KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Rabbit monoclonal anti-ubiquitin, Lys63-Specific | Millipore Sigma | Cat# 05-1308, Apu3; RRID:AB_1587580 |
| Rabbit polyclonal anti-actin | Cell Signaling | Cat# 4967; RRID:AB_330288 |
| Rabbit polyclonal anti-GAPDH | Abcam | Cat# ab9485; RRID:AB_307275 |
| Mouse monoclonal anti-myc tag | ThermoFisher | Cat# R950-25; RRID:AB_2556560 |
| Rabbit polyclonal anti-myc tag | ThermoFisher | Cat# PA1-981; RRID:AB_325961 |
| Rabbit polyclonal anti-Rad6 | Abcam | Cat# ab31917; RRID:AB_777604 |
| Rabbit monoclonal anti-Rps3/uS3 | Cell Signaling | Cat# 9538, D50G7; RRID:AB_10622028 |
| Rabbit monoclonal anti-Rpl11/uL5 | Cell Signaling | Cat# 18163, D1P5N; RRID:AB_2798794 |
| Rabbit polyclonal anti-HA tag | ThermoFisher | Cat# 71-5500, SG77; RRID:AB_2533988 |
| Rabbit polyclonal anti-Rps20/uS10 | ThermoFisher | Cat# PA5-75383; RRID:AB_2719111 |
| Mouse monoclonal anti-ubiquitin | Cell Signaling | Cat# 3936, P4D1; RRID:AB_331292 |
| Mouse monoclonal anti-Ubiquitin | Life Sensors | Cat# VU0101 |
| Mouse monoclonal anti-FLAG M2 | Sigma Aldrich | Cat# F3165; RRID:AB_259529 |
| Mouse monoclonal anti-puromycin | Millipore | Cat# MABE343, 12D10; RRID:AB_2566826 |
| Rabbit polyclonal anti-Uba1 | Enzo Life Sciences | Cat# BML-PW8395-0025; RRID:AB_2052781 |
| Mouse monoclonal anti-GFP (B-2) | Santa Cruz Biotechnology | Cat# Sc-9996; RRID:AB_627695 |
| Sheep whole anti-mouse IgG HRP-linked | Cytiva | Cat# NA931; RRID:AB_772210 |
| Donkey polyclonal anti-rabbit IgG HRP-linked | Cytiva | Cat# NA934; RRID:AB_772206 |
| Goat anti-Mouse IgG2a heavy chain HRP-linked | Abcam | Cat# ab97245; RRID:AB_10680049 |
| Bacterial and virus strains | ||
| BL21-CodonPlus (DE3)-RIL Competent Cells | Agilent Technologies | Cat#230245 |
| NEB10-beta | New England Biolabs | Cat# C3019 |
| Chemicals, peptides, and recombinant proteins | ||
| MG132 (benzyloxycarbonyl-Leu-Leu-aldehyde) | Sigma-Aldrich | Cat# 474,787; CAS: 133,407-82-6 |
| DL-Dithiothreitol ≥99.5% (RT) | Sigma-Aldrich | Cat# 43,815; CAS: 3483-12-3 |
| Iodoacetamide | Sigma-Aldrich | Cat# I1149; CAS: 144-48-9 |
| Hydrogen peroxide solution | Sigma-Aldrich | Cat# 216,763; CAS: 7722-84-1 |
| TCEP, Tris(2-carboxyethyl)phosphine hydrochloride | Sigma-Aldrich | Cat# C4706; CAS: 51,805-45-9 |
| L-Buthionine-sulfoximine | Sigma-Aldrich | Cat# B2515; CAS: 83,730-53-4 |
| Adenosine 5′-triphosphate disodium salt hydrate | Sigma-Aldrich | Cat# A2383; CAS: 34,369-07-8 |
| Dimethyl sulfoxide, DMSO | Sigma-Aldrich | Cat# D2650; CAS:67-68-5 |
| Cycloheximide | Sigma-Aldrich | Cat# 01,810; CAS: 66-81-9 |
| 3-Methyladenine, autophagy inhibitor 3-MA | Sigma-Aldrich | Cat# M9281; CAS: 5142-23-4 |
| Ubiquitin E1 Inhibitor, PYR-41 | Sigma-Aldrich | Cat# 662,105 |
| Creatine phosphate | Sigma-Aldrich | Cat# CAS: 71,519-72-7 |
| Creatine Kinase | Sigma-Aldrich | Cat# 10127566001 |
| Recombinant Ubiquitin activating enzyme E1 (human) - UBA1 | Enzo Life Sciences | Cat# BML-UW9410-0050 |
| Recombinant Ubiquitin-WT | Life Sensors | Cat# si201 |
| Recombinant Ubiquitin-K63R | Life Sensors | Cat# si218 |
| K63-Linked Di-Ubiquitin | Life Sensors | Cat# si6302 |
| Protease Inhibitor Cocktail Set I | Millipore-Sigma | Cat# 539,131 |
| cOmplete™, EDTA-free Protease Inhibitor Cocktail | Roche | Cat# 04693159001 |
| Trypsin/Lys-C Mix | Promega | Cat# V5073 |
| Recombinant eGFP | Novus Biologicals | Cat# NBP2-34923 |
| NBD (4-Chloro-7-nitrobenzofurazan) | Thermo Scientific | Cat# A14165; CAS 10199-89-0 |
| Puromycin | Sigma-Aldrich | Cat# 540,222; CAS 58-58-2 |
| MMS, Methyl metanesulfonate | Sigma-Aldrich | Cat# 129,925 |
| Hydroxyurea, 98%, Alfa Aesar | Fisher Scientific | Cat# AAA1083106 |
| L-Lysine:2HCL unlabeled | Cambridge Isotope Laboratories | Cat# ULM-8766 |
| L-Lysine:2HCL (13C6, 99%; 15N2, 99%) | Cambridge Isotope Laboratories | Cat# CNLM-291-H |
| Trypsin/Lys-C Mix, Mass Spec Grade | Promega | Cat# V5073 |
| Critical commercial assays | ||
| YeaStar RNA Kit | Zymo Research | Cat# R1002 |
| Q5 Site-Directed Mutagenesis Kit | New England Biolabs (NEB) | Cat# E0552S |
| FastStart Essential DNA Green Master | Roche Life Science | 6402712001 |
| Deposited data | ||
| Structure of Rad6~Ub | Kumar et al., 2015 | PDB:4R62 |
| Yeast Rad6 | Worthylake et al., 1998 | PDB:1AYZ |
| Yeast 80S ribosome with eEF2 | Pellegrino et al., 2018 | PDB:6GQ1 |
| Yeast translocating 80S ribosome | Flis et al., 2018 | PDB:6GZ5 |
| K63-linked ubiquitinated 80S ribosome | Zhou et al., 2020b | EMD-22198 |
| 80S ribosomes from K63R ubiquitin mutant strain | Zhou et al., 2020b | EMD-22196 |
| Ribosomes with conventional 40S beak | This study | EMD-26440 |
| Ribosomes with extended 40S beak | This study | EMD-26441 |
| SILAC RAD6/RAD6C88S redox partner | This study | PXD025726 |
| SILAC protein expression WT/rad6Δ | This study | PXD025727 |
| Experimental models: Cell lines | ||
| Human: HeLa | ATCC | Cat# CCL-2 |
| Experimental models: Organisms/strains | ||
| Yeast strains: Refer to Table S3 | This study | N/A |
| Oligonucleotides | ||
| Refer to Table S4 | this study | N/A |
| Recombinant DNA | ||
| Plasmids - Refer to Table S4 | this study | N/A |
| Software and algorithms | ||
| FlowJo | Becton Dickinson | https://www.flowjo.com |
| cryoSPARC | Punjani et al., 2017 | http://cryosparc |
| Relion 3.1 | Scheres (2012) | https://github.com/3dem/relion |
| CTFFIND4 | Rohou and Grigorieff (2015) | http://grigoriefflab.janelia.org/ctffind4 |
| Pymol | Schrödinger | http://www.pymol.org |
| Clustal Omega | EMBL-EBI | https://www.ebi.ac.uk/Tools/msa/clustalo/ |
| BioRender | Science Suite Inc. | https://biorender.com/ |
| MaxQuant | Cox and Mann, 2008 | www.maxquant.org |
| Perseus | Tyanova et al., 2016 | https://maxquant.net/perseus/ |