the “conventional” proteasome --33 components, all integral subunits --Extremely conserved among eukaryotes --20 years of biochemistry
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
UBP6 UCH37 RPN11 the proteasomal deubiquitinating enzymes 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
UBP6 UCH37 RPN11 the proteasomal deubiquitinating enzymes 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 UBP6 RPN11 John Hanna
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
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
ubp6 inhibits protein turnover in vivo & on substrates other than cyclin b Wild-type ubp6 ubr1 John Hanna (min) UB-K-URA3 Rpn5
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
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
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?
ubp6 antagonizes in vitro conjugate formation by hul5 hul5 hul5 ubp6 WT ubp6 Ub E1-Ub Bernat Crosas John Hanna
ubp6 disassembles conjugates formed by hul5 on the proteasome hul5 ubp6ubp Conjugation Bernat Crosas John Hanna - Ubp6+ Ubp Deconjugation
Canavanine WT hul5 ubp6 hul5 ubp6 Control hul5 suppresses ubp6 Bernat Crosas
NaCl (mM) WT ubp Hul5 hul5 association with the proteasome is highly sensitive to salt in ubp 6 mutants Bernat Crosas
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
% remaining Ub-K-Ura3 hul5 WT Time (min) Gcn4 hul5 WT Time (min) % remaining Alpha2 hul5 rpn4 Time (min) % remaining defective protein degradation in the hul 5 null Bernat Crosas
is hul5 an e4 enzyme? Ubp6? E3 X
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
e4 activity of hul5 on cyclin b cycB Ub-cycB APCHul5 mock hul5 ubp6 proteasomes
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
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 deubiquitin- ating 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 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
dwell time model for hul5/ubp6 Degradation Simple Dissociation Dissociation via Deubiquitination Ubp6 Hul5 Ubp6
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