3 research outputs found
Delipidation of mammalian Atg8-family proteins by each of the four ATG4 proteases
<p>During macroautophagy/autophagy, mammalian Atg8-family proteins undergo 2 proteolytic processing events. The first exposes a COOH-terminal glycine used in the conjugation of these proteins to lipids on the phagophore, the precursor to the autophagosome, whereas the second releases the lipid. The ATG4 family of proteases drives both cleavages, but how ATG4 proteins distinguish between soluble and lipid-anchored Atg8 proteins is not well understood. In a fully reconstituted delipidation assay, we establish that the physical anchoring of mammalian Atg8-family proteins in the membrane dramatically shifts the way ATG4 proteases recognize these substrates. Thus, while ATG4B is orders of magnitude faster at processing a soluble unprimed protein, all 4 ATG4 proteases can be activated to similar enzymatic activities on lipid-attached substrates. The recognition of lipidated but not soluble substrates is sensitive to a COOH-terminal LIR motif both in vitro and in cells. We suggest a model whereby ATG4B drives very fast priming of mammalian Atg8 proteins, whereas delipidation is inherently slow and regulated by all ATG4 homologs.</p
A Programmable DNA Origami Platform for Organizing Intrinsically Disordered Nucleoporins within Nanopore Confinement
Nuclear
pore complexes (NPCs) form gateways that control molecular
exchange between the nucleus and the cytoplasm. They impose a diffusion
barrier to macromolecules and enable the selective transport of nuclear
transport receptors with bound cargo. The underlying mechanisms that
establish these permeability properties remain to be fully elucidated
but require unstructured nuclear pore proteins rich in Phe-Gly (FG)-repeat
domains of different types, such as FxFG and GLFG. While physical
modeling and <i>in vitro</i> approaches have provided a
framework for explaining how the FG network contributes to the barrier
and transport properties of the NPC, it remains unknown whether the
number and/or the spatial positioning of different FG-domains along
a cylindrical, ∼40 nm diameter transport channel contributes
to their collective properties and function. To begin to answer these
questions, we have used DNA origami to build a cylinder that mimics
the dimensions of the central transport channel and can house a specified
number of FG-domains at specific positions with easily tunable design
parameters, such as grafting density and topology. We find the overall
morphology of the FG-domain assemblies to be dependent on their chemical
composition, determined by the type and density of FG-repeat, and
on their architectural confinement provided by the DNA cylinder, largely
consistent with here presented molecular dynamics simulations based
on a coarse-grained polymer model. In addition, high-speed atomic
force microscopy reveals local and reversible FG-domain condensation
that transiently occludes the lumen of the DNA central channel mimics,
suggestive of how the NPC might establish its permeability properties
Compiled Lipidomics Data_NCOMMS-16-13893-A
Raw data of MS lipidomics datasets used in Fig. 5a and Supplementary Fig. 4c. Data are from HEK cells treated with non-targeting (Control) or HS1BP3 siRNA starved for 2 hrs (mean +/- SEM., n = 6). See the Methods section for a full description of the Methods used