Folie%201

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Regulation of the Myosin-Directed Chaperone
UNC-45 by a Novel E3/E4-Multiubiquitylation
Complex in C. elegans
Cell, Vol. 118, 337349, August 6, 2004,
Thorsten Hoppe, Giuseppe Cassata, Jose M.
Barral, Wolfdieter Springer, Alex H. Hutagalung,
Henry F. Epstein, and Ralf Baumeister
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E1 ubiquitin-activating enzyme
E2 ubiquitin-conjugation enzyme
E3 ubiquitin protein ligase
E4 additional conjugation factor, catalyzes
multiubiquitin chain assembly UFD2 in yeast and
human CHIP (carboxyl terminus of
Hsp70- interacting protein) display E4 enzyme
function
CHIP is regulating the activity of the E3 enzyme
Parkin!
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Ubiquitin
Ubiquitin is a small, single domain protein of 76
residues consisting of both alpha-helix and
beta-sheet, that forms a highly stable, compact
structure.
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Structure of E4 the additional conjugation
factor
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UFD-2 and CHN-1 act in the same conjugation
pathway
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chn-1 (CHIP) mutant in C.elegans
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• chn-1(by155) C.elegans mutant phenotype
• viable, no obvious morphological defects
• reduced brood size at 20C
• at 30C F1 shows developmental arrest and
  lethality at different larval stages
• chn-1 RNAi display similar temperature
  sensitivity
• chn-1(by155)/chn-1(ok459) transheterozygotes
  like chn-1(by155) homozygotes

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CHN-1 and UFD-2 interact with UNC-45, a myosin
chaperon
A Interaction of UFD-2 and UNC-45 in vitro B In
vitro binding of UNC-45 by UFD-2 or CHN-1 UNC-45
binding of UFD-2 is 20-fold more compared to
CHN-1 C Complex of CHN-1, UFD-2, and UNC-45
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chn-1GFP
ufd-2GFP
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Interactions between CHN-1 and UNC-45 in vivo
Double mutants unc-45chn-1 unc-45 Egl phenotype
partially supressed in unc-45chn-1
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Interactions between CHN-1 and UNC-45 in vivo
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unc-45 overexpression in chn-1 mutants
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Conclusion chn-1 loss of function can not only
suppress unc-45(ts) phenotype but is also not
able to compensate for excessive amounts of UNC-45
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Is UNC-45 a substrate for CHN-1-dependent
ubiquitylation???
Activity of all 3 enzymes is required and
sufficient for UNC-45 ubiquitylation Adding
CHN-1 or UFD-2 similar results What happens when
adding both.
CHN-1 lacking the U box is not able to
ubiquitylate UNC-45
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? Dramatically stimulation of multiubiquitylation!
!
UNC-45 is ubiquitylated in vivo!
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Thats it!