Quaternary structures of Vac8 differentially regulate the Cvt and PMN pathways

Abstract

Armadillo (ARM) repeat proteins constitute a large protein family with diverse and fundamental functions in all organisms, and armadillo repeat domains share high structural similarity. However, exactly how these structurally similar proteins can mediate diverse functions remains a long-standing question. Vac8 (vacuole related 8) is a multifunctional protein that plays pivotal roles in various autophagic pathways, including piecemeal microautophagy of the nucleus (PMN) and cytoplasm-to-vacuole targeting (Cvt) pathways in the budding yeast Saccharomyces cerevisiae. Vac8 comprises an H1 helix at the N terminus, followed by 12 armadillo repeats. Herein, we report the crystal structure of Vac8 bound to Atg13, a key component of autophagic machinery. The 70-Å extended loop of Atg13 binds to the ARM domain of Vac8 in an antiparallel manner. Structural, biochemical, and in vivo experiments demonstrated that the H1 helix of Vac8 intramolecularly associates with the first ARM and regulates its selfassociation, which is crucial for Cvt and PMN pathways. The structure of H1 helix-deleted Vac8 complexed with Atg13 reveals that Vac8[Δ19-33]-Atg13 forms a heterotetramer and adopts an extended superhelical structure exclusively employed in the Cvt pathway. Most importantly, comparison of Vac8-Nvj1 and Vac8-Atg13 provides a molecular understanding of how a single ARM domain protein adopts different quaternary structures depending on its associated proteins to differentially regulate 2 closely related but distinct cellular pathways.

Snd3 controls nucleus-vacuole junctions in response to glucose signaling

“…As a functional readout for NVJ formation, we monitored PMN upon nitrogen starvation. This selective microautophagic process occurs at the nuclear-vacuolar interface and results in the pinching off of disposable parts of the nucleus into the vacuole (Krick et al, 2009;Kvam and Goldfarb, 2007;Mijaljica et al, 2007;Park et al, 2020). As expected, PMN was unaffected by the lack of Snd1 or Snd2 but completely absent in cells devoid of Snd3 (Figure S6G).…”

“…Nucleus-vacuole junctions (NVJs) establish close proximity between the vacuole and the perinuclear endoplasmic reticulum (nER) (Cadou and Mayer, 2015;Jeong et al, 2017;Kvam and Goldfarb, 2006;Pan et al, 2000;Park et al, 2020;Prinz, 2014). Only recently, its potential mammalian counterpart was described, connecting the endosomes with the ER (Eden, 2016;Hö nscher and Ungermann, 2014).…”

Theater in the Self-Cleaning Cell: Intrinsically Disordered Proteins or Protein Regions Acting with Membranes in Autophagy

“…The N-terminal IDPR partially folds into the α1-helix, the position of which depends on the binding partner. The α1-helix ranging residues 20–41 packs at the first armadillo repeat when Vac8 interacts with Atg13, but folds at positions 19–35 when Vac8 binds to Nvj1, an interactor for the formation of the nucleus–vacuole junction [ 74 ]. The disordered segment of residues 1–18 is highly accessible and includes Gly2, which is myristoylated, and Cys4, Cys5, and Cys7 ( Figure 4 F), which are palmitoylated.…”

Closing the Gap: Membrane Contact Sites in the Regulation of Autophagy

“…The main tether Vac8 was demonstrated to contribute to the cytoplasm to vacuole targeting (Cvt) pathway, a selective form of autophagy with pure anabolic functions, via binding to Atg13 [182,183]. A change in the quaternary structure of Vac8, from arch-shaped when in contact with Nvj1 to a superhelical structure when associated with Atg13, selectively supported PMN or the Cvt pathway, respectively [184]. In addition, the NVJs might play a role in lipophagy, the selective autophagic degradation of LDs occurring at sterol-enriched vacuolar microdomains [34].…”