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.