Interacting partners of Golgi‐localized small G protein Arl5b identified by a combination of in vivo proximity labelling and GFP‐Trap pull down. Issue 18 (1st August 2022)

• Record Type: Journal Article
• Authors: Houghton, Fiona J.; Makhoul, Christian; Cho, Ellie Hyun‐Jung; Williamson, Nicholas A.; Gleeson, Paul A.
• Is Part Of: FEBS letters. Volume 596:Issue 18(2022)
• Page Start: 2382
• Page End: 2399
• Publication Date: 2022-08-01
• Subjects: ACBD3/GCP60 -- Arl5b -- in vivo proximity labelling -- membrane transport -- small G proteins -- tethering factors -- trans‐Golgi network; Biochemistry -- Periodicals; Biophysics -- Periodicals; Molecular biology -- Periodicals; Biochimie -- Périodiques; Biochemistry; Biophysics; Molecular biology; Periodicals; 572.05
• DOI: 10.1002/1873-3468.14443
• Languages: English
• ISSNs: 0014-5793

Abstract

Abstract : 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. Abstract : Identifying interactomes of membrane‐associated small G proteins is challenging. Here, we applied three proteomic approaches to identify the interactive neighbourhood of constitutively active Arl5b, a small G protein of the trans ‐Golgi network (TGN). 50% of the hits were TGN/Golgi‐localised small G proteins, indicating an organised complex of small G proteins. Also identified was the tethering molecule ACDB3, and its TGN recruitment was shown to be Arl5b‐dependent.