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The Peptidyl-prolyl Isomerase Domain of the CyP-40 Cyclophilin Homolog Cpr7 Is Not Required to Support Growth or Glucocorticoid Receptor Activity in Saccharomyces cerevisiae*

Open AccessPublished:May 01, 1998DOI:https://doi.org/10.1074/jbc.273.18.10819
      CyP-40 cyclophilins are found in association with molecular chaperone Hsp90·steroid receptor complexes. The amino-terminal portion of these cyclophilins harbors the characteristic peptidyl-prolyl isomerase (PPIase) domain, whereas three copies of the tetratricopeptide (TPR) motif, a structure shown to be involved in protein-protein interactions, and a putative calmodulin-binding domain are located in the carboxyl-terminal half of the protein. The TPR domains mediate binding to Hsp90, but a requirement for the PPIase domain has not been established. To address this, we have investigated the effects of mutations that alter the PPIase domain of the Saccharomyces cerevisiae CyP-40 homolog, Cpr7. Because Cpr7 is required for rapid growth and full Hsp90 activity, a functional assessment of the PPIase domain could be performed in vivo. A mutation in the catalytic domain altering a conserved site predicted to be essential for isomerase activity did not compromise Cpr7 function. Furthermore, deletion of the entire PPIase domain did not significantly affect growth or Hsp90-mediated steroid receptor activity. These results indicate that the TPR-containing carboxyl terminus of Cpr7 is sufficient for fundamental Cpr7-dependent activity.
      The molecular chaperone Hsp90 can serve as a general chaperonein vitro when present at high concentration by preventing aggregation of a variety of substrates (
      • Wiech H.
      • Buchner J.
      • Zimmermann R.
      • Jakob U.
      ,
      • Miyata Y.
      • Yahara I.
      ,
      • Schumacher R.J.
      • Hurst R.
      • Sullivan W.P.
      • McMahon N.J.
      • Toft D.O.
      • Matts R.L.
      ,
      • Freeman B.C.
      • Morimoto R.I.
      ,
      • Schneider C.
      • Sepp-Lorenzino L.
      • Nimmesgern E.
      • Ouerfelli O.
      • Danishefsky S.
      • Rosen N.
      • Hartl F.U.
      ,
      • Thulasiraman V.
      • Matts R.L.
      ,
      • Yonehara M.
      • Minami Y.
      • Kawata Y.
      • Nagai J.
      • Yahara I.
      ,
      • Uma S.
      • Hartson S.D.
      • Chen J.J.
      • Matts R.L.
      ), but its role in vivo appears to be restricted to the maturation of a subset of proteins involved in signal transduction pathways (
      • Rutherford S.L.
      • Zuker C.S.
      ,
      • Nathan D.
      • Lindquist S.
      ,
      • Bohen S.P.
      • Kralli A.
      • Yamamoto K.R.
      ,
      • Nathan D.F.
      • Harju Vos M.
      • Lindquist S.
      ). The best characterized Hsp90-dependent substrates are members of the steroid receptor family. Maturation of steroid receptor complexes involves a dynamic assembly pathway in which the receptor first enters into an intermediate complex containing Hsp90, Hsp70, p60 (Hop), and p48 (Hip) (
      • Smith D.F.
      • Whitesell L.
      • Nair S.C.
      • Chen S.
      • Prapapanich V.
      • Rimerman R.A.
      ,
      • Dittmar K.D.
      • Pratt W.B.
      ,
      • Pratt W.B.
      • Toft D.O.
      ) and is later found in a mature complex containing Hsp90, low levels of Hsp70, p23, and one of three immunophilins (
      • Pratt W.B.
      • Toft D.O.
      ). The mature complex is required for the steroid receptor to achieve a conformation that enables it to bind and respond to hormone.
      Three immunophilins, FKBP51, FKBP52, and Cyp-40, have been identified as components of distinct steroid hormone receptor complexes. FKBP51 and FKBP52 belong to the FK506-binding protein family, whereas Cyp-40 is a cyclophilin (
      • Pratt W.B.
      • Toft D.O.
      ,
      • Marks A.R.
      ). In addition to binding to their respective immunosuppressive drugs (FK506 or cyclosporin A), most immunophilins tested thus far exhibit peptidyl-prolyl isomerase (PPIase)
      The abbreviations used are: PPIase, peptidyl-prolyl isomerase; TPR, tetratricopeptide repeat; PCR, polymerase chain reaction; ORF, open reading frame; GR, glucocorticoid receptor; kb, kilobase(s); bp, base pairs.
      1The abbreviations used are: PPIase, peptidyl-prolyl isomerase; TPR, tetratricopeptide repeat; PCR, polymerase chain reaction; ORF, open reading frame; GR, glucocorticoid receptor; kb, kilobase(s); bp, base pairs.
      activity in vitro (
      • Galat A.
      ). Because these proteins are found in Hsp90-steroid receptor complexes in organisms as diverse as mammals and yeast, it would seem likely that prolyl isomerization plays a role in governing the activity of the complex. In vitro experiments have shown that CyP-40 can act as a molecular chaperone by maintaining a substrate in a non-native folding-competent intermediate (
      • Freeman B.C.
      • Toft D.O.
      • Morimoto R.I.
      ). However, isomerization does not appear to be required for this function because treatment with cyclosporin A, which inhibits isomerase activity of cyclophilins in vitro, did not have an effect in this assay (
      • Freeman B.C.
      • Toft D.O.
      • Morimoto R.I.
      ). Furthermore, biochemical experiments have suggested that the isomerase activity of immunophilins is not required for reconstitution of the glucocorticoid receptor in mature heterocomplexes (
      • Owens-Grillo J.K.
      • Hoffmann K.
      • Hutchison K.A.
      • Yem A.W.
      • Deibel Jr., M.R.
      • Handschumacher R.E.
      • Pratt W.B.
      ). Thus, the in vivo role of the PPIase activity of immunophilins in Hsp90-dependent events, if any, remains unknown.
      In addition to the PPIase domain, Hsp90-associated immunophilins harbor three units of the tetratricopeptide repeat (TPR), a degenerate 34-amino acid motif involved in protein-protein interactions (
      • Lamb J.R.
      • Tugendreich S.
      • Hieter P.
      ). FKBP52 and CyP-40 interact directly with Hsp90 through this domain (
      • Radanyi C.
      • Chambraud B.
      • Baulieu E.-E.
      ,
      • Hoffmann K.
      • Handschumacher R.E.
      ) by binding to what appears to be a single TPR-accepting pocket on Hsp90 (
      • Owens-Grillo J.K.
      • Hoffmann K.
      • Hutchison K.A.
      • Yem A.W.
      • Deibel Jr., M.R.
      • Handschumacher R.E.
      • Pratt W.B.
      ,
      • Johnson J.L.
      • Toft D.O.
      ). Two additional components of steroid receptor-Hsp90 complexes, PP5 (a protein phosphatase) and p60, also contain TPR motifs and bind to the same site on Hsp90 as do the immunophilins (
      • Owens-Grillo J.K.
      • Czar M.J.
      • Hutchison K.A.
      • Hoffmann K.
      • Perdew G.H.
      • Pratt W.B.
      ,
      • Silverstein A.M.
      • Galigniana M.D.
      • Chen M.S.
      • Owens-Grillo J.K.
      • Chinkers M.
      • Pratt W.B.
      ). Interestingly, overexpression of the TPR domain of PP5 results in a dominant negative effect that inhibits glucocorticoid receptor-mediated transactivation (
      • Chen M.S.
      • Silverstein A.M.
      • Pratt W.B.
      • Chinkers M.
      ), supporting a function for TPR-containing proteins in steroid receptor signaling. Some of the TPR proteins that interact with Hsp90 have been proposed to target Hsp90 substrates to their cellular destination (
      • Owens-Grillo J.K.
      • Czar M.J.
      • Hutchison K.A.
      • Hoffmann K.
      • Perdew G.H.
      • Pratt W.B.
      ).
      Two Saccharomyces cerevisiae CyP-40 homologs, Cpr6 and Cpr7, have been identified (
      • Chang H.-C.J.
      • Lindquist S.
      ,
      • Duina A.A.
      • Marsh J.A.
      • Gaber R.F.
      ) and shown to interact directly with Hsp90 via their TPR-containing carboxyl termini (
      • Duina A.A.
      • Chang J.
      • Marsh J.A.
      • Lindquist S.
      • Gaber R.F.
      ). Disruption ofCPR7 confers a modest decrease in the rate of cell growth (
      • Duina A.A.
      • Marsh J.A.
      • Gaber R.F.
      ,
      • Dolinski K.
      • Muir S.
      • Cardenas M.
      • Heitman J.
      ), but this effect is severe when combined with mutations that either decrease the levels of Hsp90 or disrupt STI1, the gene encoding the yeast homolog of p60 (
      • Duina A.A.
      • Chang J.
      • Marsh J.A.
      • Lindquist S.
      • Gaber R.F.
      ). CPR7 is also required for full activity of two heterologous Hsp90 substrates, the glucocorticoid receptor (GR) and the oncogenic tyrosine kinase p60v-src (
      • Duina A.A.
      • Chang J.
      • Marsh J.A.
      • Lindquist S.
      • Gaber R.F.
      ). In this report we test the requirement for the PPIase domain in Cpr7. We conclude that fundamental functions associated with Cpr7 do not depend on its role as a prolyl isomerase but instead require the TPR-containing carboxyl terminus.

      DISCUSSION

      Two homologs of the mammalian cyclophilin CyP-40 occur in S. cerevisiae, but only one, Cpr7, has been shown to have a functional role in Hsp90-associated activity (
      • Duina A.A.
      • Chang J.
      • Marsh J.A.
      • Lindquist S.
      • Gaber R.F.
      ). Thus far, no phenotype has been associated with the deletion of CPR6(
      • Duina A.A.
      • Marsh J.A.
      • Gaber R.F.
      ,
      • Duina A.A.
      • Chang J.
      • Marsh J.A.
      • Lindquist S.
      • Gaber R.F.
      ,
      • Dolinski K.
      • Muir S.
      • Cardenas M.
      • Heitman J.
      ,
      • Warth R.
      • Briand P.-A.
      • Picard D.
      ). Interestingly, in vitro assays have detected isomerase activity for Cpr62 (
      • Dolinski K.
      • Muir S.
      • Cardenas M.
      • Heitman J.
      ,
      • Warth R.
      • Briand P.-A.
      • Picard D.
      ) but not for Cpr72 (
      • Dolinski K.
      • Muir S.
      • Cardenas M.
      • Heitman J.
      ). The results described here strongly suggest that the in vivo functions thus far associated with the yeast Cyp-40 homolog Cpr7, namely the maintenance of rapid growth rates and GR activity, are not dependent on PPIase activity but are mediated by the TPR-containing carboxyl terminus. This is consistent with the observation that pharmacological inhibition of isomerase activity does not prevent reconstitution of salt-dissociated GR heterocomplexesin vitro (
      • Owens-Grillo J.K.
      • Hoffmann K.
      • Hutchison K.A.
      • Yem A.W.
      • Deibel Jr., M.R.
      • Handschumacher R.E.
      • Pratt W.B.
      ). The TPR-containing domain is capable of mediating both physical (
      • Duina A.A.
      • Chang J.
      • Marsh J.A.
      • Lindquist S.
      • Gaber R.F.
      ) and functional (data herein) interactions with Hsp90.
      What, then, is the functional role of the PPIase domain of Cpr7? The steady-state level of Cpr7ΔPPIase was less than that of wild-type Cpr7 using the same promoters and plasmids. Thus, it is possible that the PPIase domain was adventitiously acquired in evolution as a mechanism for stabilizing the TPR-containing domain. If so, it seems unlikely that this is the only purpose of the PPIase domain because it has been highly conserved. The Cyp-40 member of the cyclophilin family is found in steroid receptor complexes from yeast to mammals. Moreover, a critical amino acid (Arg64), which is required for PPIase activity in other cyclophilins, is not required to maintain normal expression levels of Cyp-40 in yeast. Yet it too has been conserved from yeast to mammals. If stabilization of the TPR domain was the sole function of the PPIase domain, greater sequence divergence would be expected over one billion years of evolution. We favor the possibility that the PPIase domain plays a role in Hsp90-mediated events, but a subtle one. Effects that are too subtle to detect in the types of assays we have employed here can nevertheless provide sufficient advantage for selection in evolution. For example, the PPIase domain might affect the rate at which other co-chaperones join or leave the Hsp90 complex, enhance the proper localization of the complex in the cell, or act on an unidentified substrate whose function is not relevant in our assays.
      Whether such functions might involve catalyzing the isomerization of peptidyl-prolyl bonds is unclear. Although PPIase activity was not detected with Cpr7 in vitro, its activities might be restricted to particular substrates and therefore not be apparent in a standard in vitro peptide assay. Examples consistent with this scenario are presented by the bovine cyclophilin RanBP2 and the well characterized Drosophila cyclophilin homolog NinaA. RanBP2 exhibits only modest PPIase activity in vitro, but mutation of the site analogous to the R64A mutation in Cpr7 significantly decreased its function in vivo (
      • Ferreira P.A.
      • Nakayama T.A.
      • Pak W.L.
      • Travis G.H.
      ). Furthermore, although to our knowledge NinaA has not been reported to possess PPIase activity in vitro, mutations in NinaA that map to amino acid residues known to be crucial for PPIase activity in mammalian cyclophilins conferred strong ninaA mutant phenotypes in vivo (
      • Ondek B.
      • Hardy R.W.
      • Baker E.K.
      • Stamnes M.A.
      • Shieh B.-H.
      • Zuker C.S.
      ).
      In any case, with respect to growth control and GR activity in yeast, it is clear that the TPR-containing domain of Cpr7 plays a much stronger role than the PPIase domain. TPR domains have been identified in a number of proteins involved in varied cellular functions, including cell cycle control, transcriptional regulation, and steroid receptor regulation (
      • Pratt W.B.
      • Toft D.O.
      ,
      • Lamb J.R.
      • Tugendreich S.
      • Hieter P.
      ). In addition to a well established role in protein-protein interactions, recent reports have hypothesized that the TPR domains present in Hsp90-associated immunophilins are involved in subcellular targeting (
      • Owens-Grillo J.K.
      • Czar M.J.
      • Hutchison K.A.
      • Hoffmann K.
      • Perdew G.H.
      • Pratt W.B.
      ). How might the carboxyl terminus of Cpr7 function in Hsp90-dependent events? As suggested by others (
      • Owens-Grillo J.K.
      • Czar M.J.
      • Hutchison K.A.
      • Hoffmann K.
      • Perdew G.H.
      • Pratt W.B.
      ), the TPR domain of Cpr7 may indeed be involved in subcellular targeting of the Hsp90·steroid receptor complex. Alternatively, but not exclusively, we speculate that the TPR region of Cpr7 might cause a conformational change in Hsp90, enhancing its effectiveness in chaperoning steroid receptors and growth regulators into active or at least signal-responsive conformations.

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