Furthermore, full-length Tah1 interacted with Hsp82-MC but not with the Hsp82-NM fragment ( Fig. 1 B (left), full-length Tah1 interacted with Pih1 and Hsp82, which is consistent with our previous observations ( Zhao et al., 2005). Two fragments of Hsp82 were also used: Hsp82-NM (residues 1–560) and Hsp82-MC (residues 260–709). Tah1 can be divided into two folded, stable, and soluble domains (unpublished data): an N-terminal domain containing two TPR motifs (residues 1–75 Tah1-TPR) and a C-terminal region (residues 76–111 Tah1-C). 1 A) that were expressed and purified in Escherichia coli. Pull-down experiments were performed using full-length proteins as well as protein domains ( Fig. These factors are not part of the final complex but are present during the biogenesis and assembly of the snoRNPs. Other protein factors have also been found to promote the stability of snoRNAs, such as Naf1 for box H/ACA ( Dez et al., 2002 Fatica et al., 2002) and Bcd1 for Box C/D snoRNAs ( Peng et al., 2003). Association of the core proteins with box C/D or box H/ACA snoRNAs is required for the stability and proper localization of the snoRNPs ( Filipowicz and Pogacic, 2002). snoRNPs are trans-acting regulators that are responsible for cleavage and modifications of small nuclear RNA (snRNA), ribosomal RNA (rRNA), and tRNAs ( Matera et al., 2007). snoRNPs are RNP complexes made up typically of either box H/ACA or box C/D small nucleolar RNAs (snoRNAs) associated with a set of core proteins. It was also shown that depletion of yeast Rvb2 affects the localization of snoRNP core proteins ( King et al., 2001). Rvb1 and Rvb2 homologues in the mouse were found to associate with small nucleolar RNP (snoRNP) complexes and were shown to be important for snoRNP assembly and function ( Newman et al., 2000 Watkins et al., 2002, 2004). Furthermore, it was recently suggested that Rvb1 and Rvb2 play an important role in DNA damage repair and transcription ( Radovic et al., 2007).

Rvb1 and Rvb2 are essential for yeast growth ( Qiu et al., 1998) and are found to be critical components of the chromatin remodeling complexes Ino80 ( Jonsson et al., 2001) and SWR-C ( Krogan et al., 2003 Kobor et al., 2004 Mizuguchi et al., 2004) as well as of the TIP60 histone acetylase complex ( Ikura et al., 2000). Rvb1 and Rvb2 belong to the AAA + (ATPase associated with diverse cellular activities) superfamily of ATPases and show weak homology to the bacterial DNA helicase RuvB. Tah1 and Pih1 were found to interact with Hsp82 from a large-scale two-hybrid (2H) screen and were shown to affect the yeast Hsp90 chaperone activity toward the classical model substrates v-Src and glucocorticoid receptor ( Zhao et al., 2005). Both Tah1 and Pih1 were shown to be novel Hsp90 interactors. Tah1 contains two tetratricopeptide repeats (TPRs), whereas Pih1 has no known motifs ( Fig. In that work, we identified a new complex interacting with Hsp90 consisting of four proteins: Rvb1, Rvb2, Tah1, and Pih1 (also called Nop17). Previously, in an effort to globally analyze Hsp90 function and to identify novel Hsp90 substrates and cochaperones, we conducted a set of high-throughput physical and genetic interaction screens of Hsp90 in yeast ( Zhao et al., 2005).

Molecular chaperone schematic