SAC-1 Ensures Epithelial Endocytic Recycling By Restricting ARF-6 Activity

Arf6/ARF-6 is a crucial regulator of the endosomal PI(4,5)P2 pool in endocytic recycling.To further characterize ARF-6 regulation,we performed an ARF-6 interactor screen in C.elegans and identified SAC-1,the homolog of the phosphoinositide phosphatase Sac1 p in yeast,as a novel ARF-6 partner.In the absence of ARF-6,basolateral endosomes show a loss of SAC-1 staining in epithelial cells.Steady-state cargo distribution assays revealed that loss of SAC-1 specifically affected apical secretory delivery and basolateral recycling.PI(4,5)P2 levels and the endosomal labeling of ARF-6 effector UNC-16 were significantly elevated in sac-1 mutants,suggesting that SAC-1 functions as a negative regulator of ARF-6.Further analyses revealed an interaction between SAC-1 and the ARF-6-GEF BRIS-1.This interaction outcompeted ARF-6(GDP)for binding to BRIS-1 in a concentration-dependent manner.Consequently,loss of SAC-1 promotes the intracellular overlap between ARF-6 and BRIS-1.BRIS-1 knockdown resulted in a significant reduction in PI(4,5)P2 levels in SAC-1-depleted cells.Interestingly,the action of SAC-1 in sequestering BRIS-1 is independent of SAC-1’s catalytic activity.Our results suggest that the interaction of SAC-1 with BRIS-1 curbs ARF-6 activity by limiting the access of ARF-6(GDP)to its GEF BRIS-1.Objective: To study the regulatory mechanisms of phosphatidylinositol phosphatase Sac1p/SAC-1 involved in the clathrin-independent endocytic(CIE)recycling transport in C.elegans intestinal epithelial cells.Methods: It is known that the small GTPase Rab10/RAB-10 is a key regulatory factor in the clathrin-independent endocytic recycling pathway.In particular,rab-10 mutants overaccumulate h TAC-GFP in enlarged structures in the deep cytosol of intestinal epithelia.Thus,we performed a genome-wide RNAi screen using rab-10(RNAi)as the positive control to identify candidate genes whose expression knockdown lead to the h TAC-GFP overaccumulation in the intestine.After initial screen and the follow-up validation,we noted that RNAi-mediated knockdown of SAC-1 resulted in intracellular hTAC-GFP aggregation.This observation suggested that SAC-1 is required for the clathrin-independent endocytic recycling.Further screening via GST pull-down assays revealed that SAC-1 specifically interacts with the small GTPase Arf6/ARF-6,which is essential for the basolateral recycling flow of clathrin-independent cargo.We then developed a transgenic strain sac-1(ycx18),an intestine-specific CRISPR/Cas9 somatic mutant,and a large number of transgenic fluorescent-labeled animals by genetic and cell biology methods.With the utilization of laser confocal microscopy,we analyzed the ARF-6-related proteins in the intestinal cells of sac-1(ycx18)mutant animals to investigate whether and how ARF-6 is regulated.Results: In the intestinal epithelial of C.elegans,we observed that loss of SAC-1 resulted in the block of clathrin-independent recycling cargos,abnormally elevated PI(4,5)P2 levels,mislocalization of apical junctional complex and significantly accumulation of ARF-6 putative effector JIP3/UNC-16.In arf-6(tm1447)mutants,the labeling of GFP-SAC-1 in basolateral puncta was significantly diminished.Specially,we found that SAC-1 displayed a robust protein-protein interaction with BRIS-1/ARF-6-GEF through its SAC domain,whereas the catalytic activity of SAC domain is not implicated in the interaction with BRIS-1.Thus far,our experiments demonstrated that SAC-1 competes with ARF-6(GDP)for interaction with BRIS-1/ARF-GEF in a concentration-dependent manner,thereby interfering with the interaction of BRIS-1 and ARF-6(GDP)and restricting ARF-6 activity.On the other hand,SAC-1 deletion also led to defects in clathrin-dependent endocytic recycling transport and apical secretory transport in intestinal epithelia.Conclusions: Our experiments identify SAC-1 as a novel ARF-6 interactor that mediates negative feedback regulation of ARF-6 in C.elegans intestinal cells,likely with the involvement of ARF-6 GFE protein BRIS-1.