Making autophagosomes: Localized synthesis of phosphatidylinositol 3-phosphate holds the clue

Autophagy presents a topological challenge for the cell because it requires delivery of cytosolic material to the lumen of a membrane bound compartment, the lysosome. This is solved in an ingenious way by the formation of a double-membrane vesicle, the autophagosome, which captures cytosolic proteins and organelles during its transformation from a planar membrane disk into a sphere. In this way, cytosolic material first becomes lumenal and is then delivered for degradation to the lysosome. An unsolved set of questions in autophagy concerns the membrane of the autophagosome: what are the signals for its formation and what is its identity? Recently we provided some clues that may help answer these questions.¹ By following the dynamics of several phosphatidylinositol 3-phosphate (PI3P)-binding proteins during amino acid starvation (and autophagy induction) we concluded that at least some autophagosomes are formed in a starvation-induced, PI3P-enriched membrane compartment dynamically connected to the endoplasmic reticulum (ER). We termed the membranes of this compartment omegasomes (from their omega-like shape). Our data suggest that PI3P is important for providing localization clues and perhaps for facilitating the fusion step at the final stage of autophagosome formation.

Addendum to: Axe EL, Walker SA, Manifava M, Chandra P, Roderick HL, Habermann A, et al. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. J Cell Biol 2008; 182:685-701.     

Different phosphatidylinositol 3-phosphate asymmetries in yeast and mammalian autophagosomes revealed by a new electron microscopy technique

Phosphatidylinositol 3-phosphate (PtdIns3P) is a phospholipid essential for autophagy, but the detailed distribution of PtdIns3P in the membrane of autophagosomes, autophagic bodies, and other organelles is unclear due to technical difficulties. In the present study, we examined PtdIns3P distribution in autophagic membranes with an electron microscopy method called the quick-freeze freeze-fracture replica labeling method (QF-FRL), which can define the distribution of membrane lipids at the nanometer scale. In this method, membranes are split into 2 leaflets so that membrane asymmetry, i.e., differences between the 2 leaflets, can be defined unambiguously. As a result, PtdIns3P in the yeast autophagosome was found to exist much more abundantly in the lumenal leaflet (i.e., the leaflet facing the space between the outer and inner autophagosomal membranes) than in the cytoplasmic leaflet. In contrast, PtdIns3P in the mammalian autophagosome was confined to the cytoplasmic leaflet, showing an opposite asymmetry from that found in yeast. In yeast deleted for 2 cytoplasmic PtdIns3P phosphatases, Ymr1 and Sjl3, PtdIns3P distributed in an equivalent density in the 2 leaflets of the autophagosome membrane, suggesting that the asymmetry in wild-type yeast is generated as a result of unilateral PtdIns3P hydrolysis. The contrasting PtdIns3P distribution revealed in the present study suggested that formation of autophagic membranes may proceed in different ways in yeast and mammals.     

Different phosphatidylinositol 3-phosphate asymmetries in yeast and mammalian autophagosomes revealed by a new electron microscopy technique

Phosphatidylinositol 3-phosphate (PtdIns3P) is a phospholipid essential for autophagy, but the detailed distribution of PtdIns3P in the membrane of autophagosomes, autophagic bodies, and other organelles is unclear due to technical difficulties. In the present study, we examined PtdIns3P distribution in autophagic membranes with an electron microscopy method called the quick-freeze freeze-fracture replica labeling method (QF-FRL), which can define the distribution of membrane lipids at the nanometer scale. In this method, membranes are split into 2 leaflets so that membrane asymmetry, i.e., differences between the 2 leaflets, can be defined unambiguously. As a result, PtdIns3P in the yeast autophagosome was found to exist much more abundantly in the lumenal leaflet (i.e., the leaflet facing the space between the outer and inner autophagosomal membranes) than in the cytoplasmic leaflet. In contrast, PtdIns3P in the mammalian autophagosome was confined to the cytoplasmic leaflet, showing an opposite asymmetry from that found in yeast. In yeast deleted for 2 cytoplasmic PtdIns3P phosphatases, Ymr1 and Sjl3, PtdIns3P distributed in an equivalent density in the 2 leaflets of the autophagosome membrane, suggesting that the asymmetry in wild-type yeast is generated as a result of unilateral PtdIns3P hydrolysis. The contrasting PtdIns3P distribution revealed in the present study suggested that formation of autophagic membranes may proceed in different ways in yeast and mammals.     

Regulation of autophagy by phosphatidylinositol 3-phosphate

The simple phosphoinositide phosphatidylinositol 3-phosphate (PI(3)P) has been known to have important functions in endocytic and phagocytic traffic, and to be required for the autophagic pathway. In all of these settings, PI(3)P appears to create platforms that serve to recruit specific effectors for membrane trafficking events. In autophagy, PI(3)P may form the platform for autophagosome biogenesis.     

Intracellular trafficking and turnover of phosphatidylinositol 3-phosphate

Phosphatidylinositol 3-kinases (PI 3-kinases) regulate cellular functions through the 3'-phosphorylation of phosphatidylinositol (PI) and its derivatives. The PI 3-kinase product phosphatidylinositol 3-phosphate [PI(3)P] functions to recruit and activate effector proteins containing FYVE zinc finger domains. These proteins have various functions in endocytic membrane trafficking, cytoskeletal regulation and signal transduction. In order to understand the function of FYVE proteins, it is essential to study the formation, localisation, trafficking and turnover of PI(3)P. Here we review recent evidence that PI(3)P is formed on early endosomes through the activity of a PI 3-kinase which is recruited by the GTPase Rab5, and that the PI(3)P is subsequently internalised into intralumenal vesicles of multivesicular endosomes for turnover.     

How Atg18 and the WIPIs sense phosphatidylinositol 3-phosphate

The key autophagic lipid sensors are Atg18 in yeast and the WIPI proteins in mammals. Atg18 and the WIPIs belong to the PROPPIN family of proteins. PROPPINs are seven- bladed β-propellers that bind to phosphatidylinositol 3-phosphate (PtdIns3P) and phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2]. In order to understand how PROPPINs bind phosphoinositides, we have determined the crystal structure of a representative, biochemically tractable PROPPIN, Hsv2 of Kluveromyces lactis. The structure revealed that PROPPINs contain two phosphoinositide binding sites which cooperate with a hydrophobic anchoring loop in membrane binding. These three binding elements cooperate in function, as demonstrated by the incremental loss of function in Atg18 mutants impaired in combinations of the two phosphoinositide binding sites and the hydrophobic loop.     

Inportance of phosphatidylinositol 3-phosphate in sporulation of Schizosaccharomyces pombe

In Schizosaccharomyces pombe, Pik3p phosphorylates phosphatidylinositol (PI) to produce PI 3-P, which is further phosphorylated by Ste12p to yield PI 3,5-P2. Pik3p is required for both conjugation and sporulation. To test which of PI 3-P and PI 3,5-P2 is required for sporulation, diploid cells defective in production of PI 3,5-P2 were used. They underwent sporulation almost normally provided that the osmotic pressure of the medium was controlled, suggesting that not PI 3,5-P2 but PI 3-P was important. Electron microscopic analysis confirmed normal sporulation in the absence of PI 3,5-P2 although the forespore membrane was found to be less dense in these cells.     

FYVE finger proteins as effectors of phosphatidylinositol 3-phosphate.

Phosphatidylinositol 3-phosphate (PtdIns(3)P), generated via the phosphorylation of phosphatidylinositol by phosphatidylinositol 3-kinase (PI 3-kinase), plays an essential role in intracellular membrane traffic. The underlying mechanism is still not understood in detail, but the recent identification of the FYVE finger as a protein domain that binds specifically to PtdIns(3)P provides a number of potential effectors for PtdIns(3)P. The FYVE finger (named after the first letter of the four proteins containing it; Fab1p, YOTB, Vac1p and EEA1) is a double-zinc binding domain that is conserved in more than 30 proteins from yeast to mammals. It is found in several proteins involved in intracellular traffic, and FYVE finger mutations that affect zinc binding are associated with the loss of function of several of these proteins. The interaction of FYVE fingers with PtdIns(3)P may serve three alternative functions: First, to recruit cytosolic FYVE finger proteins to PtdIns(3)P-containing membranes (in concert with accessory molecules); second, to enrich for membrane bound FYVE finger proteins into PtdIns(3)P containing microdomains within the membrane; and third, to modulate the activity of membrane bound FYVE finger proteins.     

Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells

Phosphatidylinositol 3-kinase (PI3K) regulates several vital cellular processes, including signal transduction and membrane trafficking. In order to study the intracellular localization of the PI3K product, phosphatidylinositol 3-phosphate [PI(3)P], we constructed a probe consisting of two PI(3)P-binding FYVE domains. The probe was found to bind specifically, and with high affinity, to PI(3)P both in vitro and in vivo. When expressed in fibroblasts, a tagged probe localized to endosomes, as detected by fluorescence microscopy. Electron microscopy of untransfected fibroblasts showed that PI(3)P is highly enriched on early endosomes and in the internal vesicles of multivesicular endosomes. While yeast cells deficient in PI3K activity (vps15 and vps34 mutants) were not labelled, PI(3)P was found on intralumenal vesicles of endosomes and vacuoles of wild-type yeast. vps27Delta yeast cells, which have impaired endosome to vacuole trafficking, showed a decreased vacuolar labelling and increased endosome labelling. Thus PI(3)P follows a conserved intralumenal degradation pathway, and its generation, accessibility and turnover are likely to play a crucial role in defining the early endosome and the subsequent steps leading to multivesicular endosome formation.     

Insulin stimulates phosphatidylinositol 3-phosphate production via the activation of Rab5

Phosphatidylinositol 3-phosphate (PI(3)P) plays an important role in insulin-stimulated glucose uptake. Insulin promotes the production of PI(3)P at the plasma membrane by a process dependent on TC10 activation. Here, we report that insulin-stimulated PI(3)P production requires the activation of Rab5, a small GTPase that plays a critical role in phosphoinositide synthesis and turnover. This activation occurs at the plasma membrane and is downstream of TC10. TC10 stimulates Rab5 activity via the recruitment of GAPEX-5, a VPS9 domain-containing guanyl nucleotide exchange factor that forms a complex with TC10. Although overexpression of plasma membrane-localized GAPEX-5 or constitutively active Rab5 promotes PI(3)P formation, knockdown of GAPEX-5 or overexpression of a dominant negative Rab5 mutant blocks the effects of insulin or TC10 on this process. Concomitant with its effect on PI(3)P levels, the knockdown of GAPEX-5 blocks insulin-stimulated Glut4 translocation and glucose uptake. Together, these studies suggest that the TC10/GAPEX-5/Rab5 axis mediates insulin-stimulated production of PI(3)P, which regulates trafficking of Glut4 vesicles.     

Mycobacterium tuberculosis reprograms waves of phosphatidylinositol 3-phosphate on phagosomal organelles

The potent human pathogen Mycobacterium tuberculosis persists in macrophages within a specialized, immature phagosome by interfering with the pathway of phagolysosome biogenesis. The molecular mechanisms underlying this process remain to be fully elucidated. Here, using four-dimensional microscopy, we detected on model phagosomes, which normally mature into phagolysosomes, the existence of cyclical waves of phosphatidylinositol 3-phosphate (PI3P), a membrane trafficking regulatory lipid essential for phagosomal acquisition of lysosomal characteristics. We show that mycobacteria interfere with the dynamics of PI3P on phagosomal organelles by altering the timing and characteristics of the PI3P waves on phagosomes. The default program of cyclical PI3P waves on model phagosomes is composed of an initial stage (phase I), represented by a strong PI3P burst occurring only upon the completion of phagosome formation, and a subsequent stage (phase II) of recurring PI3P waves on maturing phagosomes with the average periodicity of 20 min. Mycobacteria alter this program in two ways: (i) by inducing, in a cholesterol-dependent fashion, a neophase I* of premature PI3P production, coinciding with the process of mycobacterial entry into the macrophage, and (ii) by inhibiting the calmodulin-dependent phase II responsible for the acquisition of lysosomal characteristics. We conclude that the default pathway of phagosomal maturation into the phagolysosome includes temporally organized cyclical waves of PI3P on phagosomal membranes and that this process is targeted for reprogramming by mycobacteria as they prevent phagolysosome formation.     

Phosphatidylinositol-5-phosphate activation and conserved substrate specificity of the myotubularin phosphatidylinositol 3-phosphatases

Phosphoinositides control many different processes required for normal cellular function. Myotubularins are a family of Phosphatidylinositol 3-phosphate (PtdIns3P) phosphatases identified by the positional cloning of the MTM1 gene in patients suffering from X-linked myotubular myopathy and the MTMR2 gene in patients suffering from the demyelinating neuropathy Charcot-Marie-Tooth disease type 4B. MTM1 is a phosphatidylinositol phosphatase with reported specificity toward PtdIns3P, while the related proteins MTMR2 and MTMR3 hydrolyze both PtdIns3P and PtdIns(3,5)P2. We have investigated MTM1 and MTMR6 and find that they use PtdIns(3,5)P2 in addition to PtdIns3P as a substrate in vitro. The product of PtdIns(3,5)P2 hydrolysis, PtdIns5P, causes MTM1 to form a heptameric ring that is 12.5 nm in diameter, and it is a specific allosteric activator of MTM1, MTMR3, and MTMR6. A disease-causing mutation at arginine 69 of MTM1 falling within a putative pleckstrin homology domain reduces the ability of the enzyme to respond to PtdIns5P. We propose that the myotubularin family of enzymes utilize both PtdIns3P and PtdIns(3,5)P2 as substrates, and that PtdIns5P functions in a positive feedback loop controlling their activity. These findings highlight the importance of regulated phosphatase activity for the control of phosphoinositide metabolism.     

Essential roles of PIKfyve and PTEN on phagosomal phosphatidylinositol 3-phosphate dynamics

PtdIns(3)P (phosphatidylinositol 3-phosphate) is a signaling molecule important for phagosome maturation. The major role of Vps34 in production of phagosomal PtdIns(3)P has been indicated. However, the fate of the newly generated PtdIns(3)P has not been well described. Here we show that elimination of PtdIns(3)P from phagosomal membrane was significantly delayed in RAW264.7 macrophages lacking PTEN or PIKfyve. In the PTEN-deficient cells treated with a PIKfyve inhibitor, degradation of PtdIns(3)P was almost lost, indicating that PTEN and PIKfyve are two major players in phagosomal PtdIns(3)P metabolism.     

The discovery of a 3-phosphomonoesterase that hydrolyzes phosphatidylinositol 3-phosphate in NIH 3T3 cells

Phosphatidylinositol 3-phosphate (PtdIns(3)P), a recently described phospholipid, has been linked to polyoma virus-induced cellular transformation and platelet-derived growth factor-mediated mitogenesis. PtdIns(3)P, in contrast to phosphatidylinositol, phosphatidylinositol 4-phosphate (PtdIns(4)P), and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), is resistant to hydrolysis by bovine brain phospholipase C gamma. We present here the identification of a phosphomonoesterase activity from the soluble fraction of NIH 3T3 cells which removes the phosphate from the D-3 position of PtdIns(3)P. This enzyme is specific as it has little or no activity on the monoester phosphates of PtdIns(4)P, PtdIns(4,5)P2, or inositol 1,3-bisphosphate and is tentatively designated phosphatidylinositol 3-phosphatase (PtdIns 3-phosphatase). The enzyme does not require added metal ions for activity and is maximally active in the presence of EDTA. It is inhibited by Ca2+, Mg2+, Zn2+, and the phosphatase inhibitor VO4(3-). In addition, there is no phospholipase C activity toward PtdIns(3)P in the soluble fraction of NIH 3T3 cells. In view of the absence of a phospholipase C activity that hydrolyzes PtdIns(3)P, we propose that PtdIns(3)P is not a precursor for a soluble inositol phosphate messenger but that it instead may act directly to control certain cellular processes or as a precursor for other phosphatidylinositols. PtdIns 3-phosphatase may thus terminate a metabolic signal or regulate precursor levels for other phosphatidylinositols that are phosphorylated in the D-3 position.     

Production of phosphatidylinositol 5-phosphate by the phosphoinositide 3-phosphatase myotubularin in mammalian cells

MTM1, the gene encoding myotubularin (MTM1), is mutated in the X-linked myotubular myopathy (XLMTM), a severe genetic muscular disorder. MTM1 is a phosphoinositide phosphatase hydrolyzing phosphatidylinositol 3-phosphate (PtdIns(3)P) in yeast and in vitro. Because this lipid is implicated in the regulation of vesicular trafficking, we used established cell lines from XLMTM patients to evaluate whether the lack of endogenous MTM1 expression could affect PtdIns(3)P labeling patterns. Our results showed that the vesicular trafficking related to early endosomes was not significantly affected in the XLMTM cell lines compared with control cells. However, in addition to PtdIns(3)P, we found that MTM1 can hydrolyze phosphatidylinositol 3,5-bisphosphate both in vitro and in mammalian cells. Using a mass assay, we demonstrated that the product generated is phosphatidylinositol 5-phosphate (PtdIns(5)P), a recently discovered phosphoinositide, the function of which is still unknown. In L6 myotubes overexpressing MTM1, hyperosmotic shock induced an increase in the mass level of PtdIns(5)P that was reduced by 50% upon overexpression of the MTM1 inactive mutant D278A. These data demonstrate for the first time a role for MTM1 in the production of PtdIns(5)P in mammalian cells, suggesting that the lack of transformation of phosphatidylinositol 3,5-bisphosphate into PtdIns(5)P might be an important component in the etiology of myotubular myopathy.     

Distinct Golgi populations of phosphatidylinositol 4-phosphate regulated by phosphatidylinositol 4-kinases

Phosphatidylinositol 4-phosphate (PI4P) regulates biosynthetic membrane traffic at multiple steps and differentially affects the surface delivery of apically and basolaterally destined proteins in polarized cells. Two phosphatidylinositol 4-kinases (PI4Ks) have been localized to the Golgi complex in mammalian cells, type III PI4Kbeta (PI4KIIIbeta) and type II PI4Kalpha (PI4KIIalpha). Here we report that PI4KIIIbeta and PI4KIIalpha localize to discrete subcompartments of the Golgi complex in Madin-Darby canine kidney (MDCK) cells. PI4KIIIbeta was enriched in early Golgi compartments, whereas PI4KIIalpha colocalized with markers of the trans-Golgi network (TGN). To understand the temporal and spatial control of PI4P generation across the Golgi complex, we quantitated the steady state distribution of a fluorescent PI4P-binding domain relative to cis/medial Golgi and TGN markers in transiently transfected MDCK cells. The density of the signal from this PI4P reporter was roughly 2-fold greater in the early Golgi compartments compared with that of the TGN. Furthermore, this ratio could be modulated in vivo by overexpression of catalytically inactive PI4KIIIbeta and PI4KIIalpha or in vitro by the PI4KIIIbeta inhibitor wortmannin. Our data suggest that both PI4KIIIbeta and PI4KIIalpha contribute to the compartmental regulation of PI4P synthesis within the Golgi complex. We discuss our results with respect to the kinetic effects of modulating PI4K activity on polarized biosynthetic traffic in MDCK cells.