Proximity Interactome Map of the Vac14–Fig4 Complex Using BioID

Conversion between
phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-bisphosphate
on endosomal membranes is critical for the maturation of early endosomes
to late endosomes/lysosomes and is regulated by the PIKfyve–Vac14–Fig4
complex. Despite the importance of this complex for endosomal homeostasis
and vesicular trafficking, there is little known about how its activity
is regulated or how it interacts with other cellular proteins. Here,
we screened for the cellular interactome of Vac14 and Fig4 using proximity-dependent
biotin labeling (BioID). After independently screening the interactomes
of Vac14 and Fig4, we identified 89 high-confidence protein hits shared
by both proteins. Network analysis of these hits revealed pathways
with known involvement of the PIKfyve–Vac14–Fig4 complex,
including vesicular organization and PI3K/Akt signaling, as well as
novel pathways including cell cycle and mitochondrial regulation.
We also identified subunits of coatomer complex I (COPI), a Golgi-associated
complex with an emerging role in endosomal dynamics. Using proximity
ligation assays, we validated the interaction between Vac14 and COPI
subunit COPB1 and between Vac14 and Arf1, a GTPase required for COPI
assembly. In summary, this study used BioID to comprehensively map
the Vac14–Fig4 interactome, revealing potential roles for these
proteins in diverse cellular processes and pathways, including preliminary
evidence of an interaction between Vac14 and COPI. Data are available via ProteomeXchange with the identifier PXD027917.