Abstract
The chaperonin containing tailless complex polypeptide 1 (CCT) is a multi-subunit molecular chaperone. It is found in the cytoplasm of all eukaryotic cells, where the oligomeric form plays an essential role in the folding of predominantly the cytoskeletal proteins actin and tubulin. Both the CCT oligomer and monomeric subunits also display functions that extend beyond folding, which are often associated with microtubules and actin filaments. Here, we assess the functional significance of the CCTδ V390F mutation, reported in several cancer cell lines. Upon transfection into B16F1 mouse melanoma cells, GFP-CCTδV390F incorporates into the CCT oligomer more readily than GFP-CCTδ. Furthermore, unlike GFP-CCTδ, GFP-CCTδV390F does not interact with the dynactin complex component, p150Glued. As CCTδ has previously been implicated in altered migration in wound healing assays, we assessed the behaviour of GFP-CCTδV390F and other mutants of CCTδ, previously used to assess functional interactions with p150Glued, in chemotaxis assays. We developed the assay system to incorporate a layer of the inert hydrogel GrowDex® to provide a 3D matrix for chemotaxis assessment and found subtle differences in the migration of B16F1 cells, depending on the presence of the hydrogel.
Highlights
Chaperonin containing tailless complex polypeptide 1 (CCT) is a molecular chaperone that promotes the folding of substrates in an ATP-dependent manner
The CCTδV390F mutation is located at the border between the apical and intermediate domains (Fig. 1a) and is shown mapped onto the structure of the alpha thermosome chain (PDB: 1A6D) (Fig. 1b)
We assessed if the V390F substitution could have an impact upon CCT oligomer assembly in B16F1 mouse melanoma cells
Summary
Chaperonin containing tailless complex polypeptide 1 (CCT) is a molecular chaperone that promotes the folding of substrates in an ATP-dependent manner. The eight CCT subunit genes are essential in yeast (reviewed by Stoldt et al 1996), and CCT is found in the cytoplasm of all eukaryotes. All CCT subunits share a common domain architecture, consisting of a highly conserved equatorial domain containing an ATP-binding pocket, a less conserved apical substrate binding domain and a linker domain. Equivalent mutants in the ATP-binding site of all eight CCT subunits give rise to different phenotypes in yeast consistent with the possibility of CCT subunits having distinct monomer functions (Amit et al 2010). In yeast, a non-stoichiometric level of CCT subunits is observed, consistent with at least some CCT subunits being available in increased levels as monomers (Matalon et al 2014). For some CCT subunits, individual monomeric functions have been established, for example, in cell
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