1219 Research Article Introduction Mammalian cells contain three distinct types of phosphoinositide 3-OH kinase (PI 3-kinase) (Fruman et al., 1998; Vanhaesebroeck et al., 2001). Type III PI 3-kinase catalyzes the phosphorylation of phosphatidylinositol at the D- 3 position of the inositol ring, generating phosphatidylinositol 3-phosphate [PtdIns(3)P] (Odorizzi et al., 2000). The prototype for this type of enzyme, Vps34, was first identified in Saccharomyces cerevisiae, where it is one of several gene products required for delivery of soluble proteins to the vacuole (Schu et al., 1993; Herman et al., 1992) and for autophagic sequestration of cytoplasmic proteins during starvation (Kihara et al., 2001b; Wurmser and Emr, 2002). Under both circumstances, association of Vps34 with cellular membranes depends on a myristoylated regulatory subunit, Vps15 (Kihara et al., 2001b; Stack et al., 1995). In mammalian cells, Vps34 interacts with p150, a regulatory subunit similar to Vps15 (Panaretou et al., 1997). PtdIns(3)P is required for membrane recruitment of several proteins implicated in the regulation of vesicular transport and intracellular protein sorting (Corvera, 2001; Simonsen et al., 2001). Some of these proteins (e.g. sorting nexins) contain a PX phosphoinositide-binding domain (Song et al., 2001; Cheever et al., 2001; Kanai et al., 2001; Xu et al., 2001), whereas others contain a structural motif termed the FYVE domain that binds to PtdIns(3)P with high affinity (Corvera et al., 1999; Fruman et al., 1999; Wurmser et al., 1999). Specific FYVE-domain proteins [early endosomal antigen 1 (EEA1), Rabenosyn-5, Rabip4] interact with Rab GTPases that control vesicle docking and fusion in the early endocytic pathway (Nielsen et al., 2000; Simonsen et al., 1998; Cormont et al., 2001; Kauppi et al., 2002). Interestingly, some Rabs that function in the early (Rab5) and late (Rab7) steps of the endocytic pathway also interact with the p150 subunit of the mammalian Vps34 complex, suggesting that the synthesis of PtdIns(3)P and the recruitment of FYVE-domain proteins The human type III phosphatidylinositol 3-kinase, hVps34, converts phosphatidylinositol (PtdIns) to phosphatidylinositol 3-phosphate [PtdIns(3)P]. Studies using inhibitors of phosphatidylinositide 3-kinases have indicated that production of PtdIns(3)P is important for a variety of vesicle-mediated trafficking events, including endocytosis, sorting of receptors in multivesicular endosomes, and transport of lysosomal enzymes from the trans-Golgi network (TGN) to the endosomes and lysosomes. This study utilizes small interfering (si)RNA- mediated gene silencing to define the specific trafficking pathways in which hVps34 functions in human U-251 glioblastoma cells. Suppression of hVps34 expression reduced the cellular growth rate and caused a striking accumulation of large acidic phase-lucent vacuoles that contain lysosomal membrane proteins LAMP1 and LGP85. Analysis of these structures by electron microscopy suggests that they represent swollen late endosomes that have lost the capacity for inward vesiculation but retain the capacity to fuse with lysosomes. Morphological perturbation of the late endosome compartment was accompanied by a reduced rate of processing of the endosomal intermediate form of cathepsin D to the mature lysosomal form. There was also a reduction in the rate of epidermal growth factor receptor (EGFR) dephosphorylation and degradation following ligand stimulation, consistent with the retention of the EGFR on the limiting membranes of the enlarged late endosomes. By contrast, the suppression of hVps34 expression did not block trafficking of cathepsin D between the TGN and late endosomes, or endocytic uptake of fluid-phase markers, or association of a PtdIns(3)P-binding protein, EEA1, with early endosomes. LAMP1-positive vacuoles were depleted of PtdIns(3)P in the hVps34-knockdown cells, as judged by their inability to bind the PtdIns(3)P probe GFP-2xFYVE. By contrast, LAMP1-negative vesicles continued to bind GFP-2xFYVE in the knockdown cells. Overall, these findings indicate that hVps34 plays a major role in generating PtdIns(3)P for internal vesicle formation in multivesicular/late endosomes. The findings also unexpectedly suggest that other wortmannin-sensitive kinases and/or polyphosphoinositide phosphatases may be able to compensate for the loss of hVps34 and maintain PtdIns(3)P levels required for vesicular trafficking in the early endocytic pathway or the TGN. Key words: Endosome, Vps34, Phosphatidylinositol 3-kinase, Trafficking, Golgi, Lysosome Summary Gene silencing reveals a specific function of hVps34 phosphatidylinositol 3-kinase in late versus early endosomes Erin E. Johnson, Jean H. Overmeyer, William T. Gunning and William A. Maltese* Department of Biochemistry and Cancer Biology, Medical University of Ohio, Toledo, OH 43614, USA *Author for correspondence (e-mail: wmaltese@meduohio.edu) Accepted 9 December 2005 Journal of Cell Science 119, 1219-1232 Published by The Company of Biologists 2006 doi:10.1242/jcs.02833 Journal of Cell Science