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Many biomolecules undergo liquid-liquid phase separation to form liquid-like condensates that mediate diverse cellular functions.sup.1,2. Autophagy is able to degrade such condensates using autophagosomes--double-membrane structures that are synthesized de novo at the pre-autophagosomal structure (PAS) in yeast.sup.3-5. Whereas Atg proteins that associate with the PAS have been characterized, the physicochemical and functional properties of the PAS remain unclear owing to its small size and fragility. Here we show that the PAS is in fact a liquid-like condensate of Atg proteins. The autophagy-initiating Atg1 complex undergoes phase separation to form liquid droplets in vitro, and point mutations or phosphorylation that inhibit phase separation impair PAS formation in vivo. In vitro experiments show that Atg1-complex droplets can be tethered to membranes via specific protein-protein interactions, explaining the vacuolar membrane localization of the PAS in vivo. We propose that phase separation has a critical, active role in autophagy, whereby it organizes the autophagy machinery at the PAS. The pre-autophagosomal structure in yeast is a liquid-like condensate of Atg proteins whose phase separation may have a critical, active role in autophagy., Author(s): Yuko Fujioka [sup.1] , Jahangir Md. Alam [sup.1] , Daisuke Noshiro [sup.1] [sup.2] , Kazunari Mouri [sup.3] , Toshio Ando [sup.2] , Yasushi Okada [sup.3] [sup.4] , Alexander I. [...]

The vacuole hydrolase aminopeptidase 1 (Ape1) is a cargo protein transported to the vacuole by the cytosol-to-vacuole targeting (Cvt) pathway during conditions of growth and by autophagy during conditions of starvation. After transport to the vacuole, Ape1 is processed into mature Ape1 (mApe1). mApe1 has been expressed, purified and crystallized in two crystal forms. Form I belongs to space group P21, with unit-cell parameters a = 120.6, b = 219.5, c = 133.1 Å, β = 116.5°. Form II belongs to space group R3, with unit-cell parameters a = 141.2, c = 349.4 Å. Diffraction data were collected from these crystals to a resolution of 2.5 Å for form I and 1.83 Å for form II. Self-rotation functions and the volume-to-weight ratio values suggest that forms I and II contain 12 and four mApe1 molecules per asymmetric unit, respectively, and that mApe1 exists as a tetrahedral dodecamer in both crystal forms. [ABSTRACT FROM AUTHOR]

The phagocyte NADPH oxidase is a multisubunit enzyme responsible for the production of reactive oxygen species. p47phox is a cytosolic component of the NADPH oxidase and plays an important role in the assembly of the activated complex. The structural determination of the tandem SH3 domains of p47phox is crucial for elucidation of the molecular mechanism of the activation of p47phox. We determined the X-ray crystal structure of the tandem SH3 domains with the polybasic/autoinhibitory region (PBR/AIR) of p47phox. The GAPPR sequence involved in PBR/AIR forms a left-handed polyproline type-II helix (PPII) and interacts with the conserved SH3 binding surfaces of the SH3 domains simultaneously. These SH3 domains are related by a 2-fold pseudosymmetry axis at the centre of the binding groove and interact with the single PPII helix formed by the GAPPR sequence with opposite orientation. In addition, a number of intra-molecular interactions among the SH3 domains, PBR/AIR and the linker tightly hold the architecture of the tandem SH3 domains into the compact structure and stabilize the autoinhibited form synergistically. Phosphorylation of the serine residues in PBR/AIR could destabilize and successively release the intra-molecular interactions. Thus, the overall structure could be rearranged from the autoinhibitory conformation to the active conformation and the PPII ligand binding surfaces on the SH3 domains are now unmasked, which enables their interaction with the target sequence in p22phox. [ABSTRACT FROM AUTHOR]

Autophagy mediates the bulk degradation of cytoplasmic components in lysosomes/vacuoles. Five autophagy-related (Atg) proteins are involved in a ubiquitin-like protein conjugation system. Atg12 is conjugated to its sole target, Atg5, by two enzymes, Atg7 and Atg10. The Atg12-Atg5 conjugates form a multimeric complex with Atg16. Formation of the Atg12-Atg5-Atg16 ternary complex is crucial for the functions of these proteins on autophagy. Here, the expression, purification and crystallization of the Atg12-Atg5 conjugate bound to the N-terminal region of Atg16 (Atg16N) are reported. The Atg12-Atg5 conjugates were formed by co-expressing Atg5, Atg7, Atg10 and Atg12 in Eschericia coli. The Atg12-Atg5-Atg16N ternary complex was formed by mixing purified Atg12-Atg5 conjugates and Atg16N, and was further purified by gel-filtration chromatography. Crystallization screening was performed by the free-interface diffusion method. Using obtained microcrystals as seeds, large crystals for diffraction data collection were obtained by the sitting-drop vapour-diffusion method. The crystal contained one ternary complex per asymmetric unit, and diffracted to 2.6 A resolution. [ABSTRACT FROM AUTHOR]

p40 phox is a cytosolic component of the phagocyte NADPH oxidase, which is responsible for production of the superoxide that kills invasive microorganisms. Full-length p40 phox was expressed in Escherichia coli, purified and crystallized by the sitting-drop vapour-diffusion method at 293 K using polyethylene glycol 20 000 as a precipitant. Diffraction data were collected to 3.0 Å resolution at 100 K using synchrotron radiation. The crystal belongs to space group C2221, with unit-cell parameters a = 146.27, b = 189.81, c = 79.88 Å. This crystal was estimated to contain two or three protein molecules per asymmetric unit from the acceptable range of volume-to-weight ratio values. [ABSTRACT FROM AUTHOR]

Presents a letter to the editor about Sequence-specific resonance assignments of the tandem 5H3 domains.

Autophagy is a bulk degradation process in eukaryotic cells; autophagosomes enclose cytoplasmic components for degradation in the lysosome/vacuole. Autophagosome formation requires two ubiquitin-like conjugation systems, the Atg12 and Atg8 systems, which are tightly associated with expansion of autophagosomal membrane. Previous studies have suggested that there is a hierarchy between these systems; the Atg12 system is located upstream of the Atg8 system in the context of Atg protein organization. However, the concrete molecular relationship is unclear. Here, we show using an in vitro Atg8 conjugation system that the Atg12-Atg5 conjugate, but not unconjugated Atg12 or Atg5, strongly enhances the formation of the other conjugate, Atg8-PE. The Atg12-Atg5 conjugate promotes the transfer of Atg8 from Atg3 to the substrate, phosphatidylethanolamine (PE), by stimulating the activity of Atg3. We also show that the Atg12-Atg5 conjugate interacts with both Atg3 and PE-containing liposomes. These results indicate that the Atg12-Atg5 conjugate is a ubiquitin-protein ligase (E3)-like enzyme for Atg8-PE conjugation reaction, distinctively promoting protein-lipid conjugation. [ABSTRACT FROM AUTHOR]

The phagocyte NADPH oxidase is a multisubunit enzyme responsible for the generation of superoxide anions (O2...) that kill invading microorganisms, p47phox is a cytosolic subunit of the phagocyte NADPH oxidase, which plays a crucial role in the assembly of the activated NADPH oxidase complex. The molecular shapes of the p47phox tandem SH3 domains either with or without a polybasic/autoinhibitory region (PBP/AIR) at the C terminus were studied using small angle x-ray scattering. The tandem SH3 domains with PBR/AIR formed a compact globular structure, whereas the tandem SH3 domains lacking the PBP/AIR formed an elongated structure. Alignment anisotropy analysis by NMR based on the residual dipolar couplings revealed that the tandem SH3 domains with PBP/AIR were in good agreement with a globular module corresponding to the split half of the intertwisted dimer in crystalline state. The structure of the globular module was elucidated to represent a solution structure of the tandem SH3 domain in the autoinhibited form, where the PBR/AIR bundled the tandem SH3 domains and the linker forming a closed structure. Once PBP/AIR is released by phosphorylation, rearrangements of the SH3 domains may occur, forming an open structure that binds to the cytoplasmic proline-rich region of membrane-bound p22phox. [ABSTRACT FROM AUTHOR]