GCUNC45 is the first Hsp90 co-chaperone to show alpha/beta isoform specificity

Abstract

Hsp90 is an essential molecular chaperone required for the normal functioning of many key regulatory proteins in eukaryotic cells. Vertebrates have two closely related isoforms of cytosolic Hsp90 (Hsp90alpha and Hsp90beta). However, specific functions for each isoform are largely unknown, and no Hsp90 co-chaperone has been reported to distinguish between the two isoforms. In this study, we show that the Hsp90 co-chaperone GCUNC45 bound preferentially to the beta isoform of Hsp90 in vitro. GCUNC45 efficiently blocked the progression of progesterone receptor chaperoning in an in vitro functional system when Hsp90beta was used, but did so with much less efficacy when Hsp90alpha was used. Knockdown experiments in HeLa cells showed that GCUNC45 is required for the normal cellular distribution of Hsp90beta, but not Hsp90alpha. This is the first example of a co-chaperone with isoform selectivity, and this approach may open novel avenues to understanding the functional differences between Hsp90 isoforms.

FIGURE 1.

Comparison of chicken (c) Hsp90α and human (h) Hsp90α and Hsp90β in chaperoning PR in vitro. PR hormone-binding activity was reconstituted with purified Hsp70, Hsp40, Hop, and p23 and the addition of increasing amounts of the indicated Hsp90 proteins. [³H]Progesterone binding was measured and is expressed in cpm as the mean of triplicate samples.

FIGURE 2.

GCUNC45 inhibition of PR chaperoning is specific to the Hsp90 isoform used. PR hormone-binding activity was reconstituted as described in the legend to Fig. 1 using Hsp90α or Hsp90β with the addition of increasing amounts of GCUNC45. The progesterone-binding activity of PR was measured and is expressed as cpm of bound [³H]progesterone for the mean of triplicate samples.

FIGURE 3.

Direct binding of various Hsp90 proteins to GCUNC45. A, immobilized GCUNC45 was used to pull down 13.5 μg of yeast (Y) Hsp82, 11.5 μg of chicken (c) Hsp90α, 10 μg of human (h) Hsp90α, or 8 μg of human Hsp90β. Loads lanes show 20% of the total amount used in the reaction (stained with Coomassie Blue). Note that although the loads differed somewhat, they are biased against the conclusions of the experiment. B, the binding of increasing amounts of human Hsp90α or Hsp90β to resin-bound GCUNC45 is shown directly and as determined by Scatchard analysis. AU, arbitrary densitometric units. C and D, p23 or Hop, respectively, was immunoadsorbed to a specific antibody and used to bind increasing amounts of Hsp90α or Hsp90β as indicated.

FIGURE 4.

GCUNC45 is required for the normal distribution of Hsp90β in HeLa cells. A, Western blot analysis of GCUNC45, PRB, Hsp90α, and Hsp90β expression in HeLa/PRB cells treated with 50 nm control siRNA (Control) or siRNA directed against GCUNC45 for 72 h. B, confocal images of immunostained Hsp90α (red) and Hsp90β (green) in cells grown on glass coverslips from the same plate as in A.