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Abstract
A variety of signaling proteins form heterocomplexes with and are regulated by the heat shock protein chaperone hsp90. These complexes are formed by a multiprotein machinery, including hsp90 and hsp70 as essential and abundant components and Hop, hsp40, and p23 as non-essential cochaperones that are present in much lower abundance in cells. Overexpression of signaling proteins can overwhelm the capacity of this machinery to properly assemble heterocomplexes with hsp90. Here, we show that the limiting component of this assembly machinery in vitro in reticulocyte lysate and in vivo in Sf9 cells is p23. Only a fraction of glucocorticoid receptors (GR) overexpressed in Sf9 cells are in heterocomplex with hsp90 and have steroid binding activity, with the majority of the receptors present as both insoluble and cytosolic GR aggregates. Coexpression of p23 with the GR increases the proportion of cytosolic receptors that are in stable GR.hsp90 heterocomplexes with steroid binding activity, a strictly hsp90-dependent activity for the GR. Coexpression of p23 eliminates the insoluble GR aggregates and shifts the cytosolic receptor from very large aggregates without steroid binding activity to approximately 600-kDa heterocomplexes with steroid binding activity. These data lead us to conclude that p23 acts in vivo to stabilize hsp90 binding to client protein.
Highlights
Several transcription factors and a variety of protein kinases involved in signal transduction are regulated by the abundant, ubiquitous and essential protein chaperone hsp901 [1]
The steroids bind deep in a hydrophobic cleft that appears to be collapsed in the absence of ligand [11], and the hsp90/hsp70-based chaperone machinery opens the binding cleft of the glucocorticoid receptors (GR) ligand binding domain (LBD) such that it can be accessed by steroid (Ref. 12 and references therein)
Addition of purified hsp90, hsp70, Hop, or YDJ-1 to reticulocyte lysate does not enhance its ability to activate the GR to the steroid binding state, but addition of purified p23 yields a substantial increase in GR1⁄7hsp90 heterocomplexes with steroid binding activity (Fig. 1B)
Summary
Several transcription factors and a variety of protein kinases involved in signal transduction are regulated by the abundant, ubiquitous and essential protein chaperone hsp901 [1]. P23 and Glucocorticoid Receptor Folding of full steroid binding activity [14] This led to the conclusion that factors other than hsp and hsp are limiting in insect cells but can be supplied in vitro by reticulocyte lysate. Increasing p23 increases the fraction of cytosolic receptors that are stably bound to hsp, and p23 coexpression eliminates the formation of receptor aggregates These observations argue strongly that the effects of p23 expression on a client protein function must be interpreted in terms of the ability of p23 to enhance the activity of hsp as opposed to direct chaperone effects in which p23 interacts with a partially denatured protein to affect its folding state. From a protein engineering viewpoint, coexpression of p23 may prove to be very useful in the production of the physiologically regulated state of client proteins that normally are present in persistent heterocomplexes with hsp
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