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Transmembrane phospholipid translocation mediated by Atg9 is involved in autophagosome formation.

ABSTRACT: The mechanism of isolation membrane formation in autophagy is receiving intensive study. We recently found that Atg9 translocates phospholipids across liposomal membranes and proposed that this functionality plays an essential role in the expansion of isolation membranes. The distribution of phosphatidylinositol 3-phosphate in both leaflets of yeast autophagosomal membranes supports this proposal, but if Atg9-mediated lipid transport is crucial, symmetrical distribution in autophagosomes should be found broadly for other phospholipids. To test this idea, we analyzed the distributions of phosphatidylcholine, phosphatidylserine, and phosphatidylinositol 4-phosphate by freeze-fracture electron microscopy. We found that all these phospholipids are distributed with comparable densities in the two leaflets of autophagosomes and autophagic bodies. Moreover, de novo-synthesized phosphatidylcholine is incorporated into autophagosomes preferentially and shows symmetrical distribution in autophagosomes within 30 min after synthesis, whereas this symmetrical distribution is compromised in yeast expressing an Atg9 mutant. These results indicate that transbilayer phospholipid movement that is mediated by Atg9 is involved in the biogenesis of autophagosomes.

SUBMITTER: Orii M

PROVIDER: S-EPMC7809878 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Transmembrane phospholipid translocation mediated by Atg9 is involved in autophagosome formation.

Orii Minami M Tsuji Takuma T Ogasawara Yuta Y Fujimoto Toyoshi T

The Journal of cell biology 20210301 3

The mechanism of isolation membrane formation in autophagy is receiving intensive study. We recently found that Atg9 translocates phospholipids across liposomal membranes and proposed that this functionality plays an essential role in the expansion of isolation membranes. The distribution of phosphatidylinositol 3-phosphate in both leaflets of yeast autophagosomal membranes supports this proposal, but if Atg9-mediated lipid transport is crucial, symmetrical distribution in autophagosomes should ...[more]

PMID: 33439214

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Project description:Second harmonic generation (SHG) measurements using SHG-active dye molecules have recently attracted attention as a method to detect the formation of pores in phospholipid bilayers. The bilayers, in which the dye molecules are embedded in the outer leaflet, exhibit a noncentrosymmetric structure, generating SHG signals. However, when pores form, these dye molecules translocate through the pores into the inner leaflet, leading to a more centrosymmetric structure and the subsequent loss of the SHG signals. A decrease in the SHG signals has been experimentally observed in membranes subjected to electrical stimuli. However, the characteristics of the interleaflet translocation of SHG-active dye molecules through pores remain unclear, hindering quantitative estimation of the membrane conditions, such as the pore size and density, based on the SHG signal reduction. In this study, we investigated the interleaflet translocation characteristics of Ap3, an SHG-active dye molecule, using molecular dynamics (MD) simulations and two-dimensional random-walk (RW) simulations. The MD simulations revealed that Ap3 molecules only translocate between the leaflets along the pore sidewalls. We determined the lateral diffusion coefficient of Ap3 within the membrane plane and its propensity for interleaflet movement at the pore wall. Based on these movement characteristics, the RW model successfully reproduced the characteristic time scale of the interleaflet translocation observed in the MD simulations. By varying the pore size and density in the RW simulations, we estimated that the characteristic time scale of interleaflet translocation depends on the -0.31 power of the pore radius and the -1.13 power of the pore density. Using these findings, we estimated the number of pores that probably formed in membranes during previous electroporation experiments. These results indicate the potential of optical measurement of the dye molecule movement for the indirect quantitative estimation of the pore size and number, which are challenging to measure optically.

Dataset Information

Transmembrane phospholipid translocation mediated by Atg9 is involved in autophagosome formation.

Publications

Transmembrane phospholipid translocation mediated by Atg9 is involved in autophagosome formation.

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