Genetic Analysis Reveals Domain Interactions of Arabidopsis Hsp 100/ClpB and Cooperation with the Small Heat Shock Protein Chaperone System

• Published in: The Plant cell Vol. 17; no. 2; pp. 559 - 571
• Main Authors: Ung Lee, Chris Wie, Mindy Escobar, Williams, Ben, Hong, Suk-Whan, Vierling, Elizabeth
• Format: Journal Article
• Language: English
• Published: American Society of Plant Biologists 01.02.2005
• Subjects: Adenosine triphosphatases; Alleles; Genetic mutation; Genetic suppression; Heat stress disorders; Heat tolerance; Hypocotyls; Phenotypes; Proteins; Seedlings
• ISSN: 1040-4651; 1532-298X

We have defined amino acids important for function of the Arabidopsis thaliana Hsp100/ClpB chaperone (AtHsp101) in acquired thermotolerance by isolating recessive, loss-of-function mutations and a novel semidominant, gain-of-function allele [hot1-4 (A499T)]. The hot1-4 allele is unusual in that it not only fails to develop thermotolerance to 45°C after acclimation at 38°C, but also is sensitive to 38°C, which is a permissive temperature for wild-type and loss-of-function mutants. hot1-4 lies between nucleotide binding domain 1 (NBD1) and NBD2 in a coiled-coil domain that is characteristic of the Hsp100/ClpB proteins. We then isolated two classes of intragenic suppressor mutations of hot1-4: loss-of-function mutations (Class 1) that eliminated the 38°C sensitivity, but did not restore thermotolerance function to hot1-4, and Class 2 suppressors that restored acquired thermotolerance function to hot1-4. Location of the hot1-4 Class 2 suppressors supports a functional link between the coiled-coil domain and both NBD1 and the axial channel of the Hsp100/ClpB hexamer. In addition, the strongest Class 2 suppressors restored solubility of aggregated small heat shock proteins (sHsps) after heat stress, revealing genetic interaction of the Hsp100/ClpB and sHsp chaperone systems. These results also demonstrate that quantitative phenotypes can be used for in vivo genetic dissection of protein mechanism in Arabidopsis.