Curvature-sensitive trans-assembly of human Atg8-family proteins in autophagy-related membrane tethering

Abstract

In macroautophagy, de novo formation of the double membrane-bound organelles, termed autophagosomes, is essential for engulfing and sequestering the cytoplasmic contents to be degraded in the lytic compartments such as vacuoles and lysosomes. Atg8-family proteins have been known to be responsible for autophagosome formation via membrane tethering and fusion events of precursor membrane structures. Nevertheless, how Atg8 proteins act directly upon autophagosome formation still remains enigmatic. Here, to further gain molecular insights into Atg8-mediated autophagic membrane dynamics, we study the two representative human Atg8 orthologs, LC3B and GATE-16, by quantitatively evaluating their intrinsic potency to physically tether lipid membranes in a chemically defined reconstitution system using purified Atg8 proteins and synthetic liposomes. Both LC3B and GATE-16 retained the capacities to trigger efficient membrane tethering at the protein-to-lipid molar ratios ranging from 1:100 to 1:5,000. These human Atg8mediated membrane tethering reactions require transassembly between the membrane-anchored forms of LC3B and GATE-16 and can be reversibly and strictly controlled by the membrane attachment and detachment cycles. Strikingly, we further uncovered distinct membrane curvature dependences of LC3B-and GATE-16-mediated membrane tethering reactions: LC3B can drive tethering more efficiently than GATE-16 for highly-curved small vesicles (e.g. 50 nm in diameter), although GATE-16 turns out to be a more potent tether than LC3B for flatter large vesicles (e.g. 200 and 400 nm in diameter). Our findings establish curvature-sensitive trans-assembly of human Atg8-family proteins in reconstituted membrane tethering, which recapitulates an essential subreaction of the biogenesis of autophagosomes in vivo.

The small GTPase Arf6 functions as a membrane tether in a chemically-defined reconstitution system

“…The intrinsic membrane tethering activities of human Arf-family small GTPases were assayed by monitoring turbidity changes of liposome solutions in the presence of purified Arf proteins, as described for the turbidity assays with human Rab-family small GTPases (Tamura and Mima, 2014; Inoshita and Mima, 2017; Mima, 2018; Segawa et al, 2019; Ueda et al, 2020) and Atg8-family proteins (Taniguchi et al, 2020). Purified His12-Arf1, His12-Arf6, or Rab5a-His12 (0.5 – 3 μM final) and DOGS-NTA-bearing liposomes (200-nm diameter; final 1 mM total lipids), which had been separately preincubated (30°C, 10 min), were mixed in RB150 containing 5 mM MgCl 2 and 1 mM DTT (160 μl each), applied to a 10-mm path-length cell (105.201-QS, Hellma Analytics), and immediately subjected to monitoring the turbidity changes with measuring the optical density changes at 400 nm (ΔOD400) for 5min with 10-sec intervals in a DU720 spectrophotometer at room temperature.…”

Membrane tethering potency of Rab-family small GTPases is defined by the C-terminal hypervariable regions

“…Although we used the protein stoichiometry of synaptic vesicles as a reasonable model for calculating the physiological Rab protein density on the membrane (Takamori et al, 2006), it is possible that the Rab densities on other membrane compartments such as early endosomes are variable and different from the current estimations. Regarding other putative membrane tethers in a reconstituted system, their tethering activities have been examined at the protein-to-lipid ratios similar to those tested in the present experiments (1:100-1:5,000, mol/mol; Figure 2), which include the typical ratios of 1:400 for golgin GMAP-210 (Drin et al, 2008), 1:330 for Vps21p (Lo et al, 2012), 1:2,000 for HOPS (Ho and Stroupe, 2015;Ho and Stroupe, 2016), 1:800 for Atg8p (Nair et al, 2011), and 1:500 for human Atg8 orthologs (Taniguchi et al, 2020). It should also be noted that, assuming that Rab molecules are a spherical 25-kDa protein with a radius of 2.0 nm (Erickson, 2009), membrane-bound Rab proteins occupy only 1.9% of the outer surface areas of the 200-nm liposomes when tested at the 1:2,000 Rab-to-lipid ratio.…”

“…Liposome solutions (200-nm diameter; 1 mM total lipids in final) and purified Rab-His12 proteins (final 0.2-10 M), which had been preincubated separately at 30°C for 5 min, were mixed in RB150 containing 5 mM MgCl 2 and 1 mM DTT, transferred to a 10-mm path-length cuvette (105.201-QS, Hellma Analytics), and immediately subjected to measurement of the optical density changes at 400 nm (OD400) in a DU720 spectrophotometer (Beckman Coulter) for 5 min with 10-sec intervals at room temperature. The OD400 data obtained from the kinetic turbidity assays were analyzed by curve fitting using the ImageJ2 software (National Institutes of Health) and the logistic function formula, y = a/(1+b*exp(-c*x)), where y and x correspond to the OD400 value and the time (min), respectively (Segawa et al, 2019;Taniguchi et al, 2020). The maximum capacities of Rab-mediated liposome tethering were defined as the theoretical maximum OD400 values of the fitted sigmoidal curves at t = ∞ and thus calculated as "a" from the logistic formula above.…”

“…Fluorescence microscopy-based imaging assays for Rabmediated liposome tethering were performed using a LUNA-FL automated fluorescence cell counter (Logos Biosystems), as described (Segawa et al, 2019;Taniguchi et al, 2020). Liposomes bearing fluorescence-labeled Rh-PE or FL-PE lipids (200-nm diameter; final 2 mM total lipids) and Rab-His12 proteins (final 0.5-8 M), which had been separately preincubated (30°C, 10 min), were mixed in RB150 with 5 mM MgCl 2 and 1 mM DTT, incubated without agitation (30°C, 2 hours), and then applied to a LUNA cell-counting slide (L12001, Logos Biosystems; 15 l per well).…”

“…Bright field images, Rh-fluorescence images, and FL-fluorescence images of the Rab-mediated liposome tethering reactions in the slides were obtained and processed by the LUNA-FL cell counter. Particle sizes of Rab-dependent liposome clusters observed in the fluorescence images were analyzed using the ImageJ2 software with setting the lower intensity threshold level to 150, the upper intensity threshold level to 255, and the minimum particle size to 10 pixel 2 which corresponds to approximately 10 m 2 (Segawa et al, 2019;Taniguchi et al, 2020).…”