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the “conventional” proteasome

the “conventional” proteasome. --33 components, all integral subunits. --Extremely conserved among eukaryotes. --20 years of biochemistry. major proteasome-associated proteins of budding yeast. RPN11. UBP6. UCH37. the proteasomal deubiquitinating enzymes. Proposed roles

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the “conventional” proteasome

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

  2. major proteasome-associated proteins of budding yeast

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

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

  5. recombinant ubp6 “corrects the defect” ofubp6null proteasomes John Hanna

  6. RPN11 UBP6 UCH37 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

  9. inhibition by ubp6 occurs on the proteasome John Hanna

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

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

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

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

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

  15. 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) comments

  16. 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 comments

  17. 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 comments

  18. major proteasome-associated proteins of budding yeast

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

  20. Dhul5 Dubp6 Dhul5 Dubp6 WT E1-Ub Ub ubp6 antagonizes in vitro conjugate formation by hul5 Bernat Crosas John Hanna

  21. Deconjugation - Ubp6 + Ubp6 0 0 30 30 60 60 90 90 120 120 0 0 30 30 60 60 90 90 120 120 203 115 93 48 34 28 21 7.2 ubp6 disassembles conjugates formed by hul5 on the proteasome Conjugation hul5ubp6 ubp6 Bernat Crosas John Hanna

  22. Control Canavanine WT Dhul5 Dubp6 Dhul5Dubp6 Dhul5 suppressesDubp6 Bernat Crosas

  23. 25 50 75 100 125 150 175 200 hul5 association with the proteasome is highly sensitive to salt in ubp6 mutants NaCl (mM) Hul5 WT Hul5 Dubp6 Bernat Crosas

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

  25. 100 100 hul5D hul5D rpn4D % remaining % remaining rpn4D WT Gcn4 Alpha2 10 10 0 10 20 30 0 10 20 Time (min) Time (min) 100 hul5D % remaining WT Ub-K-Ura3 10 0 20 40 Time (min) defective protein degradation in the hul5 null Bernat Crosas

  26. E3X Ubp6? is hul5 an e4 enzyme?

  27. Ub4 hul5 can exhibit proteasome-dependent e4 activity E1 ubch5 Hul5 Ub4-8 Holo Ub Ubn Ub2 Bernat Crosas Christa Bücker Ub

  28. e4 activity of hul5 on cyclin b hul5D ubp6D proteasomes mock Hul5 APC mock Hul5 Ub-cycB cycB 0 60 0 30 0 30 0 30 0 30

  29. 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

  30. Traditional view: decision to degrade a protein made by E3 beforeengagement of substrate by the proteasome--proteasome is passive

  31. Traditional view: decision to degrade a protein made by E3 beforeengagement of substrate by the proteasome--proteasome is passive Suggestion of a new discriminatory step in which substrates are actively scrutinized by the proteasome

  32. Traditional view: decision to degrade a protein made by E3 beforeengagement 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

  33. Traditional view: decision to degrade a protein made by E3 beforeengagement 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 • These activities allow active control the final and only irreversible • step in protein breakdown

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

  35. 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

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