Complementation Assays for Co-chaperone Function

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 2693; p. 105
• Main Authors: Edkins, Adrienne L, Blatch, Gregory L
• Format: Journal Article
• Language: English
• Published: United States 2023
• Subjects: Heat shock proteins; J domain proteins; DnaJ; Molecular chaperones; Protein folding; Hsp40
• ISSN: 1940-6029
• DOI: 10.1007/978-1-0716-3342-7_9

The development of mutant microorganisms lacking J domain proteins (JDPs; formerly called Hsp40s) has enabled the development of complementation assays for testing the co-chaperone function of JDPs. In these assays, an exogenously expressed novel JDP is tested for its ability to functionally substitute for a non-expressed or nonfunctional endogenous JDP(s) by reversing a stress phenotype. For example, the in vivo functionality of prokaryotic JDPs can be tested on the basis of their ability to reverse the thermosensitivity of a dnaJ cbpA mutant strain of the bacterium Escherichia coli (OD259). Similarly, the in vivo functionality of eukaryotic JDPs can be assessed in a thermosensitive ydj1 mutant strain of the yeast Saccharomyces cerevisiae (JJ160). Here we outline the use of these thermosensitive microorganisms in complementation assays to functionally characterize a JDP from the bacterium, Agrobacterium tumefaciens (AgtDnaJ), and a JDP from the trypanosomal parasite, Trypanosoma cruzi (TcJ2).