GABARAP-mediated targeting of PI4K2A/PI4KIIα to autophagosomes regulates PtdIns4P-dependent autophagosome-lysosome fusion

Abstract

For decades, phosphatidylinositol 4-phosphate (PtdIns4P) was considered primarily as a precursor in the synthesis of phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P₂). More recently, specific functions for PtdIns4P itself have been identified, particularly in the regulation of intracellular membrane trafficking. PI4K2A/PI4KIIα (phosphatidylinositol 4-kinase type 2 α), one of the 4 enzymes that catalyze PtdIns4P production in mammalian cells, promotes vesicle formation from the trans-Golgi network (TGN) and endosomes. We recently identified a novel function for PI4K2A-derived PtdIns4P, as a facilitator of autophagosome-lysosome (A-L) fusion. We further showed that that this function requires the presence of the autophagic adaptor protein GABARAP (GABA[A] receptor-associated protein), which binds to PI4K2A and recruits it to autophagosomes. The mechanism whereby GABARAP-PI4K2A-PtdIns4P promotes A-L fusion remains to be defined. Based on other examples of phosphoinositide involvement in membrane trafficking, we speculate that it acts by recruiting elements of the membrane docking and fusion machinery.

Phosphoinositides control nearly every aspect of intracellular membrane trafficking, including multiple steps of the complex autophagy pathway. In degradative endocytosis and autophagy, attention has focused primarily on the role of phosphatidylinositol 3-phosphate. In contrast, little is known about the role of PtdIns4P in autophagy. PtdIns4P, and its downstream metabolite PtdIns(4,5)P₂, are the most abundant of the 7 phosphoinositides in vertebrates. In mammals, PtdIns4P is produced by phosphorylation of phosphatidylinositol (PtdIns) by 4 PtdIns 4-kinase (PtdIns4K) isoforms: PI4K2A and PIK42B and PI4KA/PI4KIIIα and PI4KB/PI4KIIIβ. PI4KA (yeast Stt4) is largely cytosolic, but a portion is recruited to the plasma membrane, where it contributes to replenishment of PtdIns(4,5)P2 pools turned over by receptor activation. PI4KB (yeast Pik1), is predominantly recruited to the Golgi and participates in the formation of transport vesicles. Prior to our study, PI4KB was the only PtdIns4K isoform implicated in autophagy. Here we discuss our recent finding that PtdIns4P, generated by PI4K2A, is critical for autophagosome-lysosome (A-L) fusion.

Using anti-PtdIns4P antibodies and the PtdIns4P reporter, GFP-OSBP-PH (a GFP-tagged version of the PtdIns4P-binding pleckstrin homology domain of OSBP [oxysterol binding protein]), we first confirmed previous reports demonstrating that PtdIns4P is particularly abundant in the perinuclear Golgi region under nutrient-rich conditions. Brief nutrient deprivation induces the formation of PtdIns4P-containing peripheral vesicles, more than half of which were identified as autophagosomes by the presence of GFP-LC3. PI4K2A, which is also enriched in the Golgi under nutrient-rich conditions, undergoes a major redistribution from the Golgi to LC3-positive vesicles upon autophagy induction, and depletion of PI4K2A significantly reduces the concentration of PtdIns4P in autophagosomes. Unlike the type III PI4K enzymes, which are recruited to membranes from the cytosol, nearly the entire cellular pool of PI4K2A is constitutively membrane bound due to palmitoylation of multiple cysteines in a CCPCC motif within the catalytic domain. Mutation of the 4 cysteines in this motif prevents palmitoylation of the kinase, as well as its translocation to autophagosomes.

In examining mechanisms of PI4K2A recruitment to autophagosomes, we followed up on a prior report that PI4K2A binds to the autophagic adaptor protein, GABARAP. GABARAP and its close relatives, GABARAPL1 and GABARAPL2/GATE-16, comprise one subfamily of the Atg8 family of ubiquitin-like proteins. These proteins are anchored into autophagosomal membranes by post-translational conjugation to phosphatidylethanolamine. We confirmed by co-immunoprecipitation assays that PI4K2A interacts with both GABARAP and GABARAPL2, but not with LC3, the other major subfamily of Atg8 proteins. Depletion of GABARAP prevents PI4K2A translocation to autophagosomes, whereas depletion of PI4K2A does not affect GABARAP localization. Thus, GABARAP functions upstream of PI4K2A in autophagosome targeting. In view of the established role of PI4K2A-PtdIns4P in Golgi budding, it is interesting that both GABARAP and GABARAPL2 were first characterized as intracellular trafficking factors. Because both PI4K2A and GABARAP are lipidated and enriched in the TGN, we speculate that they comigrate to autophagosomes on Golgi-derived transport vesicles (Fig. 1).

Effects of siRNA-mediated depletion of either GABARAP or PI4K2A point to a role for the 2 proteins in A-L fusion. Both siGABARAP and siPI4K2A cells display abnormally large autophagosomes, impaired degradation of LC3 and SQSTM1/p62, and impaired autophagosome acidification, which occurs upon fusion with lysosomes. Acidification of lysosomes per se is unaffected. We further showed that PI4K2A-derived PtdIns4P is required for A-L fusion, as fusion defects in siPI4K2A cells are rescued by expression of siRNA-resistant wild-type, but not kinase-dead, PI4K2A. Remarkably, introduction ("shuttling") of PtdIns4P also rescues A-L fusion. Importantly, A-L fusion is not rescued by shuttling of PtdIns(4,5)P₂, demonstrating that PtdIns4P does not function merely as a metabolic precursor in this process.

The mechanism(s) underlying GABA-RAP-PI4K2A-PtdIns4P-dependent A-L fusion are, at this point, completely open to investigation. A possible scenario is one in which PtdIns4P, and perhaps PI4K2A itself, contributes to the recruitment of priming, tethering, and/or fusion machinery to autophagosomes. Current evidence supports a model in which the small GTPase RAB7, associated with both lysosome and autophagosome membranes, recruits the hetero-hexameric HOPS (homotypic fusion and protein sorting) complex, which tethers the 2 organelles together. The HOPS complex also interacts with STX17 (syntaxin 17), a SNARE protein that inserts into completed autophagosomes (Fig. 1). STX17 forms a 4-helix bundle with VAMP8 on the lysosome and SNAP29 that is recruited from the cytosol.

The GABARAP-PI4K2A complex may generate a localized pool of PtdIns4P on autophagosomes that contributes to the activation of RAB7, the recruitment of the HOPS complex, and/or the stabilization of STX17 binding to the autophagosome membrane. Another candidate PI4K2A-PtdIns4P partner in autophagosome clearance is PLEKHM1 (pleckstrin homology domain containing, family M [with RUN domain] member 1), a multivalent adaptor protein that regulates both A-L and endosome-lysosome fusion. PLEKHM1 binds directly to RAB7, the HOPS complex, and GABARAP (Fig. 1), further implicating GABARAP in the A-L fusion process. The association of PI4K2A with the PLEKHM1 complex has not yet been examined, but may occur via its interaction with GABARAP, which has a strong propensity to oligomerize. Another intriguing possibility, which remains to be tested, is whether PtdIns4P binds to the PH domains found within PLEKHM1. Biochemical analyses will be required to identify which, if any, of these proteins are PtdIns4P effectors in the A-L fusion process.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This work was funding by HS | NIH | National Institute of General Medical Sciences (NIGMS) grant RO1GM66110, Cancer Prevention and Research Institute of Texas (CPRIT) grant RP120718-P1, and HHS | NIH | National Cancer Institute (NCI) grants RO1CA109618 and RO1CA84254.