Our long-term goal in this proposal is to understand how endosomes differentially balance opposing activities (recycling versus degradation) within a single endosome, especially with respect to regulation by the only known endosomal DNA-J domain protein RME-8. We also seek to understand how RME-8 contributes to neurodegenerative disease. To gain new insights into the mechanisms that balance opposing endosomal functions, we have pioneered the use of the coelomocyte cells of the nematode C. elegans for the direct visualization of endosomal microdomains, and then applied powerful molecular genetic techniques to identify and decipher metazoan- specific mechanisms that control endosome function and functional microdomain separation. We then extend this work into mammalian systems. In previous studies, we identified the J-domain protein RME-8 as a metazoan specific endosome regulator. We further showed that RME-8 functions with Retromer component SNX-1/Snx1, and the chaperone Hsc70, to promote the recycling of retromer cargo in C. elegans and mammals. Most recently, we showed that RME-8 allows Retromer to negatively regulate ESCRT assembly on endosomes, a process required to prevent mixing of recycling and degradative microdomains. Here we propose to define how SNX-1 regulates RME-8, testing a de-repression model for RME-8 activation. We then focus on mechanistic questions of how RME-8 inhibits ESCRT microdomain expansion through the endosomal flat clathrin lattice and newly identified RME-8 interacting proteins. Finally, we use primary mouse neurons and in vivo C. elegans analysis to decipher neuronal functions of RME-8 as revealed by analysis of a familial Parkinson’s associated allele of RME-8. We propose to define how the disease allele affects RME-8 function in endosomal recycling and microdomain maintenance, and determine how RME-8 regulates long-distance axonal transport of endosomes, an additional transport step in neurons required for endocytic cargo sorting. A better mechanistic understanding of these regulatory mechanisms of endosomes will be profoundly important in identifying therapeutic targets to combat diseases associated with endolysosomal dysfunction.