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Regulation of the Myosin-Directed Chaperone UNC-45 by a Novel

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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Regulation of the Myosin-Directed Chaperone UNC-45 by a Novel

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  1. 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

  2. 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!

  3. 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.

  4. Structure of E4 – the additional conjugation factor

  5. Interaction of CHN-1 and UFD-2 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

  6. chn-1 (CHIP) mutant in C.elegans

  7. chn-1(by155) C.elegans mutant phenotype • viable, no obvious morphological defects • reduced brood size at 20°C • at 30°C: 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

  8. 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

  9. chn-1::GFP ufd-2::GFP

  10. Interactions between CHN-1 and UNC-45 in vivo Double mutants unc-45;chn-1 unc-45: Egl phenotype – partially supressed in unc-45;chn-1

  11. Interactions between CHN-1 and UNC-45 in vivo

  12. unc-45 overexpression in chn-1 mutants

  13. 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

  14. 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

  15.  Dramatically stimulation of multiubiquitylation!! UNC-45 is ubiquitylated in vivo!

  16. That’s it!

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