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 Atg8-mediated membrane-tethering reactions require trans-assembly 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.

中文翻译：

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自噬相关的膜束缚中人类Atg8家族蛋白的曲率敏感反组装。

在宏观自噬中，称为自噬体的双膜结合细胞器的从头形成对于吞噬和隔离在液位区如液泡和溶酶体中降解的胞质内容物至关重要。已知Atg8家族蛋白通过膜束缚和前体膜结构的融合事件负责自噬体的形成。尽管如此，Atg8蛋白如何直接作用于自噬体仍然是个谜。在这里，为了进一步获得分子对Atg8介导的自噬膜动力学的分子见解，我们通过使用纯化的Atg8在化学成分确定的重组系统中定量评估它们对束缚脂质膜的内在潜力，研究了两个代表性的人类Atg8直系同源物LC3B和GATE-16。蛋白质和合成脂质体。LC3B和GATE-16都保留了在蛋白质与脂质的摩尔比为1：100至1：5,000的条件下触发有效的膜束缚的能力。这些人类Atg8介导的膜栓系反应需要在膜锚定形式的LC3B和GATE-16之间进行反组装，并且可以通过膜附着和分离循环来可逆地严格控制。令人惊讶的是，我们进一步发现了LC3B和GATE-16介导的膜束缚反应的明显膜曲率依赖性：尽管GATE-16，对于高度弯曲的小囊泡（例如，直径为50 nm），LC3B可以比GATE-16更有效地驱动束缚对于更平坦的大囊泡（例如直径200和400 nm），它比LC3B更有效。