Proteomic Analysis of ARF1‐ and ARF3‐Proximal Proteins at the Trans‐Golgi Network

ABSTRACT Despite its fundamental importance, the molecular mechanisms of post‐Golgi transport remain incompletely understood. Carrier formation of post‐Golgi transport depends on ARF3 and ARF1, very close paralogs; the former localizes on the trans ‐Golgi network (TGN) whereas the latter localizes on TGN and cis‐Golgi. Identifying ARF1/3 interactors on the TGN is critical for elucidating post‐Golgi transport mechanisms. To this end, we used GBF1 M832L cells, in which ARFs on TGN can be dispersed by the addition of Brefeldin A (BFA) via inhibition of trans ‐Golgi‐localized ARF nucleotide exchange factors BIG1/2, without disturbing cis‐localizing ARFs or Golgi structure. We performed proteomic analyses of ARF1 and ARF3 proximal proteins on TGN using TurboID boosted by membrane permeable biotin methyl ester in BFA‐treated and untreated GBF1 M832L cells. By comparing these conditions, proximal proteins specific to TGN‐localizing ARF1 or ARF3, and those common to both, were identified. The ARF1‐specific proximal protein SMAP1 and the ARF1/3 common proximal protein LRBA are BIG1/2‐dependently localized to the Golgi, whereas the ARF3‐specific proximal protein Rab3IL1 is BIG1/2‐independently localized on recycling endosomes. This study establishes an approach to selectively control post‐Golgi transport and map ARF‐dependent interaction networks at the TGN, providing a framework for understanding the molecular mechanisms underlying carrier formation.