1. the “conventional” proteasome --33 components, all integral subunits --Extremely conserved among eukaryotes --20 years of biochemistry

2. Proposed roles in proteasome matura- tion tion and DNA repair Blm10 Tethers RP and CP togetherEcm29 Ubiquitin-protein ligaseHul5 Deubiquitinating enzyme Ubp6 Delivers conjugates to proteasomeRad23 major proteasome-associated proteins of budding yeast

3. UBP6 UCH37 RPN11 the proteasomal deubiquitinating enzymes Proposed roles regenerating ubiquitin allowing substrate translocation editing of bad substrates Possible liability premature deubiquitination

4. in vitro breakdown of cyclin b conjugates (min) Unmodified Cyclin B John Hanna Nate Hathaway Randy King Don Kirkpatrick Steve Gygi

5. recombinant ubp6 “corrects the defect” of ubp6 null proteasomes John Hanna

6. UBP6 UCH37 RPN11 the proteasomal deubiquitinating enzymes Proposed roles regenerating ubiquitin allowing substrate translocation editing of bad substrates Possible liability premature deubiquitination

7. proteasome inhibition by ubp6 is noncatalytic John Hanna

8. two distinct activities of ubp6 work on the same substrate: proteasome inhibition and chain trimming John Hanna

10. inhibition by ubp6 occurs on the proteasome John Hanna

11. ubp6 inhibits its sister deubiquitinating enzyme rpn11 Lid Base CP UBP6 RPN11 John Hanna

12. ubp6 inhibits its sister deubiquitinating enzyme rpn11 Lid Base CP UBP6 RPN11 Proteasome inhibition by Ubp6 is accompanied by a switch in the mode of deubiquitination by the proteasome

13. ubp6 inhibits its sister deubiquitinating enzyme rpn11 Lid Base CP UBP6 RPN11 Proteasome inhibition by Ubp6 is accompanied by a switch in the mode of deubiquitination by the proteasome Inhibition of Rpn11 by Ubp6 may be direct or indirect

14. ubp6 inhibits protein turnover in vivo & on substrates other than cyclin b Wild-type ubp6 ubr1 John Hanna 0 7 14 0 7 14 (min) UB-K-URA3 Rpn5

15. comments The ubiquitin-proteasome pathway is under strong inhibitory control

16. comments The ubiquitin-proteasome pathway is under strong inhibitory control Two deubiquitinating enzymes on the proteasome: one (Rpn11) promotes degradation (while excising chains at/near their base) the other (Ubp6) inhibits (while probably trimming chains from the distal end)

17. comments The ubiquitin-proteasome pathway is under strong inhibitory control Two deubiquitinating enzymes on the proteasome: one (Rpn11) promotes degradation (while excising chains at/near their base) the other (Ubp6) inhibits (while probably trimming chains from the distal end) Ubp6 inhibitory effect is conserved evolutionarily

18. comments The ubiquitin-proteasome pathway is under strong inhibitory control Two deubiquitinating enzymes on the proteasome: one (Rpn11) promotes degradation (while excising chains at/near their base) the other (Ubp6) inhibits (while probably trimming chains from the distal end) Ubp6 inhibitory effect is conserved evolutionarily Two inhibitory effects of Ubp6--noncatalytic and catalytic

19. Proposed roles in proteasome matura- tion tion and DNA repair Blm10 Tethers RP and CP togetherEcm29 Ubiquitin-protein ligaseHul5 Deubiquitinating enzyme Ubp6 Delivers conjugates to proteasomeRad23 major proteasome-associated proteins of budding yeast

20. hul5 confers ubiquitin-conjugating activity on the proteasome Holo WT Holo hul5 Ub Ctrl E1+ Ub E1-Ub Bernat Crosas Does it work against Ubp6?

21. ubp6 antagonizes in vitro conjugate formation by hul5  hul5  hul5  ubp6 WT  ubp6 Ub E1-Ub Bernat Crosas John Hanna

22. ubp6 disassembles conjugates formed by hul5 on the proteasome hul5 ubp6ubp6 60090301200609030120 28 203 115 93 48 34 21 7.2 Conjugation Bernat Crosas John Hanna - Ubp6+ Ubp6 60090301200609030120 Deconjugation

23. Canavanine WT  hul5  ubp6  hul5  ubp6 Control  hul5 suppresses  ubp6 Bernat Crosas

24. NaCl (mM) WT  ubp6 252001001257550175150 Hul5 hul5 association with the proteasome is highly sensitive to salt in ubp 6 mutants Bernat Crosas

25. Hul5 Ubp6 Hul5 and Ubp6 antagonize each other in a specific subpopulation of proteasomes, establishing a dynamic state for proteasome-bound multiubiquitin chains working model

26. 10 100 02040 % remaining Ub-K-Ura3 hul5  WT Time (min) 10 100 0203010 Gcn4 hul5  WT Time (min) % remaining 10 100 01020 Alpha2 hul5  rpn4  Time (min) % remaining defective protein degradation in the hul 5 null Bernat Crosas

27. is hul5 an e4 enzyme? Ubp6? E3 X

28. ++++++ -+++++ --++++ +++-++ ++++-+ +++++- E1E1 ubch5 Hul5 Ub 4-8 Holo Ub hul5 can exhibit proteasome-dependent e 4 activity Ub 2 Ub Ub 4 Ub n Bernat Crosas Christa Bücker

29. e4 activity of hul5 on cyclin b 60030 000 0 cycB Ub-cycB APCHul5 mock hul5  ubp6  proteasomes

30. Summary of findings Ubp6 inhibits the proteasome noncatalytically Ubp6 appears inhibit proteasomes catalytically Ubp6 inhibits Rpn11, directly or indirectly Ubp6 opposes the activity of Hul5

31. Traditional view: decision to degrade a protein made by E3 before engagement of substrate by the proteasome--proteasome is passive

32. Suggestion of a new discriminatory step in which substrates are actively scrutinized by the proteasome

33. Traditional view: decision to degrade a protein made by E3 before engagement of substrate by the proteasome--proteasome is passive Suggestion of a new discriminatory step in which substrates are actively scrutinized by the proteasome This step is mediated by functional interactions between deubiquitin- ating enzymes and ubiquitin ligases resident in the proteasome: Ubp6, Hul5, and Rpn11

34. These activities allow active control the final and only irreversible step in protein breakdown Traditional view: decision to degrade a protein made by E3 before engagement of substrate by the proteasome--proteasome is passive Suggestion of a new discriminatory step in which substrates are actively scrutinized by the proteasome This step is mediated by functional interactions between deubiquitinating enzymes and ubiquitin ligases resident in the proteasome: Ubp6, Hul5, and Rpn11

35. dwell time model for hul5/ubp6 Degradation Simple Dissociation Dissociation via Deubiquitination Ubp6 Hul5 Ubp6

36. lab members Bernat Crosas Suzanne Elsasser Kelly Gay John Hanna Maurits Kleijnen Soyeon Park Marion Schmidt Jeroen Roelofs Yoshiko Tone Phoebe Zhang cell bio collaborators Don Kirkpatrick Steve Gygi Nate Hathaway Randy King Currently: Inst. of Molecular Biology, Barcelona