Genetic analysis reveals domain interactions of Arabidopsis Hsp100/ClpB and cooperation with the small heat shock protein chaperone system.
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 degrees C after acclimation at 38 degrees C, but also is sensitive to 38 degrees 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 degrees 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.
Lee
Ung
U
Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721, USA.
Wie
Chris
C
Escobar
Mindy
M
Williams
Ben
B
Hong
Suk-Whan
SW
Vierling
Elizabeth
E
D000483:Alleles; D017360:Arabidopsis / Q000235:genetics* / Q000254:growth & development; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018869:Heat-Shock Response; D018546:Hypocotyl / Q000254:growth & development / Q000378:metabolism; D009154:Mutation; D010641:Phenotype; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D016963:Thermus thermophilus / Q000235:genetics / Q000378:metabolism
D029681:Arabidopsis Proteins; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins