ARC Syndrome-Linked Vps33B Protein Is Required for Inflammatory Endosomal Maturation and Signal Termination.

Toll-like receptors (TLRs) and other pattern-recognition receptors (PRRs) sense microbial ligands and initiate signaling to induce inflammatory responses. Although the quality of inflammatory responses is influenced by internalization of TLRs, the role of endosomal maturation in clearing receptors and terminating inflammatory responses is not well understood. Here, we report that Drosophila and mammalian Vps33B proteins play critical roles in the maturation of phagosomes and endosomes following microbial recognition. Vps33B was necessary for clearance of endosomes containing internalized PRRs, failure of which resulted in enhanced signaling and expression of inflammatory mediators. Lack of Vps33B had no effect on trafficking of endosomes containing non-microbial cargo. These findings indicate that Vps33B function is critical for determining the fate of signaling endosomes formed following PRR activation. Exaggerated inflammatory responses dictated by persistence of receptors in aberrant endosomal compartments could therefore contribute to symptoms of ARC syndrome, a disease linked to loss of Vps33B.

Drosophila acinus encodes a novel regulator of endocytic and autophagic trafficking.

Endosomal trafficking affects many cellular pathways from cell signaling to metabolism, but little is known about how these effects are coordinated. In a genetic screen for mutants affecting endosomal trafficking, we identified Drosophila acinus (dacn; hook-like). Its mammalian homolog Acinus has been implicated in RNA processing and chromatin fragmentation during apoptosis. Loss-of-function analysis of dacn revealed two distinct functions. First, dacn is required for stabilization of early endosomes, thus modulating levels of Notch and Egfr signaling. Second, loss of dacn interferes with cellular starvation responses by inhibiting autophagosome maturation. By contrast, overexpression of dacn causes lethality due to enhanced autophagy. We show that this enhanced autophagy is independent of the Tor pathway. Taken together, our data show that dacn encodes a regulator of endosomal and autophagosomal dynamics, modulating developmental signaling and the cellular response to starvation.

The full-of-bacteria gene is required for phagosome maturation during immune defense in Drosophila.

Arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome is a fatal recessive disorder caused by mutations in the VPS33B or VPS16B genes. Both encode homologues of the Vps33p and Vps16p subunits of the HOPS complex necessary for fusions of vacuoles in yeast. Here, we describe a mutation in the full-of-bacteria (fob) gene, which encodes Drosophila Vps16B. Flies null for fob are homozygous viable and fertile. They exhibit, however, a defect in their immune defense that renders them hypersensitive to infections with nonpathogenic bacteria. fob hemocytes (fly macrophages) engulf bacteria but fail to digest them. Phagosomes undergo early steps of maturation and transition to a Rab7-positive stage, but do not mature to fully acidified phagolysosomes. This reflects a specific requirement of fob in the fusion of phagosomes with late endosomes/lysosomes. In contrast, cargo of autophagosomes as well as endosomes exhibit normal lysosomal delivery in fob cells. These findings suggest that defects in phagosome maturation may contribute to symptoms of ARC patients including recurring infections.

Drosophila Vps16A is required for trafficking to lysosomes and biogenesis of pigment granules.

Mutations that disrupt trafficking to lysosomes and lysosome-related organelles cause multiple diseases, including Hermansky-Pudlak syndrome. The Drosophila eye is a model system for analyzing such mutations. The eye-color genes carnation and deep orange encode two subunits of the Vps-C protein complex required for endosomal trafficking and pigment-granule biogenesis. Here we demonstrate that dVps16A (CG8454) encodes another Vps-C subunit. Biochemical experiments revealed a specific interaction between the dVps16A C-terminus and the Sec1/Munc18 homolog Carnation but not its closest homolog, dVps33B. Instead, dVps33B interacted with a related protein, dVps16B (CG18112). Deep orange bound both Vps16 homologs. Like a deep orange null mutation, eye-specific RNAi-induced knockdown of dVps16A inhibited lysosomal delivery of internalized ligands and interfered with biogenesis of pigment granules. Ubiquitous knockdown of dVps16A was lethal. Together, these findings demonstrate that Drosophila Vps16A is essential for lysosomal trafficking. Furthermore, metazoans have two types of Vps-C complexes with non-redundant functions.