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Interacting partners of Golgi‐localized small G protein Arl5b identified by a combination of in vivo proximity labelling and GFP‐Trap pull down
Author(s) -
Houghton Fiona J.,
Makhoul Christian,
Cho Ellie HyunJung,
Williamson Nicholas A.,
Gleeson Paul A.
Publication year - 2022
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1002/1873-3468.14443
Subject(s) - golgi apparatus , green fluorescent protein , endosome , microbiology and biotechnology , in vivo , labelling , transport protein , in vitro , biology , chemistry , biochemistry , gene , genetics , endoplasmic reticulum , intracellular
The small G protein Arl5b is localised on the trans ‐Golgi network (TGN) and regulates endosomes‐to‐TGN transport. Here, we combined in vivo and in vitro techniques to map the interactive partners and near neighbours of Arl5b at the TGN, using constitutively active, membrane‐bound Arl5b(Q70L)‐GFP in stably expressing HeLa cells, and the proximity labelling techniques BioID and APEX2 in parallel with GFP‐Trap pull down. From MS analysis, 22 Golgi proteins were identified; 50% were TGN‐localised Rabs, Arfs and Arls. The scaffold/tethering factors ACBD3 (GCP60) and PIST (GOPC) were also identified, and we show that Arl5b is required for TGN recruitment of ACBD3. Overall, the combination of in vivo labelling and direct pull downs indicates a highly organised complex of small G proteins on TGN membranes.