Abstract
SNX18 and SNX9 are members of a subfamily of SNX (sorting nexin) proteins with the same domain structure. Although a recent report showed that SNX18 and SNX9 localize differently in cells and appear to function in different trafficking pathways, concrete evidence regarding whether they act together or separately in intracellular trafficking is still lacking. Here, we show that SNX18 has a similar role to SNX9 in endocytic trafficking at the plasma membrane, rather than having a distinct role. SNX18 and SNX9 are expressed together in most cell lines, but to a different extent. Like SNX9, SNX18 interacts with dynamin and stimulates the basal GTPase activity of dynamin. It also interacts with neuronal Wiskott-Aldrich syndrome protein (N-WASP) and synaptojanin, as does SNX9. SNX18 and SNX9 can form a heterodimer and colocalize in tubular membrane structures. Depletion of SNX18 by small hairpin RNA inhibited transferrin uptake. SNX18 successfully compensates for SNX9 deficiency during clathrin-mediated endocytosis and vice versa. Total internal reflection fluorescence microscopy in living cells shows that a transient burst of SNX18 recruitment to clathrin-coated pits coincides spatiotemporally with a burst of dynamin and SNX9. Taken together, our results suggest that SNX18 functions with SNX9 in multiple pathways of endocytosis at the plasma membrane and that they are functionally redundant.
Highlights
Endocytic pathways take membrane receptors and extracellular components into the cell, leading to activation of intracellular signaling pathways
We show that SNX18 and sorting nexin 9 (SNX9) are functionally redundant, and that SNX18 has a similar role to SNX9 in clathrinmediated endocytosis, rather than a distinct role in endosomal trafficking
SNX18 interacts with dynamin, neuronal Wiskott-Aldrich syndrome protein (N-WASP) and synaptojanin – well-known binding partners of SNX9
Summary
Endocytic pathways take membrane receptors and extracellular components into the cell, leading to activation of intracellular signaling pathways. Clathrin-mediated endocytosis begins by gathering membrane proteins and lipids through interactions with cytosolic adaptors and accessory factors, which bind to clathrin and initiate polymerization of triskeletal clathrin molecules into a spherical basket of clathrincoated pits (CCPs) (Schmid et al, 2006; Schmid and McMahon, 2007). The accessory proteins, such as amphiphysin, endophilin and sorting nexin 9 (SNX9), are concentrated at the membrane, inducing membrane curvature and the formation of intermediate tubular membrane structures (Farsad et al, 2001; Masuda et al, 2006; Peter et al, 2004; Shin et al, 2008; Takei et al, 1999). The SH3 domain is involved in interactions with endocytic proteins and actin regulatory proteins such as dynamin and synaptojanin (Evergren et al, 2007; Hill et al, 2001; Kim et al, 2005; Soulet et al, 2005)
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