Mitochondrial apoptosis is mediated by BAK and BAX, two proteins that induce mitochondrial outer membrane permeabilization, leading to cytochrome c release and activation of apoptotic caspases. In the absence of active caspases, mitochondrial DNA (mtDNA) triggers the innate immune cGAS/STING pathway, causing dying cells to secrete type I interferon. How cGAS gains access to mtDNA remains unclear. We used live-cell lattice light-sheet microscopy to examine the mitochondrial network in mouse embryonic fibroblasts. We found that after BAK/BAX activation and cytochrome c loss, the mitochondrial network broke down and large BAK/BAX pores appeared in the outer membrane. These BAK/BAX macropores allowed the inner mitochondrial membrane to herniate into the cytosol, carrying with it mitochondrial matrix components, including the mitochondrial genome. Apoptotic caspases did not prevent herniation but dismantled the dying cell to suppress mtDNA-induced innate immune signaling.
MuB is an ATP-dependent nonspecific DNA-binding protein that regulates the activity of the MuA transposase and captures target DNA for transposition. Mechanistic understanding of MuB function has previously been hindered by MuB's poor solubility. Here we combine bioinformatic, mutagenic, biochemical, and electron microscopic analyses to unmask the structure and function of MuB. We demonstrate that MuB is an ATPase associated with diverse cellular activities (AAA+ ATPase) and forms ATP-dependent filaments with or without DNA. We also identify critical residues for MuB's ATPase, DNA binding, protein polymerization, and MuA interaction activities. Using single-particle electron microscopy, we show that MuB assembles into a helical filament, which binds the DNA in the axial channel. The helical parameters of the MuB filament do not match those of the coated DNA. Despite this protein-DNA symmetry mismatch, MuB does not deform the DNA duplex. These findings, together with the influence of MuB filament size on strand-transfer efficiency, lead to a model in which MuB-imposed symmetry transiently deforms the DNA at the boundary of the MuB filament and results in a bent DNA favored by MuA for transposition.Phage Mu | nucleoprotein filament D NA transposons are ubiquitous in the genomes of all forms of life and play important evolutionary roles in generating gene diversity and in shaping genomic landscapes (1). Although typically transposons exhibit no strong sequence selectivity for the target DNA site, certain transposons avoid self-destructive insertion (reviewed in ref.2), a phenomenon called "target immunity" because the presence of a copy of the transposon renders nearby DNA sites "immune" to additional insertion by the same transposon (3-8). MuB plays critical roles in this selfimmunity in the bacteriophage Mu transposition process.Phage Mu is one of the most complex and efficient transposable elements (reviewed in refs. 3 and 4). Two phage-coded proteins, MuA and MuB, are essential for efficient Mu transposition. MuA is the transposase responsible for synapsing the two Mu end sequences and for all of the DNA cutting and joining steps in the initial stages of transposition. However, transposition is inefficient in the absence of MuB, and the residual Mu insertion that takes place uses only DNA target sites near or within the transposing element, often leading to self-destruction (9-11). MuB is a small (35-kDa) ATP-dependent nonspecific DNA-binding protein with relatively low ATPase activity (10,12,13). Upon ATP binding, MuB polymerizes preferentially on DNA, but in the absence of DNA it still can form polymers of variable sizes (14, 15). When observed by total internal reflection fluorescence microscopy, GFP-MuB-ATP binds along the DNA molecule forming many short separate segments of polymers, and, as more GFP-MuB is added, the protein-covered segments elongate to form an apparently continuous polymer that fully coats the DNA. Hydrolysis of ATP reverses this process, triggering disassembly of the MuB polymer (16-18)...
Fucosylation of the inner-most Nacetylglucosamine (GlcNAc) of N-glycans by fucosyltransferase 8 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple modification can dramatically affect the activities and half-lives of glycoproteins, effects that are relevant to understanding the invasiveness of some cancers, the development of monoclonal antibody therapeutics, and the etiology of a congenital glycosylation disorder. The acceptor substrate preferences of FUT8 are well characterized and provide a framework for understanding N-glycan maturation in the Golgi; however the structural basis for these substrate preferences and the mechanism through which catalysis is achieved remain unknown. Here, we describe several structures of mouse and human FUT8 in the apo state and in complex with guanosine diphosphate (GDP), a mimic of the donor substrate, and with a glycopeptide acceptor substrate at 1.80-2.50 Å resolutions. These structures provide insights into a unique conformational change associated with donor substrate binding, common strategies employed by fucosyltransferases to coordinate GDP, features that define acceptor substrate preferences, and a likely mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also revealed how FUT8 dimerization plays an important role in defining the acceptor substrate-binding site. Collectively, this information significantly builds on our understanding of the core fucosylation process. https://www.jbc.org/cgi/
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