Regulation of endocytic recycling in Caenorhabditis elegans

Eukaryotic endocytic pathway is important for the uptake, sorting, and the subsequential recycling or degradation processes of various cargos. It has been shown that the RME-1/EHD1 is a critical regulator of endocytic recycling. In C. elegans and MDCK cells, small GTPase RAB-10 has been specifically implicated in clathrin-independent cargo recycling. Alternatively, some cargos will be recycled from sorting endosomes to Golgi, and retromer complex was shown to be crucial for this transport in yeast and mammalian cells. In our studies, we analyzed RME-1 and RAB-10 regulated recycling pathway, exploring additional players in the process. First, we demonstrated that ALX-1 is required for endocytic recycling of specific basolateral cargo. The interaction of ALX-1 with RME-1 is required for this recycling process. In our yeast two-hybrid screen for RAB-10-interacting proteins, EHBP-1 and Arf6 GAP/CNT-1 were recovered and revealed to function together with RAB-10 regulating clathrin-independent cargo recycling. Loss of either EHBP-1 or CNT-1 produced rab-10-like cargo transport defects. Furthermore, we showed that EHBP-1 functions, as an unconventional effector, upstream of RAB-10. Nevertheless, similar to canonical Rab effectors, CNT-1 requires RAB-10 for the proper endosome localization. Collectively, our results demonstrate the functional connections of ALX-1/RME-1 and RAB-10/EHBP-1/CNT-1, provided insights into the detail mechanisms of RME-1 and RAB-10 recycling regulation. We also studied the retromer regulated retrograde transport in C. elegans. We demonstrated the physical interaction of RME-8 with retromer component ALX-1. Additionally, we showed that loss-of-function in rme-8 or snx-1, or depletion of C. elegans Hsc70 (HSP-1) by RNAi, disrupts endosome to Golgi transport of the retromer-dependent cargo protein MIG-14. Furthermore, we identified a previously unsuspected mechanism for the regulation of endosomal clathrin that is required for retrograde transport. We showed that loss of either RME-8, SNX-1 or HSP-1 leads to endosomal clathrin dynamics defect. Our work indicates that, through RME-8 and Hsc70, the retromer acts to limit clathrin accumulation, a prerequisite for the recycling of retrograde cargo.Ph.D.Includes abstractVitaIncludes bibliographical referencesby Anbing Sh