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 membranetethering 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.
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 Escherichia coli CrfC protein is an important regulator of nucleoid positioning and equipartition. Previously we revealed that CrfC homo-oligomers bind the clamp, a DNA-binding subunit of the DNA polymerase III holoenzyme, promoting colocalization of the sister replication forks, which ensures the nucleoid equipartition. In addition, CrfC localizes at the cell pole-proximal loci via an unknown mechanism. Here, we demonstrate that CrfC localizes to the distinct subnucleoid structures termed nucleoid poles (the cell pole-proximal nucleoid-edges) even in elongated cells as well as in wild-type cells. Systematic analysis of the nucleoid-associated proteins (NAPs) and related proteins revealed that HU, the most abundant NAP, and SlmA, the nucleoid occlusion factor regulating the localization of cell division apparatus, promote the specific localization of CrfC foci. When the replication initiator DnaA was inactivated, SlmA and HU were required for formation of CrfC foci. In contrast, when the replication initiation was inhibited with a specific mutant of the helicase-loader DnaC, CrfC foci were sustained independently of SlmA and HU. H-NS, which forms clusters on AT-rich DNA regions, promotes formation of CrfC foci as well as transcriptional regulation of crfC. In addition, MukB, the chromosomal structure mainetanice protein, and SeqA, a hemimethylated nascent DNA region-binding protein, moderately stimulated formation of CrfC foci. However, IHF, a structural homolog of HU, MatP, the replication terminus-binding protein, Dps, a stress-response factor, and FtsZ, an SlmA-interacting factor in cell division apparatus, little or only slightly affected CrfC foci formation and localization. Taken together, these findings suggest a novel and unique mechanism that CrfC localizes to the nucleoid poles in two steps, assembly and recruitment, dependent upon HU, MukB, SeqA, and SlmA, which is stimulated directly or indirectly by H-NS and DnaA. These factors might concordantly affect specific nucleoid substructures. Also, these nucleoid dynamics might be significant in the role for CrfC in chromosome partition.
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