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.

The structure of a highly-conserved picocyanobacterial protein reveals a Tudor domain with an RNA-binding function.

Cyanobacteria of the Prochlorococcus and marine Synechococcus genera are the most abundant photosynthetic microbes in the ocean. Intriguingly, the genomes of these bacteria are strongly divergent even within each genus, both in gene content and at the amino acid level of the encoded proteins. One striking exception to this is a 62-amino-acid protein, termed Prochlorococcus/ Synechococcushyper-conserved protein (PSHCP). PSHCP is not only found in all sequenced Prochlorococcus and marine Synechococcus genomes, but it is also nearly 100% identical in its amino acid sequence across all sampled genomes. Such universal distribution and sequence conservation suggest an essential cellular role of PSHCP in these bacteria. However, its function is unknown. Here, we used NMR spectroscopy to determine its structure, finding that 53 of the 62 amino acids in PSHCP form a Tudor domain, whereas the remainder of the protein is disordered. NMR titration experiments revealed that PSHCP has only a weak affinity for DNA, but an 18.5-fold higher affinity for tRNA, hinting at an involvement of PSHCP in translation. Isothermal titration calorimetry experiments further revealed that PSHCP also binds single-stranded, double-stranded, and hairpin RNAs. These results provide the first insight into the structure and function of PSHCP, suggesting that PSHCP appears to be an RNA-binding protein that can recognize a broad array of RNA molecules.

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.