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

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.