Primary endocytic vesicles containing transmembrane cargo fuse with the early/sorting endosome, from which cargo can be sorted for delivery to the plasma membrane, the Golgi, or the lysosome. In many cases endosome to Golgi transport is controlled by the retromer coat complex, consisting of the cargo selective complex (Vps35, Vps29, and Vps26) and members of the sorting nexin family such as Snx1 and Snx6. The J-domain Hsc70 co-chaperone RME-8 associates with Snx1 in worms and human cells is required for efficient endosome-to-Golgi transport. The sorting of cargo bound for degradation into intralumenal vesicles occurs at least in part on the same endosomes. This process requires ESCRT complexes, including ESCRT-0, consisting of Hrs and Stam. Previous work indicated that the retromer component Snx1 can also bind to Hrs, suggesting that Snx1 may act as part of a competitive mechanism that balances the relative activities of retromer and ESCRT, possibly via maintenance of distinct endosomal subdomains. Taking advantage of the normally very large endosomes of the C. elegans coelomocyte, we have been investigating the nature of retromer - ESCRT interaction in vivo. We find that C. elegans ESCRT-0 component HGRS-1 (Hrs), and retromer component SNX-1, often reside on the same large early endosomes of the coleomocyte, but in clearly distinct subdomains. Such subdomains are motile but remain mostly distinct as they move around the endosome. HGRS-1(Hrs) positive subdomains are also complementary to those labeled by retrograde cargo MIG-14 or functional RME-8. Consistent with our hypothesis that ESCRT-0 and retromer compete for territory on the endosomal limiting membrane, we find subdomain separation is greatly impaired in snx-1 null mutants, rme-8 ts-mutants at the restrictive temperature, or upon expression of RME-8 protein containing critical point mutations in the J-domain. Under such conditions HGRS-1/Hrs labeled microdomains increase in both in size and intensity. Conversely, RNAi-mediated depletion of degradative component HGRS-1/Hrs results in over-accumulation of retrograde cargo MIG-14. Importantly, we find the loss of another sorting nexin, SNX-3, that does not interact with RME-8 but is required for MIG-14 recycling, does not alter the apparent subdomain architecture. We demonstrate this the effect of rme-8ts and snx-1 null mutants is not specific to retrograde cargo MIG-14, but also holds true for C. elegans TGN-38-GFP or human CIMPR-GFP expressed in the coelomocyte. Given that the same 50 amino acids of SNX-1 binds to both HGRS-1 and RME-8, we propose SNX-1 is the fulcrum of this competitive interaction. .

Affiliation:
- MBB, Rutgers University, New Brunswick, NJ