Interacting partners of Golgi-localized small G protein Arl5b identified by a combination of in vivo proximity labelling and GFP-Trap pull down

FEBS Letters

article

Fiona J. Houghton¹  Christian Makhoul¹  Ellie Hyun-Jung Cho¹  Nicholas A. Williamson¹  Paul A. Gleeson¹ 

[1] The Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne;Biological Optical Microscopy Platform, The University of Melbourne;Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne

关键词: ACBD3/GCP60;    Arl5b;    in vivo proximity labelling;    membrane transport;    small G proteins;    tethering factors;    trans-Golgi network;   

DOI  :  10.1002/1873-3468.14443

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown