Fig 2: The E3 ubiquitin ligase activities of Vps11/18 influence signal-regulated transcription. a Immunoblot of a native protein gel of Vps-C complexes of HEK293T cells with either a shRNA-mediated knock-down or overexpression of Vps11/18. The simultaneous overexpression of all Vps-C core components was used as a positive control. A scrambled shRNA (shSC) and a shRNA targeting the bacterial β-galactosidase (shLacZ) were used as negative controls for the knock-downs. The immunoblot was probed with a Vps33A antibody. In parallel, an immunoblot was performed on the same samples separated under denaturing conditions to determine the total amount of Vps33A. The indicated Vps-C complexes/Vps33A ratios were measured with the software ImageJ and are representatives of two immunoblots. b Scheme of the RING domain mutants of Vps11/18. A cross inside the RING domain indicates defective point mutants. c In vivo ubiquitination assay of the RING mutants as in Fig. 1b. d–o Luciferase reporter assays for the indicated transcription factors, performed in HEK293T cells overexpressing either Vps11/18 or their corresponding RING domain mutants. For ERα, PR, GR, and CREB signaling pathways, the transcriptional activities were induced by a treatment with E2, progesterone (P4), dexamethasone (Dex) or a cocktail of forskolin and isobutylmethylxanthine (FI) to increase intracellular cAMP levels, respectively; NFκB and AP-1 were induced by phorbol myristate acetate (PMA), HIF-1 and p53 with cobalt (II) chloride (CoCl2). RLU, relative light units. Bar graphs show the averages ± s.e.m. with n ≥ 3 biologically independent experiments. Asterisks indicate significant differences with the corresponding negative controls with p-values < 0.05; the dots above some of the bars in j indicate differences with p-values of 0.05–0.06. Statistical significance was determined with unpaired and two-sided Student’s t-tests. Source data are provided as a Source Data file

Fig 3: Vps11/18 carry E3 ubiquitin ligase activities involved in signal transduction. a Scheme of the class C Vps proteins that constitute the core of the HOPS and CORVET complexes. The RING domain is highlighted in purple. b In vivo ubiquitination assay with HEK293T cells overexpressing the indicated Vps proteins along with HA-tagged ubiquitin. Ubiquitinated proteins were immunoblotted (IB) with an HA antibody; GAPDH is the internal loading control. c Experimental strategy to quantify the ubiquitinome upon overexpression of Vps11/18 in HEK293T cells using stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry; see text for more details. d, e Volcano plots showing the differentially ubiquitinated sites (log2 ratios) between cells transfected with the empty expression vector and cells overexpressing either Vps11 (d) or Vps18 (e) and their statistical significance (−log10(p-value) on the y-axis); values above the black dotted line are significantly regulated; significantly downregulated sites (blue dots) are at the top left of the blue threshold line while significantly upregulated sites (red dots) are at the top right of the red threshold line. f Table of the number of protein substrates of Vps11/18 with one or several differentially regulated ubiquitination sites. g Venn-diagram of the differentially ubiquitinated proteins in Vps11- or Vps18-overexpressing cells compared to the empty vector condition (at the top) with the zoomed in shared proteins ordered by hierarchical clustering (at the bottom); arrows point to E3 ubiquitin ligases that are more ubiquitinated upon Vps11 or Vps18 overexpression. h GO-based clustering of the interactome of the proteins associated with a significant change of the ubiquitinome upon Vps11/18 overexpression. Each node represents a GO term with a color code showing the relative changes of the corresponding ubiquitinome upon Vps11 (inner circle) or Vps18 (outer circle) overexpression (blue or red for enriched in control cells over cells overexpressing Vps11/18 or the other way around, respectively). The number of proteins with a particular GO term is represented by the size of its corresponding node. Source data are provided as a Source Data file

Fig 4: Vps11/18 prevent membrane-associated ERα signaling by ubiquitinating PELP1. a Combined interactome of ERα (ESR1) and Vps11/18 (blue nodes) generated with Cytoscape using the plugin PpiMapBuilder (https://goo.gl/GusMZG). The common interactors are in yellow. Proteins in white nodes do not directly interact with Vps11/18 and nodes with a bold red line represent the proteins that are associated with the membrane ERα pathway. b ERα reporter assays with HEK293T cells infected with the indicated shRNA constructs and overexpressing either wild-type ERα (Wt), an ERα palmitoylation mutant (ERα C447S) or the combination of full-length ERα C447S with the ligand binding domain of ERα (LBD) by itself. Cells were treated with E2 (mean ± s.e.m. with n = 3 biologically independent experiments). c Immunoblot of an immunoprecipitation experiment with extracts from MDA-MB-134 cells. A control immunoprecipitation was performed in parallel with a control IgG (Ctrl IP). The blots were probed for the proteins shown on the right. d Immunoblots of extracts from HEK293T and MDA-MB-134 cells overexpressing Vps11/18 for an extended period of 6 days. e Immunoblots of immunoprecipitated ubiquitinated proteins. Extracts from HEK293T cells overexpressing Vps11/18 along with HA-tagged ubiquitin were immunoprecipitated with an HA antibody and probed either for endogenous PELP1 (left) or BCAR1 (right). A control immunoprecipitation was performed in parallel with a control IgG (Ctrl IP). f ERα reporter assays with HEK293T cells overexpressing Vps11/18 along with wild-type (Wt) PELP1 or the indicated point mutants of PELP1. g In vivo ubiquitination assays as in e with exogenously expressed wild-type and mutant PELP1 as indicated. h Luciferase reporter assays for PR in HEK293T cells overexpressing Vps11/18 along with wild-type or mutant PELP1 (mean ± s.e.m. with n = 3 biologically independent experiments). P4, progesterone. i Quantitative RT-PCR analysis of the mRNA levels of the ERα target gene GREB1 in MDA-MB-134 cells overexpressing Vps11/18 along with the indicated proteins (mean ± s.e.m. with n = 3 biologically independent experiments). Asterisks indicate significant differences with the corresponding negative controls with p-values < 0.05. Statistical significance was determined with unpaired and two-sided Student’s t-tests. Source data are provided as a Source Data file

Fig 5: Regulation of ERα activity by Vps11/18 is independent of intracellular trafficking pathways. a ERα reporter gene assays with HEK293T cells overexpressing Vps11/18 in combination with wild-type (Wt) or K44A mutant (Mut) dynamin II (mean ± s.e.m. with n = 3 biologically independent experiments). b Assays as in a but with knock-downs with the indicated shRNA constructs (mean ± s.e.m. with n = 3 biologically independent experiments). c Transferrin uptake assays in HEK293T cells overexpressing Vps11/18 along with cyan fluorescent protein (CFP). Scale bar indicates 20 µm. d Transferrin uptake assays with immunostaining of EEA1 or LAMP1 in HEK293T cells overexpressing Vps11/18 and blue fluorescent protein (BFP). Scale bar indicates 20 µm. e Assays as in a with knock-downs of HRS or TSG101 (mean ± s.e.m. with n = 3 biologically independent experiments). f–i Assays as in a under the following conditions: treatments to inhibit (Wortmannin, 3-methyladenine (3-MA)) or to stimulate (serum starvation, rapamycin) autophagy (f and g, respectively); knock-down of SQSTM1 (h); treatment with the EGFR inhibitor AG1478 (i) (mean ± s.e.m. with n ≥ 3 biologically independent experiments). j, k Immunoblots displaying PKA substrates and protein levels with or without FI (j) or phospho-ERK1/2 and ERK1/2 protein levels with or without FBS (k) in HEK293T cells overexpressing Vps11/18; FI, cocktail of forskolin and isobutylmethylxanthine to increase intracellular cAMP levels; FBS, fetal bovine serum. l Assays as in a with overexpression of the transcriptional coactivators SRC1 or CARM1 (mean ± s.e.m. with n = 3 biologically independent experiments). Asterisks indicate significant differences with the corresponding negative controls with p-values < 0.05. Statistical significance was determined with unpaired and two-sided Student’s t-tests. Source data are provided as a Source Data file