FAT10 and NUB1L bind the VWA domain of Rpn10 and Rpn1 to enable proteasome-mediated proteolysis

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1 SUPPLEMENTARY INFORMATION FAT10 and NUB1L bind the VWA domain of Rpn10 and Rpn1 to enable proteasome-mediated proteolysis Neha Rani, Annette Aichem, Gunter Schmidtke, Stefan Kreft, and Marcus Groettrup

2 Supplementary Figure S1 Amino acid sequence alignments of VWA domains from different species and of human FAT10 and ubiquitin. (a) Multiple alignment of amino acid sequences of the VWA domain of various orthologs of Rpn10. The degree of sequence similarity is highlighted using the cyan-to-red color code using ALINE software. The percent identity of the sequences is approximately 25% as calculated in Jalview. (b) Amino acid sequence alignment of the N-terminal UBL domain of hfat10 and ubiquitin showing the non-alignment of Leu8 and Val70 present in ubiquitin (annotated with stars). (c) The alignment of the C-terminal UBL domain of hfat10 and ubiquitin amino acid sequence showing the non-alignment of Leu8, Ile44 and Val70 of ubiquitin (annotated with stars).

3 Supplementary Figure S2 NUB1L accelerated the degradation of FAT10 in the presence of hrpn10. (a) Graph representing the quantitative assessment of the FAT10 protein bands in the western blot shown in Fig. 5a and b (data from at least two sets of experiments). More than 75% of FAT10 was degraded in about 2 h in the presence of NUB1L (black bars). (b) Cycloheximide chase of FAT10 showing that hrpn10 can reconstitute the degradation of FAT10 (white bars) which is accelerated in the presence of NUB1L (black bars). Both graphs represent the mean values +/- range from two sets of experiments.

4 Supplementary Figure S3 The Asp11 residue in the VWA domain is important for the degradation of FAT10. Cycloheximide chase analysis of FAT10 stability in rpn10 cells reconstituted with Rpn10 or Rpn10-D11A (yeast and human). HA-tagged FAT10 was visualized by anti-ha western blotting whereas FLAG-tagged proteins, scrpn10, hrpn10, scrpn10-d11a and hrpn10-d11a were detected via FLAG-specific antibody. Pgk1 served as a loading control. (a) Stabilization of FAT10 in rpn10 yeast strain reconstituted with scrpn10-d11a. (b) Stabilization of FAT10 in rpn10 yeast strain reconstituted with hrpn10- D11A. The experiments were repeated at least two times with similar outcome.

5 Supplementary Table S1. Yeast strains. The table representing the yeast strains used in this study along with the designation and genotype. Strains Genotype References AH109 MATa, trp1-901, leu2-3,112, ura3-52, his3-200, gal4, gal80, LYS2::GAL1 UAS -GAL1 TATA - HIS3,GAL2 UAS -GAL2 TATA -ADE2, URA3::MEL1 UAS - MEL1 TATA -lacz, MEL1 Clontech NMY51 MATa his3 200 trp1-901 leu2-3,112 Dualsystems LYS2::(lexAop)4-HIS3 (lexaop)8-ade2 GAL4 ura3::(lexaop)8-lacz Biotech MHY500 MATa, his3-200, leu2-3,112 ura3-52, lys trp1-1 NRY5 MHY500 with rpn10 ::kanmx6 This study NRY51 MHY500 with pdr5 ::hphmx4 This study NRY53 MHY500 with rpn10 ::kanmx6, pdr5 ::hphmx4 This study WCG4a MATa, his3-11,15 leu2-3,112 ura3 Can S GAL2 50 YHI29/1 pre1-1 51

6 Supplementary Table S2. Gene cloning. The table showing the genes cloned into different vectors along with the restriction sites and primers used. Insert Vector Restriction site Reference for Primers vectors FAT10 pgadt7 BamHI and XhoI Clontech Laboratories FP: 5 GGC GGA TCC AT A TGG CTC CCA ATG CTT CCT GCC T 3 RP: 5 GGC CTC GAG TCA CCC TCC AAT ACA ATA AGA TGC CA 3 NUB1L pgadt7 NdeI and ClaI Clontech Laboratories FP: 5 GGC CAT ATG ATG GCA CAA AAG AAA TAT CTT CAA GC 3 RP: 5 GGC ATC GAT TTA GTT TTT CTT TGT TGC TGA CTT CC 3 hrpn10 YEplac181 EcoRI and KpnI 52 FP: 5 GGC GAA TTC ATG GTG TTG GAA AGC ACT 3 RP: 5 GGC GGT ACC TCA CTT CTT GTC TTC CTC 3 VWA pet26b NdeI and HindIII Novagen FP: 5 GGC CAT ATG GTG TTG GAA AGC ACT ATG GT 3 RP: 5 GGC AAG CTT TCA AAT CGG AGA ACT GAT GA 3 VWA YEplac181 EcoRI and KpnI 52 FP: 5 GGC GAA TTC ATG GTG TTG GAA AGC ACT AT 3 RP: 5 GGC GGT ACC TCA AAT CGG AGA ACT GAT GA 3 UIM1UIM2 YEplac181 EcoRI and KpnI 52 FP: 5 GGC GAA TTC ATG CTG GGT CTT

7 GGT GC 3 RP: 5 CCG GGT ACC TCA GGC ATC AAT GTC TGC T 3 hrpn10 (UIMs mutated) YEplac181 EcoRI and KpnI 52 FP: 5 GGC GAA TTC ATG CTG GGT CTT GGT GC 3 RP: 5 CCG GGT ACC TCA GGC ATC AAT GTC TGC T 3 FAT10 p416met25 SpeI and SalI 53 FP: 5 GGC ACT AGT ATG GCC TAC CCC TAC GAC GT 3 RP: 5 GGC GTC GAC TCA CCC TCC AAT ACA ATA AGA 3 NUB1L p414gpd SpeI and SalI 53 FP: 5 GGC ACT AGT ATG GCC TAC CCC TAC GAC GT 3 RP: 5 GGC GTC GAC TTA GTT TTT CTT TGT TGC TGA CT 3 scrpn10 pet45b(+) KpnI and HindIII Novagen FP: 5 GGC GGT ACC ATG GTA TTG GAA GCT ACA GTG 3 RP: 5 GGC AAG CTT CTA TTT GTC TTG GTG TTG TTC 3 NUB1L UBA pgex2tks NotI and XbaI GE Healthcare (pgex2tk modified) FP: 5 GGC TCT AGA ATG GCA CAA AAG AAA TAT CTT CAA GC 3 RP: 5 GGC GCG GCC GC TTA GTT TTT CTT TGT TGC TGA CTT CC 3 NUB1L UBL pgex2tks NotI and XbaI GE Healthcare FP: 5 GGC TCT AGA ATG GCA CAA AAG AAA TAT CTT CAA GC 3 RP: 5 GGC GCG GCC GC TTA GTT TTT CTT TGT TGC

8 TGA CTT CC 3 FAT10 K0 p416met25 SpeI and SalI 53 FP: 5 GGC ACT AGT ATG GCC TAC CCC TAC GAC GT 3 RP: 5 GGC GTC GAC TCA CCC TCC AAT ACA ATA AGA 3 Rpn10-D11A YEplac181 EcoRI and KpnI 52 FP: 5 GCT ACA GTG TTA GTG ATT GCT AAT TCA GAG TAC TCT CG 3 RP: 5 TGT AGC CGA GAG TAC TCT GAA TTA GCA ATC ACT AAC AC 3 hrpn10-d11a YEplac181 EcoRI and KpnI 52 FP: 5 GTG TGT GTG GCC AAC AGT GAG TAT ATG CGG AAT GG 3 RP: 5 CCA TTC CGC ATA TAC TCA CTG TTG GCC ACA CAC ACC 3

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