Transcript Document
the “conventional” proteasome --33 components, all integral subunits --Extremely conserved among eukaryotes --20 years of biochemistry major proteasome-associated proteins of budding yeast Blm10 Ecm29 Proposed roles in proteasome maturation tion and DNA repair Tethers RP and CP together Hul5 Ubiquitin-protein ligase Ubp6 Deubiquitinating enzyme Rad23 Delivers conjugates to proteasome the proteasomal deubiquitinating enzymes RPN11 UCH37 UBP6 Proposed roles regenerating ubiquitin allowing substrate translocation editing of bad substrates Possible liability premature deubiquitination in vitro breakdown of cyclin b conjugates (min) Unmodified Cyclin B John Hanna Nate Hathaway Randy King Don Kirkpatrick Steve Gygi recombinant ubp6 “corrects the defect” of ubp6 null proteasomes John Hanna the proteasomal deubiquitinating enzymes RPN11 UCH37 UBP6 Proposed roles regenerating ubiquitin allowing substrate translocation editing of bad substrates Possible liability premature deubiquitination proteasome inhibition by ubp6 is noncatalytic John Hanna two distinct activities of ubp6 work on the same substrate: proteasome inhibition and chain trimming John Hanna inhibition by ubp6 occurs on the proteasome John Hanna ubp6 inhibits its sister deubiquitinating enzyme rpn11 Lid Base CP John Hanna RPN11 UBP6 ubp6 inhibits its sister deubiquitinating enzyme rpn11 Lid Base RPN11 UBP6 CP Proteasome inhibition by Ubp6 is accompanied by a switch• in the mode of deubiquitination by the proteasome ubp6 inhibits its sister deubiquitinating enzyme rpn11 Lid Base RPN11 UBP6 CP 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• ubp6 inhibits protein turnover in vivo & on substrates other than cyclin b Wild-type ubp6 0 7 14 0 7 14 ubr1 (min) UB-K-URA3 Rpn5 John Hanna comments •The ubiquitin-proteasome pathway is under strong inhibitory control 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) 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 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 major proteasome-associated proteins of budding yeast Blm10 Ecm29 Proposed roles in proteasome maturation tion and DNA repair Tethers RP and CP together Hul5 Ubiquitin-protein ligase Ubp6 Deubiquitinating enzyme Rad23 Delivers conjugates to proteasome hul5 confers ubiquitin-conjugating activity on the proteasome E1-Ub Ub Does it work against Ubp6? Bernat Crosas ubp6 antagonizes in vitro conjugate formation by hul5 E1-Ub Ub Bernat Crosas John Hanna ubp6 disassembles conjugates formed by hul5 on the proteasome Conjugation hul5 ubp6 0 30 60 90 120 203 115 93 48 34 28 21 7.2 Bernat Crosas John Hanna Deconjugation ubp6 0 30 60 90 120 - Ubp6 0 30 60 90 120 + Ubp6 0 30 60 90 120 Dhul5 suppresses Dubp6 Control WT Dhul5 Dubp6 Dhul5 Dubp6 Bernat Crosas Canavanine hul5 association with the proteasome is highly sensitive to salt in ubp6 mutants NaCl (mM) 25 Bernat Crosas 50 75 100 125 150 175 200 WT Hul5 Dubp6 Hul5 working model Hul5 and Ubp6 antagonize each other in a specific subpopulation of proteasomes, establishing a dynamic state for proteasome-bound multiubiquitin chains Hul5 Ubp6 defective protein degradation in the hul5 null 100 100 % remaining % remaining hul5D WT 10 hul5D rpn4D rpn4D Gcn4 Alpha2 10 0 10 20 30 0 Time (min) 10 Time (min) 100 % remaining hul5D WT Ub-K-Ura3 10 Bernat Crosas 0 20 Time (min) 40 20 is hul5 an e4 enzyme? E3X Ubp6? hul5 can exhibit proteasome-dependent e4 activity E1 ubch5 Hul5 Ub4-8 Holo Ub Ubn Ub4 Ub2 Bernat Crosas Christa Bücker Ub + + + + + + - + + + + + - - + + + + + + + - + + + + + + - + + + + + + - e4 activity of hul5 on cyclin b hul5D ubp6D proteasomes APC mock Hul5 0 60 0 30 0 30 Ub-cycB cycB 0 30 0 30 ™ and a TI FF ( Uncompr essed) decompressor ar e needed t o see t his pict ur e. 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 Traditional view: decision to degrade a protein made by E3 before engagement of substrate by the proteasome--proteasome is passive 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 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 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 These activities allow active control the final and only irreversible step in protein breakdown dwell time model for hul5/ubp6 Ubp6 Degradation Simple Dissociation Hul5 Dissociation via Deubiquitination Ubp6 Hul5 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 QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this pict ure. Nate Hathaway Randy King Currently: Inst. of Molecular Biology, Barcelona