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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, 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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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


Regulation of the myosin directed chaperone unc 45 by a novel

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!


Regulation of the myosin directed chaperone unc 45 by a novel

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.


Regulation of the myosin directed chaperone unc 45 by a novel

Structure of E4 – the additional conjugation factor


Regulation of the myosin directed chaperone unc 45 by a novel

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


Regulation of the myosin directed chaperone unc 45 by a novel

chn-1 (CHIP) mutant in C.elegans


Regulation of the myosin directed chaperone unc 45 by a novel

  • 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


Regulation of the myosin directed chaperone unc 45 by a novel

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


Regulation of the myosin directed chaperone unc 45 by a novel

chn-1::GFP

ufd-2::GFP


Regulation of the myosin directed chaperone unc 45 by a novel

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


Regulation of the myosin directed chaperone unc 45 by a novel

Interactions between CHN-1 and UNC-45 in vivo


Regulation of the myosin directed chaperone unc 45 by a novel

unc-45 overexpression in chn-1 mutants


Regulation of the myosin directed chaperone unc 45 by a novel

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


Regulation of the myosin directed chaperone unc 45 by a novel

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


Regulation of the myosin directed chaperone unc 45 by a novel

 Dramatically stimulation of multiubiquitylation!!

UNC-45 is ubiquitylated in vivo!


Regulation of the myosin directed chaperone unc 45 by a novel

That’s it!


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