Phosphoinositide conversion in endocytosis and the endolysosomal system

Abstract

Phosphoinositides (PIs) are phospholipids that perform crucial cell functions, ranging from cell migration and signaling to membrane trafficking, by serving as signposts of compartmental membrane identity. Although phosphatidylinositol 4,5-bisphosphate, 3-phosphate, and 3,5-bisphosphate are commonly considered as hallmarks of the plasma membrane, endosomes, and lysosomes, these compartments contain other functionally important PIs. Here, we review the roles of PIs in different compartments of the endolysosomal system in mammalian cells and discuss the mechanisms that spatiotemporally control PI conversion in endocytosis and endolysosomal membrane dynamics during endosome maturation and sorting. As defective PI conversion underlies human genetic diseases, including inherited myopathies, neurological disorders, and cancer, PI-converting enzymes represent potential targets for drug-based therapies.

Keywords: autophagy; endocytosis; endosome; lysosome; mTOR complex (mTORC); phosphatidylinositol; phosphatidylinositol kinase (PI Kinase); phosphatidylinositol phosphatase; phosphatidylinositol signaling.

Figure 1.

Interconversion of PIs by kinases and phosphatases. Phosphatidylinositol can be phosphorylated by PI4K to yield PI(4)P), which can be further phosphorylated by PIP5K to PI(4,5)P₂, which serves as a substrate for class I PI 3-kinases (Class I PI3K) to produce PI(3,4,5)P₃. Phosphorylation of PI at the 3-OH position by class I PI 3-kinase (termed Vps34) (Class III PI3K) yields PI(3)P that can be further phosphorylated by PIKFYVE to produce PI(3,5)P₂. PIKFYVE may also synthesize PI(5)P from PI. Class II PI 3-kinases (Class II PI3K) synthesize PI(3,4)P₂ from PI(4)P and PI(3)P from the PI. Myotubularins (MTMs) are 3-phosphatases that hydrolyze PI(3)P and PI(3,5)P₂. OCRL, synaptojanin 1/2, PIPP, SKIP, and INPP5E are PI(4,5)P₂ 5-phosphatases, Fig4 is a 5-phosphatase for PI(3,5)P₂. PI(3,4,5)P₃ can be dephosphorylated by the 3-phosphatases PTEN and TPIP to PI(4,5)P₂ or by the 5-phosphatases OCRL and SHIP1/2 to produce PI(3,4)P₂. The 4-phosphatases Sac1–3 and INPP4A/B dephosphorylate PI(4)P and PI(3,4)P₂, respectively.

Figure 2.

PI conversion in CME and in the endolysosomal system. Clathrin-mediated endocytosis requires plasma membrane PI(4,5)P₂, which is a substrate for the PI 5-phosphatases OCRL, synaptojanin, and SHIP1/2. Class II PI3Kα generates a plasma membrane pool of PI(3,4)P₂ necessary for CCP maturation and formation of free clathrin-coated vesicles (CCV). PI(3)P, an essential feature of early endosomes, is generated primarily by the class III PI3K Vps34 complex II with a possible contribution of class II PI3Ks (encircled by dashed line), either by direct PI(3)P synthesis or indirectly via PI(3,4)P₂ hydrolysis by the PI 4-phosphatases INPP4A/B. Endosomal recycling to the cell surface requires PI(3)P hydrolysis by myotubularin phosphatases (MTMs) such as MTM1 and the concomitant generation of PI(4)P by PI4KIIα to enable exocytosis. As endosomes mature into late endosomes/MVBs, the PI(3)P 5-kinase PIKFYVE converts PI(3)P into PI(3,5)P₂. PI(3,5)P₂ turnover at MVBs and/or lysosomes is mediated by MTMs together with the PI(3,5)P₂ 5-phosphatase Fig4. Lysosomal membranes contain several PIs such as PI(3)P, PI(4)P, and PI(4,5)P₂. PI(3)P can be produced by class III PI3K/Vps34 directly at the lysosome or is obtained by fusion with autophagosomes, where PI(3)P is produced by VPS34 complex I. PI(4)P is generated by PI4KIIIβ, and PI(4,5)P₂ is hydrolyzed by OCRL. PI(4)P can be converted to PI(3,4)P₂ by the class II PI3KC2β.

Figure 3.

Subunit composition and regulation of mTORC1. mTORC1 consists of the mTOR kinase, the complex defining subunit raptor mLST8, and the two negative regulators PRAS40 and DEPTOR. Activation of mTORC1 depends on recruiting the complex to its place of activation–the lysosomal surface–by Ras-related GTP-binding protein (Rag) GTPases. mTORC1 is fully activated by the RHEB GTPase downstream of AKT-dependent pathways. Furthermore, lysosomal PI(3)P and PI(3,5)P₂ play roles in mTORC1 activation. AMPK and PI(3,4)P₂ can inhibit mTORC1 activity by regulating the binding of raptor to inhibitory 14-3-3 proteins.

Figure 4.

Interplay between lysosome position and function and nutrient signals regulated by PIs. In the absence of nutrients and growth factors, lysosomes are transported retrogradely via dynein motors linked via Rab7/RILP. Growth factor deprivation also triggers PI3KC2β recruitment to lysosomes, where it generates PI(3,4)P₂ to suppress mTORC1 activity, and facilitates perinuclear clustering of lysosomes. High nutrient and growth factor conditions cause activation of the class III PI3K Vps34. Vps34-mediated synthesis of PI(3)P results in lysosomal recruitment of the kinesin 1 adaptor FYCO1, which may supply kinesin motors to the Arl8-associated adaptor SKIP/PLEKHM2. Lysosomes undergo anterograde transport resulting in their dispersion to the cell periphery and in activation of mTORC1, e.g. by growth factor-derived Akt signaling.