In vivo, Pikfyve generates PI(3,5)P ₂ , which serves as both a signaling lipid and the major precursor for PI5P

Abstract

Mutations that cause defects in levels of the signaling lipid phosphatidylinositol 3,5-bisphosphate [PI(3,5)P 2 ] lead to profound neurodegeneration in mice. Moreover, mutations in human FIG4 predicted to lower PI(3,5)P 2 levels underlie Charcot–Marie–Tooth type 4J neuropathy and are present in selected cases of amyotrophic lateral sclerosis. In yeast and mammals, PI(3,5)P 2 is generated by a protein complex that includes the lipid kinase Fab1/Pikfyve, the scaffolding protein Vac14, and the lipid phosphatase Fig4. Fibroblasts cultured from Vac14 −/− and Fig4 −/− mouse mutants have a 50% reduction in the levels of PI(3,5)P 2 , suggesting that there may be PIKfyve-independent pathways that generate this lipid. Here, we characterize a Pikfyve gene-trap mouse ( Pikfyve β -geo/ β -geo ), a hypomorph with ∼10% of the normal level of Pikfyve protein. shRNA silencing of the residual Pikfyve transcript in fibroblasts demonstrated that Pikfyve is required to generate all of the PI(3,5)P 2 pool. Surprisingly, Pikfyve also is responsible for nearly all of the phosphatidylinositol-5-phosphate (PI5P) pool. We show that PI5P is generated directly from PI(3,5)P 2 , likely via 3′-phosphatase activity. Analysis of tissues from the Pikfyve β -geo/ β -geo mouse mutants reveals that Pikfyve is critical in neural tissues, heart, lung, kidney, thymus, and spleen. Thus, PI(3,5)P 2 and PI5P have major roles in multiple organs. Understanding the regulation of these lipids may provide insights into therapies for multiple diseases.

Up-regulation of lysosomal TRPML1 channels is essential for lysosomal adaptation to nutrient starvation

“…Nutrient deprivation inactivates Rag GTPases, which may mediate the recruitment of mTORC1 and TFEB to lysosomes (35). Nutrient deprivation also results in a rapid decrease in lysosomal PI(3,5)P 2 levels, which have been reported to affect mTOR localization and activity (24,36,37). The role of PI(3,5)P 2 in TFEB activation was investigated by using two different inhibitors of the PI(3,5)P 2 -synthesizing enzyme, PIKfyve: YM201636 (38) and Apilimod (39).…”

“…The level of PI(3,5)P 2 is reduced upon starvation, but rapidly reelevated upon readdition of AAs or growth factors (8,24,37). Although starvation initially (<0.5 h after starvation) reduces global PI(3,5)P 2 levels, thereby causing TFEB activation and subsequent ML1 up-regulation, upon prolonged starvation (>2-4 h), the efflux of lysosomal AAs that are produced during the course of lysosomal degradation may readily trigger newly synthesis (i.e., resynthesis) of PI(3,5)P 2 , thereby causing mTOR reactivation and TFEB inactivation (8,16,24). In addition, PI(3,5)P 2 levels may also increase transiently and locally in lysosomes, presumably in a nutrient-independent manner, to regulate lysosomal membrane trafficking (36).…”

“…Through transcriptional, translational, and posttranslational mechanisms, lysosomal ML1 channel activity is dramatically up-regulated to increase lysosomal Ca 2+ release, thereby promoting both autophagosome-lysosome fusion and lysosome reformation from autolysosomes. Note that upon completion of degradation during prolonged starvation, the efflux of lysosomal AAs may cause mTOR reactivation and PI(3,5)P 2 resynthesis (8,16,24). In addition, PI(3,5)P 2 levels may also increase transiently and locally in lysosomes, presumably in a nutrient-independent manner, to regulate lysosomal membrane trafficking (36).…”

“…ML1-mediated lysosomal Ca 2+ release may regulate many aspects of lysosomal trafficking, including lysosome to trans-Golgi-network (TGN) retrograde trafficking, autophagosome-lysosome fusion, lysosome reformation, and lysosomal exocytosis (15,19,22,23). Moreover, it has been demonstrated that nutrient starvation affects phosphoinositide dynamics and Ca 2+ signaling (16,18,24). Therefore, the objective of this study was to investigate nutrient regulation of endogenous ML1 channels and the role of such regulation in lysosomal adaptation.…”

Lipid Sorting and Multivesicular Endosome Biogenesis

“…4), which is found in a complex with PIKfyve (Duex et al 2006), showing that synthesis and turnover are tightly coupled. The activity of MTM/MTMR phosphatase may also generate PI(5)P (Zolov et al 2012;Oppelt et al 2013), a minor lipid that can be increased by various stimuli and might also play a role in lysosomal targeting (Fig. 3) (Ramel et al 2011).…”