Ceramide enhances binding of LC3/GABARAP autophagy proteins to cardiolipin-containing membranes

Varela, Yaiza R.; Etxaniz, Asier; Montes, L. Ruth; Goñi, Félix M.; Alonso, Arantza Pareja

doi:10.1016/j.ijbiomac.2022.07.032

Cited by 8 publications

(12 citation statements)

References 55 publications

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“…In previous work [ 11 , 12 ], two different mechanisms by which ceramide increases LC3/GABARAP binding to CL-containing bilayers were proposed: either Cer present in the liquid phase was mainly responsible or the presence of ceramide in the membrane produced a lipid restructuring, which favors the protein binding. In order to elucidate this mechanism, differential scanning calorimetry (DSC), confocal microscopy, and atomic force microscopy (AFM) were performed.…”

Section: Discussionmentioning

confidence: 99%

“…Both appear as “domains within domains” when they are analyzed using atomic force microscopy, a phenomenon that has been previously observed in samples involving SM:Cer interactions [ 38 ]. Such differentiated segregated phases had escaped our attention when preparing our previous contribution [ 11 ]. The possibility that some of them correspond to out-of-register domains consisting of two leaflets of different lipid composition deserves some further study.…”

Section: Discussionmentioning

confidence: 99%

“…The present contribution arises from our recent studies described in [ 11 , 12 ]. Those investigations were based on bilayers composed of egg sphingomyelin (eSM), dioleoyl phosphatidyl ethanolamine (DOPE), and CL.…”

Section: Introductionmentioning

confidence: 99%

“…Further studies from our group include the role of cardiolipin (CL)-LC3/GABARAP interactions in mitophagy [ 9 , 10 ] and more recently, the capacity of ceramide (Cer) to enhance the binding of LC3/GABARAP autophagy proteins to CL-containing membranes in mitophagy, the specific degradation of mitochondria via autophagy [ 11 , 12 ]. A substantial part of these investigations has been carried out on model membranes of defined lipid compositions.…”

Section: Introductionmentioning

confidence: 99%

“…Ceramide in turn is a non-phosphoryl-containing sphingolipid, characterized by an extremely low polarity [ 18 ]. The data by Varela et al [ 11 , 12 ] showed that, when incorporated into a matrix of egg SM and DOPE, CL was essential for LC3/GABARAP interaction with these bilayers and that Cer markedly increased binding. Moreover, giant unilamellar vesicles (GUV) examined under confocal fluorescence microscopy revealed that Cer segregated laterally into very rigid domains, while GABARAP bound only the more fluid regions, suggesting that the enhancing role of ceramide is exerted by the minority of ceramide molecules dispersed in the fluid phase.…”

Section: Introductionmentioning

confidence: 99%

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

Varela

González-Ramírez

Etxaniz

et al. 2023

IJMS

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Varela

González-Ramírez

Etxaniz

et al. 2023

IJMS

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

Etxaniz

Varela

Lázaro

et al. 2023

Cell. Mol. Life Sci.

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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.

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Ceramide regulation of autophagy: A biophysical approach

Varela,

Iriondo,

Goñi

et al. 2024

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biolog

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Ceramide enhances binding of LC3/GABARAP autophagy proteins to cardiolipin-containing membranes

Cited by 8 publications

References 55 publications

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

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

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

Ceramide regulation of autophagy: A biophysical approach

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