Supplementary MaterialsFigure 1source data 1: This document contains the quantification of fluorescently labeled Ubc6 (Physique 1D) as well as of Rhodamine-labeled lipids (Physique 1figure supplement 2B). 2source data 3: This file contains the quantification of the fraction of unmodified Ubc6 from three experiments as in Physique 2figure supplement 1C, as shown in?Physique 2figure supplement 1D. elife-56945-fig2-data3.xlsx (16K) GUID:?0E6856CA-E6A3-4A17-A55B-D79EC2616B9E Physique 3source data 1: This file contains the quantification of the fraction of extracted Ubc6 from three experiments as in Physique 3A,B, as shown Physique 3C,D. It also contains the quantification of the immobilization efficiency of Ubc6 (Physique 3figure supplement 1C) and of Rhodamine-labeled lipids (Physique 3figure supplement 1D), as well as the quantification of the extraction efficiency of Ubc6 altered with different ubiquitin chain lengths Bosentan Hydrate (Physique 3figure supplement 1G). Bosentan Hydrate elife-56945-fig3-data1.xlsx (30K) GUID:?6B4CA37D-ACB9-4174-802E-E4677F699858 Figure 4source data 1: This file contains the quantification of the quenched fraction of Ubc6 in samples containing Doa10 or its variants compared to samples lacking Doa10 from three experiments as in Figure 4A, as shown in Figure 4B. elife-56945-fig4-data1.xlsx (10K) GUID:?55044896-B3Compact disc-46DA-8ED4-D921D1B8AEFE Body 4source data 2: This document provides the quantification from the TEV-protected fraction of Ubc6 from 3 experiments, as shown in Body 4C. elife-56945-fig4-data2.xlsx (15K) GUID:?C0F3B7C2-417F-494A-B50C-1C2616BF4673 Figure 4source data 3: This file provides the quantification from the quenched fraction of Bosentan Hydrate Ubc6 or its variants in samples containing Doa10 in comparison to samples inadequate Doa10 Bosentan Hydrate from 3 experiments as in Figure 4D, as shown in Figure 4E. elife-56945-fig4-data3.xlsx (10K) GUID:?18DA5BBF-1011-4306-BD66-17A7ED609396 Physique 4figure product 1source data 1: This file contains numerical values for data shown in Physique 4figure product 1E. elife-56945-fig4-figsupp1-data1.xlsx (10K) GUID:?E5770593-5381-48A1-98CB-58A29C6089D5 Figure 5source data 1: This file contains the quantification of the number of ubiquitins (n) transferred per Ubc6 or Ubc6SybTM (Figure 5figure product 1D) as well as the quantification of the number of total ubiquitin transferred from three experiments as in Figure 5C, as shown in Figure 5D. elife-56945-fig5-data1.xlsx (24K) GUID:?9F29317E-09B6-4265-844B-49E320F11F8D Physique 5source data 2: This file contains the quantification of the fraction of unmodified Ubc6 from three experiments as in Physique 5E, as shown in Physique 5F. elife-56945-fig5-data2.xlsx (16K) GUID:?23CFAEB1-244F-4950-927D-007EF701E0ED Physique 5figure supplement 1source data 1: This file contains the quantification of the fraction of unmodified Ubc6 as shown in Physique 5figure supplement 1A, C and F. elife-56945-fig5-figsupp1-data1.xlsx (21K) GUID:?97C989C2-DA57-48D0-9E7D-A9D030A1DCE7 Figure 5figure supplement 2source data 1: This file contains the quantification of the fraction of unmodified Ubc6 as shown in Figure 5figure supplement 2B. elife-56945-fig5-figsupp2-data1.xlsx (16K) GUID:?444F940C-707D-403A-803F-F6496AA44E6C Physique 5figure supplement 2source data 2: This file contains the quantification of the number of total ubiquitin transferred in presence of different Doa10 variants from three experiments, as shown in Physique 5figure supplement 2C. elife-56945-fig5-figsupp2-data2.xlsx (28K) GUID:?084703CE-624C-4BD2-90B3-1018F8405380 Figure 6source data 1: This file contains the quantification of the quenched fraction of Ubc6 in samples containing Doa10 compared to samples missing Doa10 from three experiments as in Figure 6A, as shown in Figure 6B. Bosentan Hydrate elife-56945-fig6-data1.xlsx (11K) GUID:?3FAD4227-F633-49FA-AC6F-A1723E0F5928 Figure 6source data 2: This file contains the quantification of the quenched fraction of Ubc6 in samples containing Cdc48/UN compared to samples lacking Cdc48/UN from three experiments as in Figure 6C, as shown in Figure 6D. elife-56945-fig6-data2.xlsx (11K) GUID:?3E8452F5-AF75-494D-8741-D06DC32DAB13 Figure 6source data 3: This file contains the quantification of streptavidin in the top flotation fraction from three experiments as in Figure 6E, as shown in Figure 6F. elife-56945-fig6-data3.xlsx (10K) GUID:?C7F991AA-9BEA-47A2-ADFD-D1FB3F3C72A3 Transparent reporting form. elife-56945-transrepform.docx (246K) GUID:?984D55E9-7C87-4982-9B4C-536233918D6B Data Availability StatementAll data generated or analysed during this study are included in the manuscript and supporting files. Abstract In endoplasmic reticulum-associated protein degradation (ERAD), membrane proteins are ubiquitinated, extracted from Rabbit Polyclonal to DMGDH your membrane, and degraded by the proteasome. The cytosolic ATPase Cdc48 drives extraction by pulling on polyubiquitinated substrates. How hydrophobic transmembrane (TM) segments are moved from your phospholipid bilayer into cytosol, often together with hydrophilic and folded ER luminal protein parts, is not known. Using a reconstituted system with purified proteins from recognized two universally conserved membrane-embedded ubiquitin ligases that ubiquitinate ERAD substrates, Hrd1 (SYVN1 in human) (Bordallo et al., 1998; Hampton et al., 1996; Kikkert et al., 2004; Nadav et al., 2003) and Doa10 (TEB-4/MARCH6 in animals, SUD-1 in Ubc6 homologue Ubc32, and the mammalian Ube2J1, Hrd1 was identified as the ubiquitin ligase.