the conventional proteasome

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the conventional proteasome
--33 components, allintegral subunits
--Extremely conserved among eukaryotes
--20 years of biochemistry
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major proteasome-associated proteins of budding
yeast
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the proteasomal deubiquitinating enzymes
Proposed roles regenerating ubiquitin allowing
substrate translocation editing of bad
substrates Possible liability premature
deubiquitination
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in vitro breakdown of cyclin b conjugates
(min)
John Hanna Nate Hathaway Randy King Don
Kirkpatrick Steve Gygi
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recombinant ubp6 corrects the defect of ubp6
null proteasomes
John Hanna
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the proteasomal deubiquitinating enzymes
Proposed roles regenerating ubiquitin allowing
substrate translocation editing of bad
substrates Possible liability premature
deubiquitination
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proteasome inhibition by ubp6 is noncatalytic
John Hanna
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two distinct activities of ubp6 work on the same
substrateproteasome inhibition and chain
trimming
John Hanna
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(No Transcript)
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inhibition by ubp6 occurs on the proteasome
John Hanna
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ubp6 inhibits its sister deubiquitinating enzyme
rpn11
John Hanna
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ubp6 inhibits its sister deubiquitinating enzyme
rpn11

• Proteasome inhibition by Ubp6 is accompanied by a
  switchin the mode of deubiquitination by the
  proteasome

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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

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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
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comments

• The ubiquitin-proteasome pathway is under strong
  inhibitory control

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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)

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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

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major proteasome-associated proteins of budding
yeast
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hul5 confers ubiquitin-conjugating activity on
the proteasome
Does it work against Ubp6?
Bernat Crosas
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ubp6 antagonizes in vitro conjugate formation by
hul5
Bernat Crosas John Hanna
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ubp6 disassembles conjugates formed by hul5 on
the proteasome
Conjugation
hul5 ubp6
ubp6
Bernat Crosas John Hanna
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Dhul5 suppresses Dubp6
Bernat Crosas
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hul5 association with the proteasome is highly
sensitive to salt in ubp6 mutants
NaCl (mM)
Hul5
WT
Hul5
Dubp6
Bernat Crosas
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working model
Hul5 and Ubp6 antagonize each other in a specific
subpopulation of proteasomes, establishing a
dynamicstate for proteasome-bound multiubiquitin
chains
Hul5
Ubp6
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defective protein degradation in the hul5 null
Bernat Crosas
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is hul5 an e4 enzyme?
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hul5 can exhibit proteasome-dependent e4 activity
E1
ubch5
Hul5
Ub4-8
Holo
Ub
Ubn
Ub2
Bernat Crosas Christa Bücker
Ub
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e4 activity of hul5 on cyclin b
hul5D ubp6D proteasomes
mock
Hul5
APC
Hul5
mock
Ub-cycB
cycB
60
0
30
30
0
0
0
30
30
0
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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
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Traditional view decision to degrade a protein
made by E3 beforeengagement of substrate by the
proteasome--proteasome is passive
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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
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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
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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
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dwell time model for hul5/ubp6
Degradation
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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