ns, not significant (P 0.05); **, P 0.01; ***, P 0.001; ****, P 0.0001 (Tukey’s multiple comparisons test). Open in a separate window Figure S1. Nups are degraded by autophagy Ro 32-3555 upon TORC1 inactivation. is important for degradation of this nucleoporin not assembled into the NPC. Thus, this study provides the first evidence for autophagic degradation of the NPC and Nups, which we term NPC-phagy and nucleoporinophagy. Introduction Macroautophagy (hereafter autophagy) is an intracellular degradation pathway found in most eukaryotes in which different cellular components are sequestered within double-membrane vesicles called autophagosomes and transported to lytic compartments (lysosomes or vacuoles; Yang and Klionsky, 2010; Ohsumi, 2014). Although autophagic sequestration used to be deemed a nonselective process, an increasing number of studies have revealed that a wide variety of proteins and organelles are sequestered into autophagosomes in a selective manner (Kirkin, 2020; Gatica et al., 2018). In selective autophagy, proteins called autophagy receptors recognize specific cargo molecules or structures. In knockout (= 3). ns, not significant (P 0.05); **, P 0.01; ***, P 0.001; ****, P 0.0001 (Tukey’s multiple comparisons test). Open in a separate window Figure S1. Nups are degraded by autophagy upon TORC1 inactivation. (A) Chromosomal Nup genes were fused with the GFP gene, and these cells were observed under a fluorescence microscope. DIC, differential interference contrast microscopy. Scale bars, 5 m. (B) WT and = 3). ns, not significant (P 0.05); **, Ro 32-3555 P 0.01; ***, P 0.001; ****, P 0.0001 (Tukey’s multiple comparisons test). (C and D) Immunoelectron microscopy of knockout caused marked defects in degradation of all of the GFP-tagged Nups we examined (Fig. 2 A). Autophagy receptors interact with both degradation targets and Atg8 family proteins to link the targets to forming autophagosomal membranes (Gatica et al., 2018; Kirkin, 2020). These receptors contain the Atg8-family interacting motif (AIM; or the LC3 interacting region), which binds to the AIM-binding pocket (AIMBP) of Atg8 family proteins (Noda et al., 2010; Johansen and Lamark, 2020). The P52 and R67 residues are located around the AIMBP, and Rabbit Polyclonal to TBX3 an alanine substitution at these residues decreases the receptor-binding ability of Atg8 (Noda et al., 2008). Similar to knockout, this mutation also severely impaired autophagic degradation of GFP-tagged Nups (Fig. 2 B). These results suggest that a receptor-dependent mechanism mediates autophagic degradation of Ro 32-3555 Nups. Open in a separate window Figure 2. Receptor-mediated selective autophagy degrades the NPC. (A) WT, = 3). ns, not significant (P 0.05); *, P 0.05; **, P 0.01; ***, P 0.001; ****, P 0.0001 (Tukey’s multiple comparisons test). (B) = 3). ns, not significant (P 0.05); **, P 0.01; Ro 32-3555 ***, P 0.001; ****, P 0.0001 (Tukey’s multiple comparisons test). (C and D) Immunoelectron microscopy of (D) cells treated with rapamycin for 24 h was performed using anti-Nsp1 and anti-HA antibodies, respectively. The middle and right panels in C and D, respectively, are magnified view of the boxed Ro 32-3555 area in the left panels. The right panel in C shows another example from a different area. Arrowheads indicate electron-dense regions containing Nsp1 or HA signals (gold particles) in double-membrane vesicles within autophagic bodies. Scale bars, 1 m (left) and 100 nm (middle and right). V, vacuole. N, nucleus. Nups assembled into seven different NPC substructures were all degraded by autophagy (Fig. 1 B). In addition, their degradation was similarly affected by knockout and mutations in the AIMBP of Atg8 (Fig. 2, A and B). These results suggest that Nups are mainly sequestered into autophagosomes in the state of the NPC, while some Nups may also be degraded by autophagy in a form of assembly intermediates or unassembled proteins. Consistent with this prediction, immunoelectron microscopy detected signals of the pore filament component Nsp1 and the cytoplasmic filament component Nup159 (Nup159-HA) at electron-dense structures embedded in nuclear envelopeCderived, double-membrane vesicles (see next section) within autophagic bodies, which are autophagosomal inner vesicles that accumulate within the vacuolar lumen in vacuolar protease-deficient cells (Takeshige et al., 1992; Fig. 2, C and D; and Fig. S1 C). We previously reported that double-membrane vesicles form by budding from the nucleus and are selectively sequestered into autophagosomes in yeast cells treated with rapamycin (Mochida et al., 2015). This pathway is called nucleophagy and requires the outer nuclear membrane protein Atg39 as a specific receptor. Because Nups were found in double-membrane vesicles.