The microtubule-associated protein, NUD-1, exhibits chaperone activity in vitro.
Regulation of cell division requires the concerted function of proteins and protein complexes that properly mediate cytoskeletal dynamics. NudC is an evolutionarily conserved protein of undetermined function that associates with microtubules and interacts with several key regulators of mitosis, such as polo-kinase 1 (Plk1) and dynein. NudC is essential for proper mitotic progression, and homologs have been identified in species ranging from fungi to humans. In this paper, we report the characterization of the Caenorhabditis elegans NudC homolog, NUD-1, as a protein exhibiting molecular chaperone activity. All NudC/NUD-1 proteins share a conserved p23/HSP20 domain predicted by three-dimensional modeling [Garcia-Ranea, Mirey, Camonis, Valencia, FEBS Lett 529(2-3):162-167, 2002]. We demonstrate that nematode NUD-1 is able to prevent the aggregation of two substrate proteins, citrate synthase (CS) and luciferase, at stoichiometric concentrations. Further, NUD-1 also protects the native state of CS from thermal inactivation by significantly reducing the inactivation rate of this enzyme. To further determine if NUD-1/substrate complexes were productive or simply "dead-end" unfolding intermediates, a luciferase refolding assay was utilized. Following thermal denaturation, rabbit reticulocyte lysate and ATP were added and luciferase activity measured. In the presence of NUD-1, nearly all of the luciferase activity was regained, indicating that unfolded intermediates complexed with NUD-1 could be refolded. These studies represent the first functional evidence for a member of this mitotically essential protein family as having chaperone activity and facilitates elucidation of the role such proteins play in chaperone complexes utilized in cell division. C. elegans NUD-1 is a member of an evolutionary conserved protein family of unknown function involved in the regulation of cytoskeletal dynamics. NUD-1 and its mammalian homolog, NudC, function with the dynein motor complex to ensure proper cell division, and knockdown or overexpression of these proteins leads to disruption of mitosis. In this paper, we show that NUD-1 possesses ATP-independent chaperone activity comparable to that of small heat shock proteins and cochaperones and that changes in phosphorylation state functionally alter chaperone activity in a phosphomimetic NUD-1 mutant.
Faircloth
Lindsay M
LM
Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL 35487, USA.
Churchill
Perry F
PF
Caldwell
Guy A
GA
Caldwell
Kim A
KA
D000818:Animals; D017173:Caenorhabditis elegans / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002950:Citrate (si)-Synthase / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation; D008156:Luciferases / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D018832:Molecular Chaperones / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature
D029742:Caenorhabditis elegans Proteins; D008869:Microtubule-Associated Proteins; D018832:Molecular Chaperones; D010750:Phosphoproteins; D011994:Recombinant Proteins; C446241:nud-1 protein, C elegans; D008156:Luciferases; D002950:Citrate (si)-Synthase