The mammalian phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) regulates endosome-to-TGN retrograde transport

Abstract

The yeast gene fab1 and its mammalian orthologue Pip5k3 encode the phosphatidylinositol 3-phosphate [PtdIns(3)P] 5-kinases Fab1p and PIKfyve, respectively, enzymes that generates phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2]. A shared feature of fab1Δ yeast cells and mammalian cells overexpressing a kinase-dead PIKfyve mutant is the formation of a swollen vacuolar phenotype: a phenotype that is suggestive of a conserved function for these enzymes and their product, PtdIns(3,5)P2, in the regulation of endomembrane homeostasis. In the current study, fixed and live cell imaging has established that, when overexpressed at low levels in HeLa cells, PIKfyve is predominantly associated with dynamic tubular and vesicular elements of the early endosomal compartment. Moreover, through the use of small interfering RNA, it has been shown that suppression of PIKfyve induces the formation of swollen endosomal structures that maintain their early and late endosomal identity. Although internalisation, recycling and degradative sorting of receptors for epidermal growth factor and transferrin was unperturbed in PIKfyve suppressed cells, a clear defect in endosome to trans-Golgi-network (TGN) retrograde traffic was observed. These data argue that PIKfyve is predominantly associated with the early endosome, from where it regulates retrograde membrane trafficking to the TGN. It follows that the swollen endosomal phenotype observed in PIKfyve-suppressed cells results primarily from a reduction in retrograde membrane fission rather than a defect in multivesicular body biogenesis.

Core Protein Machinery for Mammalian Phosphatidylinositol 3,5-Bisphosphate Synthesis and Turnover That Regulates the Progression of Endosomal Transport

“…Thus, expression of PIKfyve K1831E resulted in early endosome enlargement, high colocalization with early endosome markers, vacuole formation, accelerated rate of endosome fusion, and reduced number of intraluminal vesicles in MVB-like structures, with changes largely depending on the duration of expression (10,21). Noteworthy, some of these changes are remarkably similar to those recently observed by PIKfyve protein depletion (20) and reduced PtdIns(3)P-to-PtdIns(3,5)P 2 conversion that is now measured in our study (Fig. 5D).…”

“…Recent studies in higher eukaryotes (15,16,20) together with our observations about the effects of PIKfyve on the performance of trafficking pathways (10,19,21,22,49,63) provide a basis for definitive conclusions. Thus, there is a consensus that perturbed PtdIns(3,5)P 2 (by dominant-negative PIKfyve K1831E or PIKfyve protein depletion) in mammalian cells does not affect to a significant degree the receptor internalization, recycling, trafficking to lysosomes and degradation, or cargo sorting in the biosynthetic pathway (20,22).…”

“…Perturbed PtdIns(3,5)P 2 synthesis, however, delays the fluid-phase transport to lysosomes at a post-early endosomal step in both mammalian and fruit fly Garland cells (16,22). In addition, in mammalian cells PIKfyve protein depletion was found to impair the early endosome-to-TGN traffic and proper endosome processing of GLUT4-containing vesicles in response to insulin (20,49,63). These data, together with the unconditional gross enlargement of mammalian early endosomes observed under these conditions, are consistent with a rate-limiting function of the PIKfyve-catalyzed PtdIns(3)P-to-PtdIns(3,5)P 2 conversion in the course of cargo transport and exit from early endosomes to later destinations, as we have suggested previously (21,22,48).…”

“…Thus, there is a consensus that perturbed PtdIns(3,5)P 2 (by dominant-negative PIKfyve K1831E or PIKfyve protein depletion) in mammalian cells does not affect to a significant degree the receptor internalization, recycling, trafficking to lysosomes and degradation, or cargo sorting in the biosynthetic pathway (20,22). By and large, similar conclusions emerge from cell studies in D. melanogaster and C. elegans PIKfyve mutants, except that receptor degradation in the fruit fly shows a greater sensitivity to the PtdIns(3,5)P 2 reduction (15,16).…”

“…By contrast, ECV/MVB formation/detachment, but not the inward invagination of the MVE limiting membrane, is reportedly dependent on annexin2 (37). Whether the enlarged size of MVEs observed in cell models with loss-of-function or expressing dominant-negative mutants of PIKfyve (10,16,19,20) is associated with arrested ECV/MVB formation/detachment (or maturation) because of perturbed PtdIns(3,5)P 2 endosome membrane remodeling has never been examined.…”