Snx-3 Mediates Retromer-Independent Tubular Endosomal Recycling By Opposing Eea-1-Facilitated Trafficking

Early endosomes are the sorting hub on the endocytic pathway, wherein sorting nexins (SNXs) play important roles for formation of the distinct membranous microdomains with different sorting functions. Tubular endosomes mediate the recycling of clathrin-independent endocytic (CIE) cargoes back toward the plasma membrane. However, the molecular mechanism underlying the tubule formation is still poorly understood. Here we screened the effect on the ARF-6-associated CIE recycling endosomal tubules for all the SNX members in Caenorhabditis elegans (C. elegans). We identified SNX-3 as an essential factor for generation of the recycling tubules. The loss of SNX-3 abolishes the interconnected tubules in the intestine of C. elegans. Consequently, the surface and total protein levels of the recycling CIE protein hTAC are strongly decreased. Unexpectedly, depletion of the retromer components VPS-26/-29/-35 has no similar effect, implying that the retromer trimer is dispensable in this process. We determined that hTAC is captured by the ESCRT complex and transported into the lysosome for rapid degradation in snx-3 mutants. Interestingly, EEA-1 is increasingly recruited on early endosomes and localized to the hTAC-containing structures in snx-3 mutant intestines. We also showed that SNX3 and EEA1 compete with each other for binding to phosphatidylinositol-3-phosphate enriching early endosomes in Hela cells. Our data demonstrate for the first time that PX domain-only C. elegans SNX-3 organizes the tubular endosomes for efficient recycling and retrieves the CIE cargo away from the maturing sorting endosomes by competing with EEA-1 for binding to the early endosomes. However, our results call into question how SNX-3 couples the cargo capture and membrane remodeling in the absence of the retromer trimer complex.Author summary Trafficking of internalized materials through the endolysosomal system is essential for the maintenance of homeostasis and signaling regulation in all eukaryotic cells. Early endosomes are the sorting hub on the endocytic pathway. After internalization, the plasma membrane lipid, proteins, and invading pathogens are delivered to early endosomes for further degradation in lysosomes or for retrieval to the plasma membrane or the trans-Golgi network for reuse. However, when, where and by what mechanism various cargo proteins are sorted from each other and into the different pathways largely remain to be explored. Here, we identified SNX-3, a PX-domain only sorting nexin family member, as a novel regulator for the tubular endosomes underlying recycling of a subset of CIE cargoes. Compared with EEA-1, the superior recruitment of SNX-3 at the CIE-derived subpopulation of endosomes is critical for preventing these endosomes from converging to the classical sorting endosomes and subsequently into the multivesicular endosomal pathway. We speculate that through a spatio-temporal interplay with the retromer, SNX-3 is involved in different recycling transport carriers. Our finding of SNX-3's role in modulating the formation of tubular endosomes provides insight into the sorting and trafficking of CIE pathways.