Lysosomal degradation of cytoplasmic components by autophagy ensures the continuous turnover of proteins and organelles and aids cellular survival during nutrient deprivation and other stress conditions. Lysosomal targeting of cytoplasmic proteins and organelles requires the concerted action of several proteins and multisubunit complexes. The core components of this machinery are conserved from yeast to humans and many of them are well-characterized; however, novel molecular players have been recently discovered and are waiting for detailed analysis. The osteopetrosis-linked PLEKHM1 protein is a lysosomal adaptor involved in autophagosome and endosome to lysosome fusion events and its role in lysosomal positioning in osteoclasts was reported together with its proposed binding partner, the relatively uncharacterized DEF8 protein. Here, we report the generation and subsequent analysis of novel mutant alleles of Drosophila plekhm1 and def8. Interestingly, the CRISPR-generated null mutations of these genes do not have any obvious effects on autophagy in Drosophila tissues, even though RNAi knockdown of these genes seems to perturb autophagy. Although these results are quite surprising and raise the possibility of compensatory changes in the case of null mutants, the new alleles will be valuable tools in future studies to understand the cellular functions of Drosophila Plekhm1 and Def8 proteins.

Proper balance of exocytosis and endocytosis is important for the maintenance of plasma membrane lipid and protein homeostasis. This is especially critical in human podocytes and the podocyte-like Drosophila nephrocytes that both use a delicate diaphragm system with evolutionarily conserved components for ultrafiltration. Here we show that the sorting nexin 25 homolog Snazarus (Snz) binds to Rab11 and localizes to Rab11-positive recycling endosomes in Drosophila nephrocytes, unlike in fat cells where it is present in plasma membrane/lipid droplet/ER contact sites. Loss of Snz leads to redistribution of Rab11 vesicles from the cell periphery and increases endocytic activity in nephrocytes. These changes are accompanied by defects in diaphragm protein distribution that resemble those seen in Rab11 gain-of-function cells. Of note, co-overexpression of Snz rescues diaphragm defects in Rab11 overexpressing cells, whereas snz knockdown in Rab11 overexpressing nephrocytes or simultaneous knockdown of snz and tbc1d8b encoding a Rab11 GAP lead to massive expansion of the lacunar system that contains mislocalized diaphragm components: Sns and Pyd/ZO-1. We find that loss of Snz enhances while its overexpression impairs secretion, which, together with genetic epistasis analyses, suggest that Snz counteracts Rab11 to maintain the diaphragm via setting the proper balance of exocytosis and endocytosis. [Media: see text] [Media: see text] [Media: see text]