LC3 subfamily in cardiolipin-mediated mitophagy: a comparison of the LC3A, LC3B and LC3C homologs

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 macroauto-phagy/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 initia-tion, identifying LC3A as a novel stakeholder in CL-mediated mitophagy.This work was supported in part by the Spanish Ministerio de Ciencia e Innovación (MCI), Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) (grant No. PGC2018-099857- B-I00), by the Basque Government (grants No. IT1625-22 and IT1270- 19), by Fundación Ramón Areces (CIVP20A6619), by Fundación Biofísica Bizkaia and by the Basque Excellence Research Centre (BERC) program of the Basque Government. MI and YV were recipients of predoctoral FPU fellowships from the Spanish Ministry of Science Innovation and Universities (FPU16/05873, FPU18/00799), UB thanks the University of the Basque Country for a predoctoral contract, JHH was supported by a Postdoctoral Fellowship from the Basque Government

Effects of a N-Maleimide-derivatized Phosphatidylethanolamine on the Architecture and Properties of Lipid Bilayers

N-maleimide-derivatized phospholipids are often used to facilitate protein anchoring to membranes. In autophagy studies, this is applied to the covalent binding of Atg8, an autophagy protein, to a phosphatidylethanolamine (PE) in the nascent autophagosome. However, the question remains on how closely the N-maleimide PE derivative (PE-mal) mimicks the native PE in the bilayer. In the present paper, spectroscopic and calorimetric techniques have been applied to vesicles containing either PE or PE-mal (together with other phospholipids) to compare the properties of the native and derivatized forms of PE. According to differential scanning calorimetry, and to infrared spectroscopy, the presence of PE-mal did not perturb the fatty acyl chains in the bilayer. Fluorescence spectroscopy and microscopy showed that PE-mal did not alter the bilayer permeability either. However, fluorescence emission polarization of the Laurdan and DPH probes indicated an increased order, or decreased fluidity, in the bilayers containing PE-mal. In addition, the infrared spectral data from the phospholipid phosphate region revealed a PE-mal-induced conformational change in the polar heads, accompanied by increased hydration. Globally considered, the results suggest that PE-mal would be a reasonable substitute for PE in model membranes containing reconstituted proteins.This work was funded in part by the Spanish Ministry of Science, Innovation, and Universities (MCIU), Agencia Estatal de Investigación (AEI), Fondo Europeo de Desarrollo Regional (FEDER) (grant No. PID2021-124461NB-I00), the Basque Government (grant No. IT1625-22), Fundación Ramón Areces (CIVP20A6619), Fundación Biofísica Bizkaia and the Basque Excellence Research Centre (BERC) program of the Basque Government. E.J.G.-R. was supported by Fundación Ramón Areces. Y.R.V. was a recipient of a pre-doctoral FPU fellowship from the Spanish Ministry of Science, Innovation, and Universities (FPU18/00799). U.B. thanks the University of the Basque Country for a pre-doctoral contract

Lipids in Mitochondrial Macroautophagy: Phase Behavior of Bilayers Containing Cardiolipin and Ceramide

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.This work was funded in part by the Spanish Ministry of Science, Innovation, and Universities (MCIU), Agencia Estatal de Investigación (AEI), Fondo Europeo de Desarrollo Regional (FEDER) (grant No. PID2021-124461NB-I00), the Basque Government (grant No. IT1625-22), Fundación Ramón Areces (CIVP20A6619), Fundación Biofísica Bizkaia, and the Basque Excellence Research Centre (BERC) program of the Basque Government. E.J.G.-R. was supported by Fundación Ramón Areces. M.N.I. and Y.R.V. were recipients of pre-doctoral FPU fellowships from the Spanish Ministry of Science, Innovation, and Universities (FPU16/05873, FPU18/00799). U.B. thanks the University of the Basque Country for a pre-doctoral contract

Effect of ATG12–ATG5-ATG16L1 autophagy E3-like complex on the ability of LC3/GABARAP proteins to induce vesicle tethering and fusion

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.This work was supported in part by the Spanish Ministerio de Ciencia e Innovación (MCI), Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) (grants No. PGC2018-099857-B-I00 and PID2021-124461NB-I00), by the Basque Government (grants No. IT1625-22 and IT1270-19), by Fundación Biofísica Bizkaia, by Fundación Ramón Areces (grant No. CIVP20A6619), and by the Basque Excellence Research Centre (BERC) program of the Basque Government. MI and YV were recipients of predoctoral FPU fellowships from the Spanish Ministry of Science, Innovation and Universities (FPU16/05873, FPU18/00799), UB thanks the University of the Basque Country for a predoctoral contract. This work was supported by Human Frontiers Science Program RGP0026/2017 (S.M.)