Structural Insights into Vac8 Complexes and Their Regulatory Roles

Abstract

Vac8, a yeast vacuolar protein with armadillo repeats, mediates various cellular processes by changing its binding partners; however, the mechanism by which Vac8 differentially regulates these processes remains poorly understood. Vac8 interacts with Nvj1 to form the nuclear–vacuole junction (NVJ) and with Atg13 to mediate cytoplasm- to-vacuole targeting (Cvt), a selective autophagy-like pathway that delivers cytoplasmic aminopeptidase I directly to the vacuole. In addition, Vac8 associates with Myo2, a yeast class V myosin, through its interaction with Vac17 for vacuolar inheritance from the mother cell to the emerging daughter cell during cell divisions. Here, this study determined the X- ray crystal structure of the Vac8–Vac17 complex and found that its interaction interfaces are bipartite, unlike those of the Vac8–Nvj1 and Vac8–Atg13 complexes. When the key amino acids present in the interface between Vac8 and Vac17 were mutated, vacuole inheritance was severely impaired in vivo. Furthermore, binding of Vac17 to Vac8 prevented dimerization of Vac8, which is required for its interactions with Nvj1 and Atg13, by clamping the H1 helix to the ARM1 domain of Vac8 and thereby preventing exposure of the binding interface for Vac8 dimerization. Consistently, the binding affinity of Vac17- bound Vac8 for Nvj1 or Atg13 was markedly lower than that of free Vac8. Likewise, free Vac17 had no affinity for the Vac8–Nvj1 and Vac8–Atg13 complexes. These results provide insights into how vacuole inheritance and other Vac8-mediated processes, such as NVJ formation and Cvt, occur independently of one another.|Phagocytosis is a cellular process critical for immune defense and tissue homeostasis, involving the engulfment of foreign particles, pathogens, and apoptotic cells. Recent studies suggest that the endoplasmic reticulum (ER) mediates phagocytosis. During the early stages of phagocytosis, the ER is positioned near the phagocytic cup at the plasma membrane, providing membrane for phagosome formation. This study investigates the role of ER structure in phagocytosis by examining COS-7 ATL2/3 double knockout (DKO) cells, which exhibit reduced three-way junctions and an unbranched ER morphology. Notably, phagocytic assays revealed a significant increase in apoptotic cell clearance in ATL2/3 DKO cells, and expressing Rtn4a to promote unbranched ER morphology slightly increased phagocytosis in both WT and ATL2/3 DKO cells. These findings suggest that ER structural dynamics may be involved in influencing the phagocytic activity.