Mechanistic insight into the nucleus–vacuole junction based on the Vac8p–Nvj1p crystal structure

Abstract

Formation of the nucleus-vacuole junction (NVJ) is mediated by direct interaction between the vacuolar protein Vac8p and the outer nuclear endoplasmic reticulum membrane protein Nvj1p. Herein we report the crystal structure of Vac8p bound to Nvj1p at 2.4-Å resolution. Vac8p comprises a flexibly connected N-terminal H1 helix followed by 12 armadillo repeats (ARMs) that form a right-handed superhelical structure. The extended 80-Å-long loop of Nvj1p specifically binds the highly conserved inner groove formed from ARM1−12 of Vac8p. Disruption of the Nvj1p-Vac8p interaction results in the loss of tight NVJs, which impairs piecemeal microautophagy of the nucleus in Saccharomyces cerevisiae. Vac8p cationic triad (Arg276, Arg317, and Arg359) motifs interacting with Nvj1p are also critical to the recognition of Atg13p, a key component of the cytoplasm-to-vacuole targeting (CVT) pathway, indicating competitive binding to Vac8p. Indeed, mutation of the cationic triad abolishes CVT of Ape1p in vivo. Combined with biochemical data, the crystal structure reveals a Vac8p homodimer formed from ARM1, and this self-association, likely regulated by the flexible H1 helix and the C terminus of Nvj1p, is critical for Vac8p cellular functions. M embrane contact sites (MCSs) between subcellular compartments play pivotal roles in cellular processes such as cooperative lipid biosynthesis, ion homeostasis, and interorganellar trafficking of molecules in eukaryotic cells (1-3). MCSs are physically formed through dynamic and direct interactions between proteins that are located in two distinct subcompartments. The nucleus-vacuole junction (NVJ), one of the best-characterized MCSs, is a physical contact site between perinuclear and vacuolar membranes and mediates essential cellular processes such as piecemeal microautophagy of the nucleus (PMN) and lipid biosynthesis in yeast (4, 5). PMN is a selective autophagic recycling process stimulated by carbon or nitrogen starvation through the target of rapamycin signaling pathway in Saccharomyces cerevisiae. Upon nutrient starvation, the region of the nucleus in the vicinity of NVJs invaginates into the vacuolar lumen and forms a bleb-like structure, which is released as a vesicle and eventually degraded by vacuolar hydrolases (5, 6). Because PMN occurs at the NVJ sites, their formation is essential for the initiation of the PMN process (7). NVJs are also involved in lipid metabolism by recruiting the two lipid-modifying enzymes, oxystereol-binding proteins homology (Osh1p) involved in nonvesicular lipid trafficking and the enoyl-CoA reductase Tsc13p that mediates the synthesis of verylong-chain fatty acids (8-11).Previous studies revealed that NVJs are generated by forming tight interactions between Vac8p, a vacuolar membrane protein, and Nvj1p, a nuclear membrane protein (12). Vac8p was initially found as a key player in vacuole inheritance by cooperating with Vac17p, actin, profilin, and Myo2p (13). In addition, Vac8p has a crucial role in mediating the processing of aminopeptidase I through in...

Quaternary structures of Vac8 differentially regulate the Cvt and PMN pathways

“…The loop (residues 35-40) bridging the H1 helix and the first ARM (ARM1) is disordered in the structure ( Figure 1D). The overall conformation of the 12 concerted ARMs of tVac8 observed in the crystal structure of the tVac8-tAtg13 complex was essentially identical to that observed in the tVac8-tNvj1 complex [24], with a root-mean-square deviation (RMSD) value of 0.88 Å for all Cα atoms. The 70-Å extended loop of Atg13 binds across the inner groove formed by the central ARMs of Vac8 ( Figure 1C).…”

“…Based on these data, we made a truncated Atg13 construct (residues 567-695; designated as tAtg13) for structural analysis of the tVac8-tAtg13 complex. tVac8 (residues 10-515) was used in our previous study ( Figure 1A) [24]. Analytical ultracentrifugation (AUC) experiment revealed that the tVac8-tAtg13 complex formed a heterodimer in solution ( Figure 1B).…”

“…While much has been learned about the Vac8-Atg13 complex and its involvement in the Cvt pathway, the molecular mechanism by which Atg13 is specifically recognized by Vac8 remains elusive. In addition, our previous study suggested that Nvj1 shares a binding interface for Vac8 with Atg13; therefore, Nvj1 or Atg13 competes for binding to Vac8 [24]. However, it is still unknown how Vac8 can discriminate between Nvj1 or Atg13, and thereby differentially regulate PMN or Cvt pathways that occur simultaneously in yeast.…”

Nuclear envelope-vacuole contacts mitigate nuclear pore complex assembly stress

“…Although NVJs have not been specifically shown to mediate lipid transfer between the vacuole and NE, interorganelle contact sites often serve as sites of lipid exchange (Kornmann et al, 2009;Elbaz-Alon et al, 2014) due to their close apposition and enrichment of lipid-exchanging factors. The ergosterol-and PI(4)P-binding protein Osh1 (Manik et al, 2017), which is found at NVJs and interacts with Nvj1 (Jeong et al, 2017;Kvam and Goldfarb, 2004;Levine and Munro, 2001), belongs to the oxysterol-binding homology protein family that can aid in lipid transfer between membranes (de Saint-Jean et al, 2011). Additionally, LDs are thought to promote lipid transfer between organelles, potentially through "lipid bridges" (Schuldiner and Bohnert, 2017).…”

“…Although morphologically distinct, vacuoles are functionally similar to metazoan lysosomes, serving as acidic organelles involved in protein degradation and nutrient storage. Several outer NE membrane proteins including Nvj1 (Pan et al, 2000) and Mdm1 (Henne et al, 2015) are present at NVJs and tether the NE to the vacuole through specific interactions with corresponding vacuolar proteins like Vac8 (Pan et al, 2000;Jeong et al, 2017) or phospholipid species such as phosphatidylinositol 3-phosphate (Henne et al, 2015). NVJs expand when cells are grown using carbon sources other than glucose (Hariri et al, 2018;Bean et al, 2018) or to promote piecemeal autophagy of the nucleus (PMN) in response to nutrient starvation , which involves autophagy-dependent degradation of specific nuclear proteins in the vacuole (Krick et al, 2008).…”