Mouse anti-Human VPS16 monoclonal antibody, clone 3G21

Of the various phosphatidylinositol 3- kinases (PI3Ks), only the class III enzyme Vps34 has been shown to regulate phagosome maturation. During studies of phagosome maturation in THP-1 cells deficient in class IA PI3K p110 alpha, we discovered that this PI3K isoform is required for vacuole maturation to progress beyond acquisition of Rab7 leading to delivery of lysosomal markers. Bead phagosomes from THP-1 cells acquired p110 alpha and contained PI3P and PI(3,4,5) P3; however, p110 alpha and PI(3,4,5) P3 levels in phagosomes from p110 alpha knockdown cells were decreased. Phagosomes from p110 alpha knock down cells showed normal acquisition of both Rab5 and EEA-1, but were markedly deficient in the lysosomal markers LAMP-1 and LAMP-2, and the lysosomal hydrolase, beta-galactosidase. Phagosomes from p110 alpha deficient cells also displayed impaired fusion with Texas Red dextran-loaded lysosomes. Despite lacking lysosomal components, phagosomes from p110 alpha deficient cells recruited normal levels of Rab7, Rab-interacting lysosomal protein (RILP) and homotypic vacuole fusion and protein sorting (HOPs) components Vps41 and Vps16. The latter observations demonstrated that phagosomal Rab7 was active and capable of recruiting effectors involved in membrane fusion. Nevertheless, active Rab7 was not sufficient to bring about the delivery of lysosomal proteins to the maturing vacuole, which is shown for the first time to be dependent on a class I PI3K.

CORVET and HOPS are protein complexes mediating the maturation of early endosomes (EEs) into late endosomes (LEs)/vacuoles. These heterohexamers share four `core' components, Vps11, Vps16, Vps18 and Vps33, and differ in two specific subunits, CORVET Vps8 and Vps3 and HOPS Vps39 and Vps41. Whereas ablating HOPS-specific components has minor growth effects, ablating any CORVET constituent severely debilitates Aspergillus nidulans growth, buttressing previous work indicating that maturation of EEs into LEs is physiologically crucial. A genetic screen revealed that impairing the slt cation homeostasis pathway rescues the growth defect resulting from inactivation of the `core' protein Vps33. Subsequent genetic analyses showed that the defect resulting from lack of any one of the five other CORVET components could similarly be rescued by sltA Delta eliminating the slt regulator SltA. Whereas double deletants lacking functionally non-equivalent components of the CORVET and HOPS complexes are rescued by sltA Delta, those lacking functionally equivalent components are not, suggesting that intermediate `hybrid' complexes previously detected in yeast are physiologically relevant. vps3 Delta, vps8 Delta, vps39 Delta and vps41 Delta result in small vacuoles. This phenotype is remediable by sltA Delta in the case of CORVET-specific, but not in the case of HOPS-specific deletants, indicating that the slt- effect on vacuolar size necessitates HOPS.