An Intracellular Probe of Hsc70:Substrate Interactions
Hsc70 is a constitutively active member of the heat shock chaperone family. It has many roles in all cell types, including prevention of protein aggregation, uncoating of clathrin vesicles, and involvement in the maturation of newly synthesised protein. ABCA1, a member of the ABC family, has been shown to have mutations that disrupt membrane localisation. The rescue of correct maturation and membrane localisation by chemical chaperones suggest a role for protein misfolding and endogenous molecular chaperones such as Hsc70 in its rescue. The mutants utilised in this study have distorted folding in such a manner that the activity is lost due to the inability to mature through trafficking to the plasma membrane where the protein is functional. In this work a fluorescent Hsc70 probe was produced and a method developed for its introduction into mammalian cells to investigate an interaction with misfolded ABCA1. Co-localisation of the Hsc70 chaperone with mislocalised ABCA1 variants were investigated with confocal microscopy. Hsc70 C574S, required for covalent attachment of a single fluorescent label, is active at about 25% the level of native protein in a refolding assay. The transduction protocol results in some fluorescent Hsc70 being internalised into HEK293T mammalian cells. Efficiency of transduction was assessed by fluorescence microscopy and flow cytometry. Between 20% and 90% of cells were found to be transduced with fluorescent protein. Labelled Hsc70 appears in the cytoplasm, distributed in the same pattern as wildtype ABCA1, suggesting co-localisation. The coincidence of location increases with the N1800H variant. However membrane location does not seem to be rescued by transduction of additional Hsc70. The Y1767D variant seems to have a lesser interaction with Hsc70, although this could be an artefact of the labelled chaperone, where endogenous untracked Hsc70 could be interacting. Hsc70 has been successfully used as an intracellular probe, detectable in HEK293T cells and displaying co-localisation with the ABCA1 protein.
Advisor: Wilbanks, Sigurd
Degree Name: Master of Science
Degree Discipline: Biochemistry
Publisher: University of Otago
Keywords: Hsc70; Hsp70; Chaperone; ABCA1
Research Type: Thesis