Abstract
The molecular chaperone Hsc70 assists in the folding of non-native proteins together with its J domain- and BAG domain-containing cofactors. In Caenorhabditis elegans, two BAG domain-containing proteins can be identified, one of them being UNC-23, whose mutation induces severe motility dysfunctions. Using reporter strains, we find that the full-length UNC-23, in contrast to C-terminal fragments, localizes specifically to the muscular attachment sites. C-terminal fragments of UNC-23 instead perform all Hsc70-related functions, like ATPase stimulation and regulation of folding activity, albeit with lower affinity than BAG-1. Interestingly, overexpression of CFP-Hsc70 can induce muscular defects in wild-type nematodes that phenocopy the knockout of its cofactor UNC-23. Strikingly, the motility dysfunction in the unc-23 mutated strain can be cured specifically by down-regulation of the antagonistic Hsc70 cochaperone DNJ-13, implying that the severe phenotype is caused by misregulation of the Hsc70 cycle. These findings point out that the balanced action of cofactors in the ATP-driven cycle of Hsc70 is crucial for the contribution of Hsc70 to muscle functionality.
Highlights
The knockout of the protein UNC-23, a cochaperone of Hsc70, leads to severe motility dysfunction in the model organism C. elegans
Bcl-2-associated athanogene (BAG)-1 consists of a C-terminal BAG domain and an N-terminal ubiquitin-like domain, and BAG-1 is the homolog of the human Bag1 with an overall amino acid identity of 30%
PXXP repeats, which are thought to interact with Src homology 3 domains [35], instead can be found in other human BAG proteins, like Bag3 and Bag4 (Fig. 1A), but the homology of their BAG domain to UNC-23 is much lower
Summary
The knockout of the protein UNC-23, a cochaperone of Hsc, leads to severe motility dysfunction in the model organism C. elegans. In Caenorhabditis elegans, two BAG domain-containing proteins can be identified, one of them being UNC-23, whose mutation induces severe motility dysfunctions. Hsc is composed of an N-terminal ATP/ADP-binding domain, a substrate-binding domain, and a C-terminal lid domain, which covers the substrate binding groove [4, 5] It acts via a regulated ATP-dependent cycle that is influenced by different cofactors, like Hsp40s/J domain-containing proteins and nucleotide exchange factors (NEFs).. In order to investigate protein localization, the coding sequence of H14N18.1, obtained from Open Biosystems (Thermo Scientific) was amplified and inserted into the reporter construct in frame to YFP with the following primers: UNC-23-YFP fusion, ATT GCA GCT AGC ATG TTT CAG AAC ATA CCA ATC AAA ATA C (forward) and CAG CCT GCT AGC TTC GCT TTG ATC ATC CAT C (reverse).
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