Defining the proximal interaction networks of Arf GTPases reveals a mechanism for the regulation of PLD1 and PI4KB

Abstract

The Arf GTPase family is involved in a wide range of cellular regulation including membrane trafficking and organelle–structure assembly. Here, we have generated a proximity interaction network for the Arf family using the miniTurboID approach combined with TMT‐based quantitative mass spectrometry. Our interactome confirmed known interactions and identified many novel interactors that provide leads for defining Arf pathway cell biological functions. We explored the unexpected finding that phospholipase D1 (PLD1) preferentially interacts with two closely related but poorly studied Arf family GTPases, ARL11 and ARL14, showing that PLD1 is activated by ARL11/14 and may recruit these GTPases to membrane vesicles, and that PLD1 and ARL11 collaborate to promote macrophage phagocytosis. Moreover, ARL5A and ARL5B were found to interact with and recruit phosphatidylinositol 4‐kinase beta (PI4KB) at trans‐Golgi, thus promoting PI4KB's function in PI4P synthesis and protein secretion.

RUFY1 binds Arl8b and mediates endosome-to-TGN CI-M6PR retrieval for cargo sorting to lysosomes

“…S1, B and C ). In agreement with our results that both RUFY1 and RUFY3 interact with Arl8b, a recent study has identified both proteins as significant hits in the proximal interaction network of Arl8a and Arl8b ( Li et al, 2022 ).…”

ARFs get the BioID treatment: what have we been missing?

“…PLD1 is a somewhat enigmatic phospholipase that hydrolyses PC to form PA, with various poorly defined roles in signal transduction, endocytosis, exocytosis and membrane trafficking (McDermott et al , 2020). Previous work has indicated that Arf1/6 can regulate PLD1 but, somewhat counter‐intuitively, Li et al (2022) now clearly show that ARL11/14 binds to a distinct region of this protein (the so‐called “loop region”) in a GTP‐dependent manner, causing an impressive activation of PLD activity. Further, knock‐out of ARL11 reduces phagocytosis of zymosan particles in a macrophage‐like cell line, and knock‐out of PLD1 prevents the ability of heterologously expressed ARL11 Q67L (a GTPase defective mutant) to stimulate phagocytosis in the same cell line.…”

“…Proximity labelling was applied by Li et al (2022) to screen the proximal interactors of 25 members of the Arf family of small GTPases. Constitutively active mutants of each GTPase were fused to miniTurbo biotin ligase and stably expressed in HEK293 cells.…”

“…In uncovering previously unknown roles for Arf family GTPases, Li et al (2022) have demonstrated the power of proximity screening assays for looking at the function of large protein families. Now, the difficult but potentially highly rewarding task is to prioritise the 1,500+ leads, dissecting out which proteins physically interact, directly or indirectly with their respective Arf and which are in the neighbourhood for a reason that can be rationally interpreted.…”

“…Li et al (2022) have now applied the miniTurboID approach coupled to TMT‐LC–MS/MS to the Arfs. By taking the pragmatic and well‐validated approach of generating stable HEK293 cell lines expressing fusions between miniTurbo and GTPase mutants of the Arf proteins, they were able to specifically search for effectors of their GTP‐bound states.…”