Regulation of LC3 lipidation by the autophagy-specific class III phosphatidylinositol-3 kinase complex

Brier, Livia W.; Ge, Liang; Stjepanović, Goran; Thelen, Ashley M.; Hurley, James H.; Schekman, Randy

doi:10.1091/mbc.e18-11-0743

Cited by 63 publications

(47 citation statements)

References 62 publications

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“…Overnight treatment with the Vps34 inhibitor Vps34-IN1 (Bago et al, 2014) abolished the basal WIPI2 puncta seen without CQ, as well as the accumulated WIPI2 puncta in the presence of CQ ( Fig 6C,D). These data show that PI(3)P is required for WIPI2 recruitment, consistent with previous reports (Dooley et al, 2014;Polson et al, 2010;Karanasios et al, 2013;Brier et al, 2019). LC3 puncta, in contrast, were still present in Vps34-IN1-treated cells ( Fig 6D), consistent with a PI(3)P-and WIPI2-independent route for LC3 recruitment.…”

Section: Lc3-positive Autophagosomes Accumulate In Knockout Rpesupporting

confidence: 92%

Multiple phosphatidylinositol(3)phosphate roles in retinal pigment epithelium membrane recycling

et al. 2020

Preprint

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The low-abundance lipid phosphatidylinositol-3-phosphate (PI(3)P) is important for membrane dynamics in autophagy, endosome processing, and phagocytosis. In retinal pigmented epithelial cells (RPE) all three pathways are important, but phagocytosis of disk membranes shed from adjacent photoreceptors is especially important for ensuring health of photoreceptors and preventing retinal degeneration. By eliminating Vps34, the kinase responsible for synthesizing PI(3)P in RPE, we have found that PI(3)P plays distinct roles in each pathway. In phagocytosis it is not required for disk engulfment or phagosome transport but is essential for recruitment and lipidation of LC3. In contrast, initiation of autophagy and LC3 recruitment to autophagosomes does not require PI(3)P, which can be bypassed by an alternative mechanism of ATG16L recruitment that does not require PI(3)P, Rab11a, or WIPI2. In all three pathways, PI(3)P is essential for fusion with lysosomes; autophagosomes, phagosomes, and Rab7-positive late endosomes, as well as enlarged lysosomes, accumulate in large numbers in the absence of Vps34, leading to cell death.

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Section: Lc3-positive Autophagosomes Accumulate In Knockout Rpesupporting

confidence: 92%

Multiple phosphatidylinositol(3)phosphate roles in retinal pigment epithelium membrane recycling

et al. 2020

Preprint

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“…This is emphasized by the data shown in Figure 4E. These data explain the observation of a PI3KC3-C1 requirement for efficient LC3 lipidation in cells (Axe et al, 2008;Itakura et al, 2008;Itakura and Mizushima, 2010;Matsunaga et al, 2009;Zhong et al, 2009) and of ERGIC-derived membranes in a cell free system (Brier et al, 2019). They also explain the strict requirement for WIPI2 for the lipidation reaction in canonical autophagy and even in STING-induced LC3 lipidation, which bypasses PI3KC3-C1 but is dependent on WIPI2 and the E3 (Gui et al, 2019).…”

Section: Discussionsupporting

confidence: 68%

A PI3K-WIPI2 positive feedback loop allosterically activates LC3 lipidation in autophagy

Fracchiolla

Chang

Hurley

et al. 2019

Preprint

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Autophagy requires the synthesis of PI(3)P and the conjugation of LC3 to the phagophore membrane. We reconstituted these two reactions and their coupling by WIPI2, and showed that positive feedback between PI3KC3-C1 and WIPI2 leads to rapid LC3 lipidation by the ATG16L1 complex. AbstractAutophagy degrades cytoplasmic cargo by its delivery to lysosomes within double membrane autophagosomes. Synthesis of the phosphoinositide PI(3)P by the autophagic PI 3-kinase complex I (PI3KC3-C1) and conjugation of ATG8/LC3 proteins to phagophore membranes by the ATG12-ATG5-ATG16L1 (E3) complex are two critical steps in autophagosome biogenesis, connected by WIPI2. Here we present a complete reconstitution of these events. On giant unilamellar vesicles (GUVs), LC3 lipidation is strictly dependent on the recruitment of WIPI2, which in turn depends on PI(3)P. Ectopically targeting E3 to membranes in the absence of WIPI2 is insufficient to support LC3 lipidation, demonstrating that WIPI2 allosterically activates the E3 complex. PI3KC3-C1 and WIPI2 mutually promote the recruitment of each other in a positive feedback loop. When both PI 3kinase and LC3 lipidation reactions were carried out simultaneously, positive feedback between PI3KC3-C1 and WIPI2 led to rapid LC3 lipidation with kinetics similar to those seen in cellular autophagosome formation. 105 132 108 125

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“…In particular, BCL2 uses its BH3 domain to associate with the BH3 domain of BECN1, facilitating BECN1 homodimerization and, in consequence, preventing BECN1 from forming the class III PtdIns3K complex I, which is responsible for generating phosphatidylinositol 3-phosphate (PtdIns3P) [95][96][97]. PtdIns3P is an essential molecule for the formation of phagophore structures and the recruitment of the enzymatic complex required for the lipidation of LC3 [17] ( Figure 4B). Therefore, the release of BECN1 from BECN1-BCL2 complexes is essential for the initiation of the autophagic response.…”

Section: Nef Enhances the Association Between Becn1 And Bcl2 To Prevementioning

confidence: 99%

“…However, upon autophagy activation, LC3-I is lipidated by an E3-like enzymatic complex (E1: ATG7, E2: ATG3, and E3: ATG12-ATG5-ATG16L1) that adds phosphatidylethanolamine (PE) to its C-terminus, converting LC3-I into LC3-II: the autophagycompetent variant ( Figure S1). This lipidation process takes place almost simultaneously with the formation of the phagophore and is dependent upon the activation status of BECN1 [15][16][17]. LC3-II is crucial for proper elongation and maturation of autophagosomes.…”

Section: Introductionmentioning

confidence: 99%

HIV-1 Nef counteracts autophagy restriction by enhancing the association between BECN1 and its inhibitor BCL2 in a PRKN-dependent manner

et al. 2020

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Macroautophagy/autophagy is an auto-digestive pro-survival pathway activated in response to stress to target cargo for lysosomal degradation. In recent years, autophagy has become prominent as an innate antiviral defense mechanism through multiple processes, such as targeting virions and viral components for elimination. These exciting findings have encouraged studies on the ability of autophagy to restrict HIV. However, the role of autophagy in HIV infection remains unclear. Whereas some reports indicate that autophagy is detrimental for HIV, others have claimed that HIV deliberately activates this pathway to increase its infectivity. Moreover, these contrasting findings seem to depend on the cell type investigated. Here, we show that autophagy poses a hurdle for HIV replication, significantly reducing virion production. However, HIV-1 uses its accessory protein Nef to counteract this restriction. Previous studies have indicated that Nef affects autophagy maturation by preventing the fusion between autophagosomes and lysosomes. Here, we uncover that Nef additionally blocks autophagy initiation by enhancing the association between BECN1 and its inhibitor BCL2, and this activity depends on the cellular E3 ligase PRKN. Remarkably, the ability of Nef to counteract the autophagy block is more frequently observed in pandemic HIV-1 and its simian precursor SIV cpz infecting chimpanzees than in HIV-2 and its precursor SIV smm infecting sooty mangabeys. In summary, our findings demonstrate that HIV-1 is susceptible to autophagy restriction and define Nef as the primary autophagy antagonist of this antiviral process.

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Regulation of LC3 lipidation by the autophagy-specific class III phosphatidylinositol-3 kinase complex

Cited by 63 publications

References 62 publications

Multiple phosphatidylinositol(3)phosphate roles in retinal pigment epithelium membrane recycling

Multiple phosphatidylinositol(3)phosphate roles in retinal pigment epithelium membrane recycling

A PI3K-WIPI2 positive feedback loop allosterically activates LC3 lipidation in autophagy

HIV-1 Nef counteracts autophagy restriction by enhancing the association between BECN1 and its inhibitor BCL2 in a PRKN-dependent manner

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