GABARAP Like-1 enrichment on membranes: Direct observation of trans-homo-oligomerization between membranes and curvature-dependent partitioning into membrane tubules

Abstract

The Atg8/LC3/GABARAP protein family has been implicated in membrane remodeling events on the growing autophagosome. In particular, each of these proteins can form a protein-lipid conjugate that has been shown in vitro to drive liposome aggregation and in some cases membrane fusion. Furthermore, yeast Atg8 has been described as a curvature sensing protein, through its natural capacity to concentrate on highly curved membranes. A key advance with yeast Atg8, was the introduction of Giant Unilamellar Vesicles (GUVs) as an in vitro support that could allow membrane deformation and tethering to be observed by simple microscopy. Further, micromanipulation of an individual GUV could be used to create local areas of curvature to follow Atg8 partitioning. Here, we use a recently developed method to decorate GUVs with the mammalian Atg8 protein GABARAPL1 and establish the generality of the observations made on yeast Atg8. Then we apply double micromanipulation, the capture and positioning of two independently prepared GUVs, to test elements of the mechanism, speed and reversibility of mammalian Atg8 protein-mediated tethering. We find that the membranes adhere through GABARAPL1/GABARAPL1 homotypic trans-interactions. On a single membrane with two regions with significantly different curvatures we observed that the regions of higher curvature can be enriched up to 10 times in GABARAPL1 compared to the planar regions. This approach has the potential to allow the formation and study of specific topographically-controlled interfaces involving Atg8-proteins and their targets on apposing membranes.

Association of γ-aminobutyric acid type A receptor-associated protein with prognosis in patients after radical pancreatic cancer treatment

“…The mechanism of this lipidation effect in autophagy closure is not fully understood, but appears to be GABARAP-speci c, as depletion of all LC3 has no effect on starvation-induced autophagy ux, but depletion of all GABARAP inhibits this effect [12,39] . In addition, GABARAP proteins can bridge some of the divisional pores left by the nal phase of autophagosome closure to facilitate organelle closure, consistent with the idea that lipidized GABARAPL1 accumulates at sites of high curvature and membrane-to-membrane juxtaposition [40] GABARAP speci cally recruits PLEKHM1 in the nal phase and mediates HOPS recruitment to drive autophagylysosomal fusion [16] , In cells lacking all GABARAP proteins after knockdown, PLEKHM1 was not recruited at the membrane [41] , Also leads to failure of mutual fusion between autophagosomes and lysosomes to degrade substrates that are encapsulated by autophagosomes [42] . The GABARAP subfamily is essential for the later stages of autophagy cell maturation [43] .…”

LC3B is lipidated to large lipid droplets during prolonged starvation for noncanonical autophagy

“…An interaction was only observed in the case of the LC3B-lipidated LD (Figures 6G and 6H). Pulling the two objects apart led to the deformation of the vesicle into a tubule connecting it to the LC3B-lipidated droplet (Figure 6G), indicating the tethering of the two interfaces, as previously observed 62 , and, eventually, their subsequent merge and physical contiguity 65 .…”

Autophagosome biogenesis: From membrane growth to closure

“…Because Atg8/ATG8 proteins can tether or fuse small liposomes in vitro ( Nair et al, 2011 ; Nakatogawa et al, 2007 ; Weidberg et al, 2011 ), one possible model is that ATG8 proteins are part of the machinery needed to fuse vesicles to drive membrane expansion. Notably, in vitro, these proteins tether membranes only in a topologically restricted trans conformation ( Motta et al, 2018 ), and they drive lipid-mixing only if the membranes exhibit strongly destabilized lipid packing ( Nair et al, 2011 ). Thus if they drive fusion in vivo, lipidated Atg8 would need to be present on both the incoming vesicle and expanding phagophore at sites where one or both of these membranes were “prone” to fuse, perhaps because of high local curvature or high surface densities of fusogenic lipids.…”