Volume 59, Issue 6, Pages 1035-1042 (September 2015) ESCRTs Cooperate with a Selective Autophagy Receptor to Mediate Vacuolar Targeting of Soluble Cargos  Xiao-Man Liu, Ling-Ling Sun, Wen Hu, Yue-He Ding, Meng-Qiu Dong, Li-Lin Du  Molecular Cell  Volume 59, Issue 6, Pages 1035-1042 (September 2015) DOI: 10.1016/j.molcel.2015.07.034 Copyright © 2015 Elsevier Inc. Terms and Conditions

Molecular Cell 2015 59, 1035-1042DOI: (10.1016/j.molcel.2015.07.034) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 1 Nbr1 Mediates Vacuolar Targeting of Two Aminopeptidases, Lap2 and Ape2 (A) Domain architecture of human NBR1 and fission yeast Nbr1. (B) Localization of fission yeast Nbr1. Wild-type cells expressing endogenously mCherry-tagged Nbr1 were grown to mid-log phase in EMM medium and examined by fluorescence microscopy. Cpy1-Venus serves as a vacuole lumen marker. DIC, differential interference contrast. (C) Identification of Lap2 and Ape2 as Nbr1-interacting proteins. YFP-FLAG-His6 (YFH)-tagged Nbr1 was purified using GFP-Trap and its associated proteins were identified by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). (D) Coimmunoprecipitation of Lap2 and Ape2 with Nbr1. Lap2 was endogenously tagged with a 13Myc tag. Endogenously TAP-tagged Nbr1 was immunoprecipitated using IgG Sepharose beads. Total cell lysates and IgG precipitates were analyzed by immunoblotting. Strains with either Lap2 or Nbr1 untagged were used as controls. (E and F) Localization of Lap2 and Ape2. Wild-type (WT), nbr1Δ, ape2Δ, and lap2Δ strains expressing endogenously mCherry-tagged Lap2 or Ape2 were grown to mid-log phase in EMM medium and examined by fluorescence microscopy. (G and H) Vacuolar processing of endogenously mCherry-tagged Nbr1 by vacuolar proteases Isp6 and Psp3 was examined by immunoblotting using an mCherry antibody. Coomassie staining of PVDF membrane served as loading control. The protein level of Nbr1-mCherry appeared to be lower in the absence of Ape2 or Lap2, perhaps due to reduced stability of free Nbr1. Scale bars, 3 μm. See also Figure S1. Molecular Cell 2015 59, 1035-1042DOI: (10.1016/j.molcel.2015.07.034) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 2 Interdependent Interactions between Nbr1, Lap2, and Ape2 Are Critical for Their Vacuolar Targeting (A) Ape2 and Nbr1 copurified with Lap2, and Lap2 and Nbr1 copurified with Ape2. Lap2-YFH expressed from the nmt1 promoter and Ape2-Venus expressed from the native promoter were purified using GFP-Trap agarose beads. Bead-bound proteins were identified by LC-MS/MS. (B) Nbr1-YFH and its copurified proteins were fractionated on a Superose-6 sizing column. The indicated fractions were resolved by SDS-PAGE and analyzed by immunoblotting. Peak positions of the molecular weight standards are indicated by arrows. (C) Coprecipitation between Lap2 and Ape2 was diminished by nbr1Δ. (D) Coprecipitation between Nbr1 and Ape2 was diminished by lap2Δ, and coprecipitation between Nbr1 and Lap2 was diminished by ape2Δ. (E) Both the ZZ fingers and the FW domain are required for the vacuolar targeting function of Nbr1 and Lap2. (F) Nbr1-N but not Nbr1-C could coprecipitate Ape2 and Lap2. (G) Nbr1-N, but not Nbr1-C, when expressed as the only copy of Nbr1 in the cells, could support the interaction between Ape2 and Lap2. (H) CXMS analysis identified crosslinks formed within and between Nbr1, Lap2, and Ape2. Intermolecular crosslinks are depicted with darker color lines. (I) Amino acids 2–6 of Ape2 are required for the vacuolar targeting of itself and Lap2. Scale bars, 3 μm. See also Figures S2 and S3 and Table S1. Molecular Cell 2015 59, 1035-1042DOI: (10.1016/j.molcel.2015.07.034) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 3 ESCRTs but Not Core Atg Proteins Are Essential for the NVT Pathway (A) Localization of Lap2 in WT, nbr1Δ, atg5Δ, and atg11Δ cells. (B) Localization of Nbr1 in WT, sst2Δ, sst4Δ, and sst6Δ cells. (C) Localization of Lap2 in WT, sst2Δ, sst4Δ, and sst6Δ cells. (D) Lap2-mCherry processing by vacuolar proteases Isp6 and Psp3 was diminished in sst2Δ, sst4Δ, and sst6Δ cells. Total lysates from indicated strains were analyzed by immunoblotting using an mCherry antibody. Coomassie staining of PVDF membrane served as loading control. Scale bars, 3 μm. See also Figure S4. Molecular Cell 2015 59, 1035-1042DOI: (10.1016/j.molcel.2015.07.034) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 4 NVT Components Colocalize with the ESCRT Machinery at MVBs and Require Ubiquitination for Vacuolar Targeting (A) NVT complex assembly was normal in ESCRT mutants. Lap2-YFP was immunoprecipitated with GFP-Trap. The coimmunoprecipitation of Nbr1-mCherry and Ape2 was probed with anti-mCherry and anti-Ape2 antibodies, respectively. (B) Lap2-mCherry exhibited cytoplasmic punctate signals colocalizing with ESCRT-0 subunits but not with Cpy1. Two such puncta are indicated by arrows. (C) Nbr1-mCitrine colocalized with puncta formed by ESCRT-0 subunits. (D) Lap2-mCitrine colocalized with puncta formed by ESCRT-0 subunits. (E) Distribution of mCherry-tagged proteins fused to either enzymatically active or inactive (∗) catalytic domain of fission yeast Ubp7 or HSV-1 UL36. (F) The vacuolar processing of Nbr1-mCherry was completely disrupted when fused with Ubp7 but not when fused with the enzymatically inactive Ubp7∗. (G) Nbr1 is ubiquitinated. Biotin-tagged ubiquitin (Tagwerker et al., 2006) was purified using streptavidin beads under a denaturing condition and copurified Nbr1 was detected by immunoblotting. Arrows point to ubiquitinated Nbr1. (H) Distribution of Lap2-mCherry in mutants defective in ubiquitin enzymes. (I) Inactivating the VHS domain of Sst4 abolished the vacuolar accumulation of Nbr1. The vacuolar accumulation of Nbr1 was abolished by mutations inactivating the VHS domain of Sst4 (I30A and L34D), but not by mutations inactivating the UIM1 domain of Sst4 (A268Q and S272D) or mutations inactivating the UIM2 domain of Sst4 (A312Q and S316D) (Ren and Hurley, 2010). (J) A model of the NVT pathway. Cytosolic hydrolases Ape2 and Lap2 are recognized by the autophagy receptor Nbr1 and assembled together into the NVT complex. Ubiquitination of Nbr1 and/or its associated proteins results in the association with the ESCRT machinery, which sequestrates the NVT complex into intralumenal vesicles (ILVs) inside of MVBs. Ape2 and Lap2 are released into the vacuole lumen after fusion between an MVB and a vacuole. Scale bars, 3 μm. See also Figure S4. Molecular Cell 2015 59, 1035-1042DOI: (10.1016/j.molcel.2015.07.034) Copyright © 2015 Elsevier Inc. Terms and Conditions