Abstract
Sgt1p is a conserved, essential protein required for kinetochore assembly in both yeast and animal cells. Sgt1p has homology to both TPR and p23 domains, sequences often found in proteins that interact with and regulate the molecular chaperone, Hsp90. The presence of these domains and the recent findings that Sgt1p interacts with Hsp90 has led to the speculation that Sgt1p and Hsp90 form a co-chaperone complex. To test this possibility, we have used purified recombinant proteins to characterize the in vitro interactions between yeast Sgt1p and Hsp82p (an Hsp90 homologue in yeast). We show that Sgt1p interacts directly with Hsp82p via its p23 homology region in a nucleotide-dependent manner. However, Sgt1p binding does not alter the enzymatic activity of Hsp82p, suggesting that it is distinct from other co-chaperones. We find that Sgt1p can form a ternary chaperone complex with Hsp82p and Sti1p, a well characterized Hsp90 co-chaperone. Sgt1p interacts with its binding partner Skp1p through its TPR domains and links Skp1p to the core Hsp82p-Sti1p co-chaperone complex. The multidomain nature of Sgt1p and its ability to bridge the interaction between Skp1p and Hsp82p argue that Sgt1p acts as a "client adaptor" recruiting specific clients to Hsp82p co-chaperone complexes.
Highlights
A large group of Hps90-associated proteins have been proposed to assist Hsp90 in client recognition or in the transition of client to its final active state
We have found that the p23 homology domain of Sgt1p is required for the direct interaction between Sgt1p and Hsp90
Sgt1p can form at least two distinct ternary complexes; in the first, Hsp90 interacts directly with Sgt1p and Sti1p and in the second, Sgt1p interacts with Skp1p through its tetracopeptide repeat (TPR) domain and Hsp90 through its p23 homology domain
Summary
Plasmids and Cloning—Construction of His-Sgt1p for baculoviral expression has been previously described [6] as has HisSkp1p [29]. Contaminating chaperones were released from Sti1p and Sgt1p protein fusions by including a final wash step with SHB-Tris buffer containing 5 mM ATP for 30 min at 4 °C, followed by three washes in SHB-Tris buffer with no nucleotide. This step reduced co-purifying proteins, it had no effect on the binding assays. For the other binding reactions shown, 10 l of GST, GST-Hsp82p, or GST-Skp1p (ϳ2 g of protein), bound to glutathione-Sepharose 4B beads (Amersham Biosciences) and purified as described above, was added to 200 l of SHB-Tris followed by addition of ϳ2 g of additional protein(s) to be assayed. Addition of 60 M geldanamycin completely inhibited the ATPase activity of our His-Hsp82p preparation (data not shown), ruling out the possibility of contaminating ATPases
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