Identification of the interacting partners of a lysosomal membrane protein in living cells by BioID technique

Dat Nguyen-Tien; Takehiro Suzuki; Toshihiko Kobayashi; Noriko Toyama-Sorimachi; Naoshi Dohmae

doi:10.1016/j.xpro.2022.101263

Identification of the interacting partners of a lysosomal membrane protein in living cells by BioID technique

STAR Protoc. 2022 Mar 31;3(2):101263. doi: 10.1016/j.xpro.2022.101263. eCollection 2022 Jun 17.

Authors

Dat Nguyen-Tien¹, Takehiro Suzuki², Toshihiko Kobayashi¹, Noriko Toyama-Sorimachi¹, Naoshi Dohmae²

Affiliations

¹ Department of Molecular Immunology and Inflammation, Research Institute, National Center for Global Health and Medicine, 1-21-1 Toyama, Shinjuku, Tokyo 162-8655, Japan.
² Biomolecular Characterization Unit, RIKEN Center for Sustainable Resource Science, Saitama 351-0198, Japan.

Abstract

The purpose of this protocol is to screen and identify the physiologically relevant interactors of a lysosomal protein in living cells. Here, we describe how to identify solute carrier family 15 member 4 (SLC15A4)-interacting proteins by BioID and mass spectrometry analysis. This protocol utilizes fusion of SLC15A4 with a mutant form of biotin ligase, BirA. The fusion protein can promiscuously biotinylate the proteins proximal to SLC15A4. The biotinylated endogenous proteins are pulled down by magnetic streptavidin beads and detected by mass spectrometry analysis. For complete details on the use and execution of this protocol, please refer to Kobayashi et al. (2021).

Keywords: Cell-based Assays; Mass Spectrometry; Molecular Biology; Protein Biochemistry; Proteomics.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Biotinylation
Lysosomal Membrane Proteins
Mass Spectrometry
Proteins* / chemistry
Streptavidin

Substances

Lysosomal Membrane Proteins
Proteins
Streptavidin