Molecular chaperones generally display a promiscuous protein binding capacity enabled by an affinity for short hydrophobic amino acid motifs that likely accounts for the shared ability to suppress non-native protein aggregation in vitro (Hendrick and Hartl, 1993
). To offset the broad binding, chaperones typically have short-lived, low affinity interactions to avoid interfering with the functional activity of a client protein. These characteristics fit well with the adopted molecular chaperone definition—a protein that has a functional effect on another protein/protein complex without becoming part of the final operative structure (Ellis, 1987
). While these evolved characteristics are likely pivotal to the cellular operations of a chaperone, these properties have hindered the identification of client proteins and impeded our ability to gain an overall understanding of the physiological networks that are served by the various molecular chaperones.
Based upon high cellular abundances and wide-ranging influences on diverse pathways Hsp70 and Hsp90 and their associated cochaperones constitute the central eukaryotic molecular chaperone system (Wegele et al., 2004
). While Hsp70 has been extensively studied (Mayer et al., 2009
), the general in vivo roles of Hsp90 are just being revealed. Although the Hsp70 system is one of the most conserved, the Hsp90 machine is a recently evolved chaperone system. Escherichia coli
contains a single Hsp90 homolog, HtpG, but does not express any apparent cochaperones that are common to eukaryotes. Nevertheless, Hsp90 has evolved into an essential eukaryotic protein, as have many of its cochaperones including Cdc37 and mammalian p23 (Johnson and Brown, 2009
). Hence, Hsp90 and its associated cofactors form a chaperone system that has been adopted to support the unique challenges of higher order organisms.
Hsp90 was originally identified in stable association with signaling proteins (i.e.
, kinases and steroid receptors) and it has been argued that its primary function is to maintain metastable factors in activatable states (Pratt and Toft, 2003
). However, signaling protein maintenance likely does not account for all duties of Hsp90. Recent studies indicate that Hsp90 serves in a broad range of cellular processes including protein transport, epigenetic status and cell cycle progression (Zhao et al., 2005
; Millson et al., 2005
; McClellan et al., 2007
). Thus, Hsp90 has a central role in cell homeostasis, which is exemplified by its use as a therapeutic target for a variety of cancers and other diseases (Whitesell and Lindquist, 2005
). Given the breadth of pathways affected by Hsp90, an efficient means to control and guide this chaperone is needed to insure proper function and avoid detrimental pleiotropic effects that might occur if this abundant chaperone was left unchecked.
To control Hsp90 numerous cochaperones including p23, Hop, Cdc37, Sgt1 and Aha1 have coevolved with the eukaryotic Hsp90s (Johnson and Brown, 2009
). In a manner paralleling Hsp70 cochaperones, the Hsp90 cohorts can modulate Hsp90s' ATP hydrolysis activity and potentially direct substrate specificity. A number of biochemical and structural studies have dissected the ATPase cycle of Hsp90 along with the potential influence of cochaperones (Hessling et al., 2009
; Wandinger et al.
, 2009). In addition, a mechanism by which a cochaperone might guide Hsp90 to select clients has been revealed, as Cdc37 forms a tripartite complex having contacts to both Hsp90 and the substrate protein (Vaughan et al., 2006
). Besides Hsp90-dependent events, cochaperones might also regulate client proteins autonomously using their innate chaperone activities (Freeman et al., 1996
). In general, however, independent cochaperone functions have been difficult to conclude since the known client proteins are shared with Hsp90.
We have investigated the physiological pathways served by the yeast p23 cochaperone Sba1 using high-throughput genomic and proteomic approaches. We focused on Sba1 since this cochaperone is broadly expressed in most eukaryotes, is an abundant cochaperone, forms co-complexes with Hsp90 alone and with client proteins and might also work autonomously. To comprehend the Sba1 interaction network and its relation to the yeast Hsp90 molecular chaperone Hsp82, we have incorporated in-depth primary and comparative bioinformatic analyses. In addition, we have assessed the Sba1-dependence of potential target pathways using various experimental techniques and we have determined whether the reliance is conserved in higher eukaryotic cells. Taken together, the presented studies establish a broad Sba1/p23 cellular network that includes a sizeable nuclear subnet.