Regulation of the Myosin-Directed Chaperone UNC-45 by a Novel E3/E4-Multiubiquitylation Complex in C. elegans. Cell, Vol. 118, 337–349, 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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Chaperone UNC-45 by a Novel
E3/E4-Multiubiquitylation Complex in C. elegans
Cell, Vol. 118, 337–349, August 6, 2004,
Thorsten Hoppe, Giuseppe Cassata, Jose´ M. Barral, Wolfdieter Springer, Alex H. Hutagalung, Henry F. Epstein, and Ralf Baumeister
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!
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.
HSP-1 is the C.elegans ortholog of Hsp70
HSP-1 interact with CHN-1 and does not compete for UFD-2 binding
Self-ubiquitylation of UFD-2 and CHN-1
Dependent on LET-70 (E2 enzyme)
UFD-2 and CHN-1 act in the same conjugation pathway
chn-1 (CHIP) mutant in C.elegans
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
Double mutants unc-45;chn-1
unc-45: Egl phenotype – partially supressed in unc-45;chn-1
unc-45 overexpression in chn-1 mutants
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
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
UNC-45 is ubiquitylated in vivo!