Monitoring Editor: Sean Munro Yeast and animal homotypic fusion and vacuole protein sorting (HOPS) complexes contain conserved subunits, but HOPS-mediated traffic in animals might require additional proteins. Here we demonstrate that SPE-39 homologues, which are found only in animals, are present in RAB5-, 7- and 11-positive endosomes where they play a conserved role in lysosomal delivery and probably function via their interaction with the core HOPS complex. While C. elegans
spe-39 mutants were initially identified as having abnormal vesicular biogenesis during spermatogenesis, we show that these mutants also have disrupted processing of endocytosed proteins in oocytes and coelomocytes. C. elegans SPE-39 interacts in vitro with both VPS33A and VPS33B while RNAi of VPS33B causes
spe-39-like spermatogenesis defects. The human SPE-39 ortholog, C14orf133, also interacts with VPS33 homologues and both coimmunoprecipitates and cosediments with other HOPS subunits. SPE-39 knockdown in cultured human cells altered the morphology of syntaxin 7-, 8-, and 13-positive endosomes. These effects occurred concomitantly with delayed mannose 6-phosphate receptor-mediated cathepsin D delivery and degradation of internalized epidermal growth factor receptors. Our findings establish that SPE-39 proteins are a previously unrecognized regulator of lysosomal delivery and that C. elegans spermatogenesis is an experimental system useful for identifying conserved regulators of metazoan lysosomal biogenesis.