

Studying the biochemical function of membrane transporters is important in understanding the biology of transporter-laden organelles such as lysosomes and vacuoles. We present a protocol for overexpressing, purifying, and reconstituting a vacuole membrane transporter Ypq1 into proteoliposomes and describe steps to measure transport activity using radioactive substrates. The protocols established here can be used to study other vacuolar or lysosomal membrane transporters. For complete details on the use and execution of this protocol, please refer to Arines et al..

摘要

研究膜转运蛋白的生化功能对于理解诸如溶酶体和液泡等富含转运蛋白的细胞器的生物学特性至关重要。我们提供了一种将液泡膜转运蛋白Ypq1过表达、纯化并重构到蛋白脂质体中的方案，并描述了使用放射性底物测量转运活性的步骤。此处建立的方案可用于研究其他液泡或溶酶体膜转运蛋白。有关本方案使用和执行的完整详细信息，请参考阿林斯等人的文献。