Mechanistic Insights into Inter-Organellar Membrane Contact Sites Based on Crystal Structures

Abstract

Eukaryotic cells contain various membrane-bound subcellular compartments called organelles. For cell survival, organelles perform specific functions respectively or cooperate through communication. Recently, as the development of electron microscopy, it has been discovered that organelles are located closely and make contact sites with about 10 nm distance between two membranes, which is called membrane contact sites (MCSs). MCSs are constructed and maintained stably or transiently by tethering structures mainly composed of proteins. It has been revealed that MCSs are important sites for various cellular functions such as intracellular signaling, lipid and ion exchange, organelle trafficking, and inheritance. Nucleus-vacuole junctions (NVJs) are one of the firstly identified MCSs in the budding yeast Saccharomyces cerevisiae. The formation of NVJs depends on the nuclear membrane protein Nvj1 and vacuolar protein Vac8. NVJs formed by the interaction between Vac8 and Nvj1 are critical for mediating piecemeal microautophagy of the nucleus (PMN), one of the selective microautophagy pathways in the yeast. Herein, we report the crystal structure of Vac8-Nvj1 complex. Vac8 comprises the H1 helix at the N terminus, followed by 12 armadillo repeats (ARMs). The 80 Å extended loop of Nvj1 binds to the conserved inner groove of Vac8 ARM domain in an antiparallel manner. Disruption of the Vac8-Nvj1 interaction results in the loss of NVJ formation and further PMN in Saccharomyces cerevisiae. Vac8 cationic triad (Arg276, Arg317, and Arg359) motifs interacting with Nvj1 are also critical to the recognition of Atg13, a key component of the cytoplasm-to-vacuole targeting (CVT) pathway, indicating competitive binding to Vac8. Indeed, the crystal structure of Vac8-Atg13 complex reveals that the 70 Å extended loop of Atg13 binds to the ARM domain of Vac8 in a similar manner with Nvj1. The interaction between Vac8 and Atg13 is also essential for mediating CVT pathway in Saccharomyces cerevisiae. Structural, biochemical, and in vivo experiments demonstrate that the H1 helix of Vac8 intramolecularly interacts with the first ARM and regulates its self-association, which is crucial for PMN and CVT pathway. Crystal structures also reveal that Vac8-Nvj1 and Vac8-Atg13 form heterotetramer with the different quaternary organization. The structural comparison provides a molecular understanding of how a single ARM domain protein adopts different quaternary structures depending on its binding proteins to differentially regulate two closely related but distinct cellular pathways. Next, recent research suggested that 3′-5′ exonuclease domain-containing protein 2, EXD2 plays an essential role as a new component of DNA double-strand break repair machinery in the nucleus with a conserved DEDDy superfamily 3′-5′ exonuclease domain. However, the following researches raised the possibility that EXD2 might be localized to mitochondria with unclear mitochondrial sublocation. Herein, electron microscope imaging analysis and proximity labeling reveals that EXD2 is anchored to the mitochondrial outer membrane through a conserved N-terminal transmembrane domain, while the C-terminal region is cytosolic. The crystal structure of the exonuclease domain reveals a domain-swapped dimer in which the central α5¬α7 helices are mutually crossed over, resulting in chimeric active sites. Additionally, the C-terminal segments absent in other DnaQ family exonucleases enclose the central chimeric active sites. Structural analyses of crystal structures in complex with Mn2+/Mg2+ and biochemical experiments demonstrate that unusual dimeric organization and additional C-segments stabilize the active site, facilitate discrimination between DNA and RNA substrates based on divalent cation coordination, and generate a positively charged groove that binds substrates. Based on structural studies and previously reported data, we suggest that EXD2 can play possible roles in the mitochondrial outer membrane or inter-mitochondrial membrane contact sites with its novel exonuclease activity.