Externalization of the phospholipid cardiolipin (CL) to the outer mitochondrial membrane has been proposed to act as a mitophagy trigger. CL would act as a signal for binding the LC3 macroautophagy/autophagy proteins. As yet, the behavior of the LC3-subfamily members has not been directly compared in a detailed way. In the present contribution, an analysis of LC3A, LC3B and LC3C interaction with CL-containing model membranes, and of their ability to translocate to mitochondria, is described. Binding of LC3A to CL was stronger than that of LC3B; both proteins showed a similar ability to colocalize with mitochondria upon induction of CL externalization in SH-SY5Y cells. Besides, the double silencing of LC3A and LC3B proteins was seen to decrease CCCP-induced mitophagy. Residues 14 and 18 located in the N-terminal region of LC3A were shown to be important for its recognition of damaged mitochondria during rotenone- or CCCP-induced mitophagy. Moreover, the in vitro results suggested a possible role of LC3A, but not of LC3B, in oxidized-CL recognition as a counterweight to excessive apoptosis activation. In the case of LC3C, even if this protein showed a stronger CL binding than LC3B or LC3A, the interaction was less specific, and colocalization of LC3C with mitochondria was not rotenone dependent. These results suggest that, at variance with LC3A, LC3C does not participate in cargo recognition during CL-mediated-mitophagy. The data support the notion that the various LC3-subfamily members might play different roles during autophagy initiation, identifying LC3A as a novel stakeholder in CL-mediated mitophagy. Abbreviations: ACTB/β-actin: actin beta; Atg8: autophagy-related 8; CL: cardiolipin; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; DMSO: dimethyl sulfoxide; DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DTT: DL-dithiothreitol; FKBP8: FKBP prolyl isomerase 8; GABARAP: GABA type A receptor associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GABARAPL2: GABA type A receptor associated protein like 2; GFP: green fluorescent protein; IMM: inner mitochondrial membrane; LUV/LUVs: large unilamellar vesicle/s; MAP1LC3A/LC3A: microtubule associated protein 1 light chain 3 alpha; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAP1LC3C/LC3C: microtubule associated protein 1 light chain 3 gamma; NME4/NDPK-D/Nm23-H4: NME/NM23 nucleoside diphosphate kinase 4; O/A: oligomycin A + antimycin A; OMM: outer mitochondrial membrane; PA: phosphatidic acid; PC: phosphatidylcholine; PG: phosphatidylglycerol; PINK1: PTEN induced putative kinase 1; PtdIns4P: phosphatidylinositol-4-phosphate; Rho-PE: lissamine rhodamine phosphatidylethanolamine; SUV/SUVs: small unilamellar vesicle/s

In macroautophagy, the autophagosome (AP) engulfs portions of cytoplasm to allow their lysosomal degradation. AP formation in humans requires the concerted action of the ATG12 and LC3/GABARAP conjugation systems. The ATG12–ATG5-ATG16L1 or E3-like complex (E3 for short) acts as a ubiquitin-like E3 enzyme, promoting LC3/GABARAP proteins anchoring to the AP membrane. Their role in the AP expansion process is still unclear, in part because there are no studies comparing six LC3/GABARAP family member roles under the same conditions, and also because the full human E3 was only recently available. In the present study, the lipidation of six members of the LC3/GABARAP family has been reconstituted in the presence and absence of E3, and the mechanisms by which E3 and LC3/GABARAP proteins participate in vesicle tethering and fusion have been investigated. In the absence of E3, GABARAP and GABARAPL1 showed the highest activities. Differences found within LC3/GABARAP proteins suggest the existence of a lipidation threshold, lower for the GABARAP subfamily, as a requisite for tethering and inter-vesicular lipid mixing. E3 increases and speeds up lipidation and LC3/GABARAP-promoted tethering. However, E3 hampers LC3/GABARAP capacity to induce inter-vesicular lipid mixing or subsequent fusion, presumably through the formation of a rigid scaffold on the vesicle surface. Our results suggest a model of AP expansion in which the growing regions would be areas where the LC3/GABARAP proteins involved should be susceptible to lipidation in the absence of E3, or else a regulatory mechanism would allow vesicle incorporation and phagophore growth when E3 is present.

The binding and early stages of activity of a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa on giant unilamellar vesicles (GUV) have been monitored using fluorescence confocal microscopy. Both the lipids and the enzyme were labeled with specific fluorescent markers. GUV consisted of a mixture of phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and cholesterol in equimolar ratios, to which 5–10 mol% of the enzyme end-product ceramide and/or diacylglycerol were occasionally added. Morphological examination of the GUV in the presence of enzyme reveals that, although the enzyme diffuses rapidly throughout the observation chamber, detectable enzyme binding appears to be a slow, random process, with new bound-enzyme-containing vesicles appearing for several minutes. Enzyme binding to the vesicles appears to be a cooperative process. After the initial cluster of bound enzyme is detected, further binding and catalytic activity follow rapidly. After the activity has started, the enzyme is not released by repeated washing, suggesting a “scooting” mechanism for the hydrolytic activity. The enzyme preferentially binds the more disordered domains, and, in most cases, the catalytic activity causes the disordering of the other domains. Simultaneously, peanut- or figure-eight-shaped vesicles containing two separate lipid domains become spherical. At a further stage of lipid hydrolysis, lipid aggregates are formed and vesicles disintegrate.

Cardiolipin (CL) is a key lipid for damaged mitochondrial recognition by the LC3/GABARAP human autophagy proteins. The role of ceramide (Cer) in this process is unclear, but CL and Cer have been proposed to coexist in mitochondria under certain conditions. Varela et al. showed that in model membranes composed of egg sphingomyelin (eSM), dioleoyl phosphatidylethanolamine (DOPE), and CL, the addition of Cer enhanced the binding of LC3/GABARAP proteins to bilayers. Cer gave rise to lateral phase separation of Cer-rich rigid domains but protein binding took place mainly in the fluid continuous phase. In the present study, a biophysical analysis of bilayers composed of eSM, DOPE, CL, and/or Cer was attempted to understand the relevance of this lipid coexistence. Bilayers were studied by differential scanning calorimetry, confocal fluorescence microscopy, and atomic force microscopy. Upon the addition of CL and Cer, one continuous phase and two segregated ones were formed. In bilayers with egg phosphatidylcholine instead of eSM, in which the binding of LC3/GABARAP proteins hardly increased with Cer in the former study, a single segregated phase was formed. Assuming that phase separation at the nanoscale is ruled by the same principles acting at the micrometer scale, it is proposed that Cer-enriched rigid nanodomains, stabilized by eSM:Cer interactions formed within the DOPE- and CL-enriched fluid phase, result in structural defects at the rigid/fluid nanointerfaces, thus hypothetically facilitatingLC3/GABARAP protein interaction.

Macroautophagy is a cellular degradation process characterized by the formation of the double-membrane structure termed autophagosome (AP). The process of AP formation is not fully understood, but it is thought to happen through the combined action of direct lipid transfer and incorporation of new vesicles to the edges of the growing structure. Human LC3/GABARAP autophagy-related proteins are known to induce vesicle tethering and lipid mixing in vitro, which makes them suitable for the latter expansion mechanism. Ceramide (Cer) is a sphingolipid previously described to facilitate membrane fusion. Cer has also been related to macroautophagy modulation previously, although its specific role remains unclear. Moreover, the presence of sphingolipids in the AP has been suggested by recent experiments, increasing the relevance of Cer in macroautophagy. The present work has investigated the potential role that Cer could have on the proposed fusion of new vesicles to the nascent AP membrane. Interaction of purified ATG proteins with lipid vesicles of defined composition has been quantified using fluorescence spectroscopic techniques. Our results suggest that, if present, Cer could promote the vesicle tethering and leakage-free intervesicular lipid mixing induced by GABARAP and GABARAPL1, which would in turn mediate AP membrane expansion.

Autophagy is a catabolic process in which a double-membrane organelle, the autophagosome (AP), engulfs cellular components that will be degraded in the lysosomes. ATG8 protein family members participate at various stages of AP formation. The present study compares the capacity to induce lipid-vesicle tethering and fusion of two ATG8 family members, LC3B and LC3C, with model membranes. LC3B is the most thoroughly studied ATG8 protein. It is generally considered as an autophagosomal marker and a canonical representative of the LC3 subfamily. LC3C is less studied, but recent data have reported its implication in various processes, crucial to cellular homeostasis. The results in this paper show that LC3C induces higher levels of tethering and of intervesicular lipid mixing than LC3B. As the N-terminus of LC3C is different from that of the other family members, various mutants of the N-terminal region of both LC3B and LC3C were designed, and their activities compared. It was concluded that the N-terminal region of LC3C was responsible for the enhanced vesicle tethering, membrane perturbation and vesicle-vesicle fusion activities of LC3C as compared to LC3B. The results suggest a specialized function of LC3C in the AP expansion process.