A Comparative Analysis of the Membrane Binding and Remodeling Properties of Two Related Sorting Nexin Complexes Involved in Autophagy.

The sorting nexin (SNX) proteins, Atg20 and Atg24, are involved in nonselective autophagy, are necessary for efficient selective autophagy, and are required for the cytoplasm-to-vacuole transport pathway. However, the specific roles of these proteins in autophagy are not well understood. Atg20 and Atg24 each contain a Phox homology domain that facilitates phosphoinositide binding. They also each contain an SNX-Bin/Amphiphysin/Rvs domain that forms a cup-shaped dimer, capable of binding to curved membranes and remodeling those membranes in some cases. Atg20 and Atg24 form two distinct complexes, an Atg24/Atg24 homodimer and an Atg20/Atg24 heterodimer. Despite the presence of Atg24 in both complexes, it is currently unclear if these complexes have different membrane binding and remodeling properties. Therefore, in this study, we explored the membrane binding and shaping properties of these two dimeric complexes. We found that Atg24/Atg24 and Atg20/Atg24 have distinct membrane binding preferences. Both dimers recognized membranes containing phosphatidylinositol 3-phosphate [PI(3)P] and phosphatidylinositol 3,5-bisphosphate, but Atg20/Atg24 bound to a broader array of liposomes, including those lacking phosphorylated phosphatidylinositol. In addition, we discovered that while both complexes bound to autophagosomal-like liposomes containing at least 5% PI(3)P, Atg20/Atg24 was capable of binding to autophagosomal-like liposomes lacking PI(3)P. Lastly, we observed that the Atg20/Atg24 heterodimer tubulates PI(3)P-containing and autophagosomal-like liposomes, but the Atg24/Atg24 homodimer could not tubulate these liposomes. Our findings suggest that these two dimers contain distinct membrane binding and shaping properties.

Membrane Binding and Homodimerization of Atg16 Via Two Distinct Protein Regions is Essential for Autophagy in Yeast.

Macroautophagy is a bulk degradation mechanism in eukaryotic cells. Efficiency of an essential step of this process in yeast, Atg8 lipidation, relies on the presence of Atg16, a subunit of the Atg12-Atg5-Atg16 complex acting as the E3-like enzyme in the ubiquitination-like reaction. A current view on the functional structure of Atg16 in the yeast S. cerevisiae comes from the two crystal structures that reveal the Atg5-interacting α-helix linked via a flexible linker to another α-helix of Atg16, which then assembles into a homodimer. This view does not explain the results of previous in vitro studies revealing Atg16-dependent deformations of membranes and liposome-binding of the Atg12-Atg5 conjugate upon addition of Atg16. Here we show that Atg16 acts as both a homodimerizing and peripheral membrane-binding polypeptide. These two characteristics are imposed by the two distinct regions that are disordered in the nascent protein. Atg16 binds to membranes in vivo via the amphipathic α-helix (amino acid residues 113-131) that has a coiled-coil-like propensity and a strong hydrophobic face for insertion into the membrane. The other protein region (residues 64-99) possesses a coiled-coil propensity, but not amphipathicity, and is dispensable for membrane anchoring of Atg16. This region acts as a Leu-zipper essential for formation of the Atg16 homodimer. Mutagenic disruption in either of these two distinct domains renders Atg16 proteins that, in contrast to wild type, completely fail to rescue the autophagy-defective phenotype of atg16Δ cells. Together, the results of this study yield a model for the molecular mechanism of Atg16 function in macroautophagy.

A highly conserved glutamic acid in ALFY inhibits membrane binding to aid in aggregate clearance.

Autophagy-linked FYVE protein (ALFY) is a large, multidomain protein involved in the degradation of protein aggregates by selective autophagy. The C-terminal FYVE domain of ALFY has been shown to bind phosphatidylinositol 3-phosphate (PI(3)P); however, ALFY only partially colocalizes with other FYVE domains in cells. Thus, we asked if the FYVE domain of ALFY has distinct membrane binding properties compared to other FYVE domains and whether these properties might affect its function in vivo. We found that the FYVE domain of ALFY binds weakly to PI(3)P containing membranes in vitro. This weak binding is the result of a highly conserved glutamic acid within the membrane insertion loop in the FYVE domain of ALFY that is not present in any other human FYVE domain. In addition, not only does this glutamic acid reduce binding to membranes in vitro and inhibits its targeting to membranes in vivo, but it is also important for the ability of ALFY to clear protein aggregates.

A pseudo-receiver domain in Atg32 is required for mitophagy.

Mitochondria are targeted for degradation by mitophagy, a selective form of autophagy. In Saccharomyces cerevisiae, mitophagy is dependent on the autophagy receptor, Atg32, an outer mitochondrial membrane protein. Once activated, Atg32 recruits the autophagy machinery to mitochondria, facilitating mitochondrial capture in phagophores, the precursors to autophagosomes. However, the mechanism of Atg32 activation remains poorly understood. To investigate this crucial step in mitophagy regulation, we examined the structure of Atg32. We have identified a structured domain in Atg32 that is essential for the initiation of mitophagy, as it is required for the proteolysis of the C-terminal domain of Atg32 and the subsequent recruitment of Atg11. The solution structure of this domain was determined by NMR spectroscopy, revealing that Atg32 contains a previously undescribed pseudo-receiver (PsR) domain. Our data suggests that the PsR domain of Atg32 regulates Atg32 activation and the initiation of mitophagy. ABBREVIATIONS: AIM: Atg8-interacting motif; GFP: green fluorescent protein; LIR: LC3-interacting region; NMR: nuclear magnetic resonance; NOESY: nuclear Overhauser effect spectroscopy; PDB: protein data bank; PsR: pseudo-receiver; RMSD: root-mean-square deviation.

Crystal structure of the thermochromic bis-(di-ethyl-ammonium) tetra-chlorido-cuprate(II) complex.

In the structure of the title complex salt, (Et2NH2)2[CuCl4], the asymmetric unit consists of four unique di-ethyl-ammonium cations and three unique tetra-chlorido-cuprate anions. Two of the three anions are located with their copper atoms on independent crystallographic twofold axes, while the remaining tetra-chlorido-cuprate is located at a general position of the ortho-rhom-bic space group P21212. Two of the three Cu atoms adopt a distorted square-planar/disphenoidal geometry and the third Cu atom has a regular square-planar coordination environment. The di-ethyl-ammonium cations form an extensive hydrogen-bonded network through N-H⋯Cl inter-actions with the tetra-chlorido-cuprate anions, resulting in a two-dimensional sheet-like hydrogen-bonded network parallel to the ab direction. The complex was observed to undergo a color shift from deep green at room temperature to pale yellow at temperatures above 328 K.