Cdc37 maintains cellular viability in Schizosaccharomyces pombe independently of interactions with heat-shock protein 90 Emma L Turnbull, Ina V Martin* and Peter A Fantes Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, UK Keywords Cdc37; fission yeast; heat-shock protein 90 (Hsp90); molecular chaperone; pombe Correspondence P A Fantes, Institute of Cell Biology, School of Biological Sciences, Mayfield Road, University of Edinburgh, Edinburgh EH9 3JR, UK Fax: +44 131 651 3331 Tel: +44 131 650 5669 E-mail: p.fantes@ed.ac.uk *Present address Institute of Physiology, RWTH Aachen, Uniklinikum, Pauwelsstr 30, 52074 Aachen, Germany (Received 11 May 2005, revised 17 June 2005, accepted 20 June 2005) Cdc37 is a molecular chaperone that interacts with a range of clients and co-chaperones, forming various high molecular mass complexes Cdc37 sequence homology among species is low High homology between yeast and metazoan proteins is restricted to the extreme N-terminal region, which is known to bind clients that are predominantly protein kinases We show that despite the low homology, both Saccharomyces cerevisiae and human Cdc37 are able to substitute for the Schizosaccharomyces pombe protein in a strain deleted for the endogenous cdc37 gene Expression of a construct consisting of only the N-terminal domain of S pombe Cdc37, lacking the postulated heat-shock protein (Hsp) 90-binding and homodimerization domains, can also sustain cellular viability, indicating that Cdc37 dimerization and interactions with the cochaperone Hsp90 may not be essential for Cdc37 function in S pombe Biochemical investigations showed that a small proportion of total cellular Cdc37 occurs in a high molecular mass complex that also contains Hsp90 These data indicate that the N-terminal domain of Cdc37 carries out essential functions independently of the Hsp90-binding domain and dimerization of the chaperone itself doi:10.1111/j.1742-4658.2005.04825.x Cdc37 is a molecular chaperone that was identified in two different ways First, cdc37 was identified during a screen for Saccharomyces cerevisiae mutants that arrest with a cell division cycle (cdc) phenotype [1] and secondly as a 50 kDa protein from chick cells called p50 associated with the client v-src, that was subsequently shown to share sequence homology with S cerevisiae Cdc37 [2] Cdc37 has been found to associate with client proteins involved in a range of cellular processes including cell cycle regulation, DNA and protein synthesis and signal transduction (for review see [3]) Many protein clients rely on chaperones for activation, folding and protection from degradation Client proteins of Cdc37 are predominantly protein kinases such as Cdk4 [4–6] and Raf1 [7] which bind the N-terminal domain of Cdc37 [8] Cdc37 has been identified in high molecular mass complexes in association with a wide variety of clients and other co-chaperones [6,9–11] Structurally, three domains of human Cdc37 were defined by limited proteolysis and peptide analysis and are referred to as the N-terminal, middle and C-terminal domains [8] At present there is no known role for the C-terminal domain, whereas functions for the N-terminal and middle regions have been identified The N-terminal domain of Cdc37 is the region most highly conserved among species and has been found to bind the client protein kinase, eIF2a kinase [8] Conserved residues within the N-terminal domain, serines Abbreviations cdc, cell division cycle; Cdk4, cyclin dependent kinase 4; CKII, casein kinase II; 5FOA, 5¢ fluoro-2¢-deoxyuridine; GST, glutathione S-transferase; Hsp, heat-shock protein; HA, influenza hemagglutinin epitope FEBS Journal 272 (2005) 4129–4140 ª 2005 FEBS 4129 Hsp90 binding is not essential for S pombe Cdc37 14 and 17 in S cerevisiae [12] and serine 13 in rat [13] and human [14] (equivalent to S cerevisiae serine 14), have been identified as important sites of phosphorylation by casein kinase II (CKII) Phosphorylation of these conserved serine residues by CKII is required for Cdc37 activity [12,13] There is evidence that Cdc37 and CKII maintain each other’s activity in a feedback loop of activation [12] Phosphorylation of these serine residues is important for client interactions, as the unphosphorylated form of human Cdc37 was found to have significantly reduced binding affinity towards several client kinases [13] Cdc37 has been found to display a range of chaperone activities towards bound clients Cdc37 can facilitate the assembly of protein kinases such as cyclin dependent kinase (Cdk4) and its partner cyclin D into complexes [5] Cdc37 can also promote an activation competent state of the client in vitro by cooperating with other co-chaperones such as heat-shock protein (Hsp) 70 and Hdj1 [15] Cdc37 interacts with a range of clients and co-chaperones, such as Hsp90, forming a variety of heterocomplexes There are several lines of evidence which indicate that Cdc37 functions in part with Hsp90 by delivering client protein kinases to this cochaperone Cdc37 and Hsp90 have been found in the same high molecular mass (450 kDa) complex associated with the client Cdk4 in NIH-3T3 cells [6] A complex consisting of the interacting domains of yeast Hsp90 and human Cdc37 has been crystallised and its structure determined [16] Amino acids 164–170 and 204–208 of human Cdc37 were found to form a hydrophobic patch that interacts with the N-terminal region of yeast Hsp90 [16] Human Cdc37 binds both the N-terminal domains and the adjacent linker regions of the Hsp90 dimer [17] Cdc37 binds to Hsp90 as a dimer [18] at a : molar ratio [17] Cdc37 preferentially binds a non-ATP bound form of Hsp90 and suppresses ATP turnover [18] After Cdc37 has been released from the tertiary complex with Hsp90 and the client, ATP turnover by Hsp90 is carried out as a two step process, promoting conformational changes of the Hsp90–client complex [19] Studies of the interaction between Cdc37 and Hsp90 are more advanced in mammalian systems due to the unstable nature of the tertiary complex in yeast systems [20] A genetic interaction between Cdc37 and Hsp90 has been observed in S cerevisiae, in that mutations compromised for Cdc37 and Hsp90 function are synthetically lethal [15] Identification of biochemical interactions between Hsp90 and Cdc37 in yeast systems is limited In S cerevisiae an interaction between Hsp90 and Cdc37 has been shown using recombinant glutathione S-transferase (GST)-Cdc37 in pull-down experiments [21] and in the yeast two-hybrid 4130 E L Turnbull et al assay, using a mutant form of Hsp90 in which ATP hydrolysis was inhibited [22] The fission yeast Schizosaccharomyces pombe has been used as a model eukaryote for the investigation of a variety of cellular processes, notably cell cycle control and the responses to stress Little is known about Cdc37 in S pombe The cdc37 gene is essential for viability [23], and depletion of the Cdc37 protein in shut-off experiments led to heterogeneous cell phenotypes, indicating an involvement in several cellular roles that have not been elucidated A temperature conditional cdc37 mutant was isolated as a suppressor of hyperactivation of the stress-activated mitogen activated protein kinase pathway [24], and a direct interaction between Cdc37 and the client kinase Spc1 ⁄ Sty1 was demonstrated We set out to identify which domains of S pombe Cdc37 were essential for function We generated a series of truncation mutants of cdc37 and expressed them in a cdc37Dstrain to ascertain their ability to compensate for loss of wild-type Cdc37 Surprisingly, we discovered that expression of the N-terminal domain alone can sustain cellular viability These truncated proteins not contain the postulated Hsp90-binding domain, suggesting that binding of the cochaperone Hsp90 by Cdc37 is not required for cellular viability These data indicate that Cdc37 has an essential role, independent of interactions with Hsp90 However, biochemical investigations reveal that a small proportion of total Cdc37 protein is associated with the cochaperone Hsp90 in a high molecular mass complex Results Human and S cerevisiae Cdc37 are functional homologues of S pombe Cdc37 Alignment of Cdc37 homologues from human, S cerevisiae and S pombe show low overall sequence identity (Fig 1A) Despite low overall sequence homology, specific regions of Cdc37 are more highly conserved The N-terminal domain of Cdc37 is the most highly conserved region and is involved in client interactions [8] In the N-terminal 40 amino acids there is 80% identity between the S pombe and S cerevisiae sequences and 50% identity between the S pombe and human proteins To investigate conservation of Cdc37 function between species, plasmids encoding human, S cerevisiae and S pombe Cdc37 were introduced into the S pombe strain ED1526 and expressed from pREP81 in the plasmid shuffle assay (see below) (Fig 1B) Note that expression of wild-type S pombe Cdc37 from pREP81 generates a level of Cdc37 FEBS Journal 272 (2005) 4129–4140 ª 2005 FEBS E L Turnbull et al Hsp90 binding is not essential for S pombe Cdc37 A B C Fig Comparison of Cdc37 homologues (A) Alignment of Cdc37 protein sequences from human, S cerevisiae and S pombe Black boxes indicate identical amino acids amongst all three Cdc37 homologues Grey boxes denote identical amino acids between two Cdc37 homologues (B) Human, S cerevisiae and S pombe Cdc37 were expressed from pREP81 (wild-type levels) in the S pombe strain ED1526 by plasmid shuffle to determine their ability to sustain cellular viability (C) Human, S cerevisiae and S pombe Cdc37 overexpression (from pREP1) in the plasmid shuffle S pombe strain ED1526 to determine the ability of Cdc37 homologues to rescue an S pombe cdc37D protein very similar to endogenous (data not shown) S cerevisiae CDC37 expression was able to maintain cellular viability (Fig 1B) This observation suggests that there is functional equivalence between yeast Cdc37 proteins At wild-type expression levels, human Cdc37 was unable to sustain cellular viability (Fig 1B), although increased expression from pREP1 restored cellular viability (Fig 1C) These data suggest that human Cdc37 is a functional homologue of S pombe Cdc37, although the human protein may act inefficiently in S pombe blot analysis on GST, S pombe protein extracts and GST-Cdc37 using depleted serum results in a loss of signal against S pombe protein extracts and recombinant Cdc37 (Fig 2A) The predicted molecular mass of S pombe Cdc37 is  56 kDa, but by SDS ⁄ PAGE gel it runs at  64 kDa S cerevisiae Cdc37 has a predicted molecular mass of 58.4 kDa, but was found to run on an SDS ⁄ PAGE gel at  68 kDa [25] Taken together these data indicate that the observed 64 kDa protein corresponds to S pombe Cdc37 which the antibody specifically recognizes Affinity purified S pombe Cdc37 antibody The C-terminal domain of S pombe Cdc37 is not essential for in vivo function To investigate Cdc37 in S pombe a polyclonal antibody was raised in rabbit and affinity purified The specificity of this antibody was tested by western blot analysis against GST, S pombe whole cell protein extracts and GST-Cdc37 The antibody recognized GST-Cdc37 and a 64 kDa protein from S pombe protein extracts (Fig 2A) To verify that the 64 kDa protein is indeed Cdc37, S pombe Cdc37 antibodies were depleted from the antiserum by preincubation with GST-Cdc37 conjugated to glutathione beads Western FEBS Journal 272 (2005) 4129–4140 ª 2005 FEBS Biochemical investigations using limited proteolysis and peptide analysis have defined three discrete domains in human Cdc37 (p50); an N-terminal domain consisting of amino acids 1–126, a middle region composed of residues 128–282 and a C-terminal domain of amino acids 283–378 [8] By aligning human and S pombe Cdc37 sequences we were able to map these regions onto the yeast protein as indicated in Fig 2B To identify the functional domains of Cdc37, we 4131 Hsp90 binding is not essential for S pombe Cdc37 A E L Turnbull et al C B D Fig The C-terminus is dispensable for Cdc37 function in S pombe (A) Verification of the specificity of the anti-S pombe Cdc37 IgG (Upper panel) western blots of GST, native S pombe protein extracts and GST-Cdc37 were carried out with S pombe Cdc37 antibody (Lower panel) Anti-serum depleted of Cdc37 antibodies (see text) was used in western blots against GST, native S pombe protein extracts and GST-Cdc37 (B) The protein sequence of human (p50) and S pombe Cdc37 were aligned (Fig 1A) Structural domains defined in human Cdc37 by limited proteolysis and peptide analysis [8] were mapped onto the S pombe Cdc37 protein sequence The Hsp90 binding and dimerization domains of human Cdc37 identified by crystallization studies [16] were also mapped onto S pombe Cdc37 by alignment Boxes with horizontal stripes indicate the location of the postulated Hsp90 binding domain and the box with diagonal stripes denotes the homodimerization domain (C) Truncation mutants of S pombe Cdc37 were expressed from pREP81 in the S pombe plasmid shuffle strain ED1526 to determine their ability to sustain cellular viability (D) Protein levels of Cdc37 truncation mutants were compared to endogenous Cdc37 by western blot of total cellular protein extracts with the anti-S pombe Cdc37 IgG Asterisk indicates proteolytic truncation of endogenous Cdc37 expressed truncation mutants of Cdc37 in S pombe ED1526 and tested them for function by plasmid shuffle assay Truncation mutants Cdc37(1–428), Cdc37(1–412), Cdc37(1–385), Cdc37(1–360) and Cdc37(1–351), deleted in the C-terminal domain, were able to compensate for loss of full length Cdc37 (Fig 2C) These data indicate that the C-terminal domain is not essential for Cdc37 function in S pombe The shorter truncation mutants, Cdc37(1–321), Cdc37(1–273), Cdc37(1–264) and Cdc37(1–250), were unable to support cellular viability (Fig 2C) Cells expressing these truncations phenotypically resembled those observed in Cdc37 depletion experiments, comprising morphologically heterogeneous nondividing cells [23] Surprisingly, the mutants Cdc37(1–190) and Cdc37(1–220) were able to support a low level of growth when expressed at wild-type levels (Fig 2C), although these truncations lack most of the middle and 4132 all of the C-terminal domains, including the postulated Hsp90-binding and dimerization regions [16] Mutants with truncations extending into the N-terminal domain, Cdc37(1–155), Cdc37(1–120), Cdc37(1–60) and Cdc37(1–37), were unable to promote colony formation at any temperature (Fig 2C) Mutants of S pombe Cdc37 deleted from the N-terminus for the first 20 and 40 amino acids were unable to maintain cellular viability at low or high expression levels (data not shown) The Cdc37 mutants, Cdc37(1–321), Cdc37(1–273), Cdc37(1–264) and Cdc37(1–250), truncated within the middle domain, might be unable to sustain cellular viability if the mutant proteins were unstable and present at reduced levels This has been observed for the S cerevisiae mutant cdc37-1 which is truncated at codon 360 [25] The level of Cdc37 protein in each of the truncation mutants was assayed Whole cell protein extracts were FEBS Journal 272 (2005) 4129–4140 ª 2005 FEBS E L Turnbull et al Hsp90 binding is not essential for S pombe Cdc37 made for truncation mutants expressed from pREP81 in the cdc37+ strain ED1090 and equal amounts loaded onto SDS polyacrylamide gels Western blot analysis allowed comparison in each mutant of the levels of the Cdc37 truncation protein with that of endogenous full length protein All truncation mutants except for Cdc37(1–155) yielded a truncated protein that was detected by western blot with the Cdc37 antibody (Fig 2D) In other experiments (not shown) we tested the possibility that some truncated proteins, particularly those truncated within the middle domain, might be insoluble and therefore unable to contribute to essential Cdc37 function(s) We prepared native extracts of strains expressing various Cdc37 constructs and fractionated them by centrifugation into supernatant and pellet fractions which were then analysed by western blotting However the truncated proteins showed no increase in the proportion of insoluble fraction compared with the full length Cdc37 All mutants deleted within the middle and C-terminal domains were detected by the S pombe Cdc37 antibody at levels approximately equal to endogenous Cdc37 However, Cdc37(1–190) and Cdc37(1–220) were detected by the antibody at reduced levels compared to endogenous Cdc37 This may be due to low expression levels, reduced stability of mutant proteins or poor recognition by the S pombe Cdc37 antibody whose recognition epitope(s) has not been fully characterized Therefore, phenotypes observed for Cdc37(1–190) and Cdc37(1–220) may arise from reduced protein levels Overexpression of the N-terminal domain of Cdc37 is sufficient for cellular viability Several Cdc37 truncations were overexpressed from pREP1 to determine the effect of increasing mutant protein levels on cellular viability Overexpression of the mutants Cdc37(1–321), Cdc37(1–273), Cdc37(1–264) and Cdc37(1–250), truncated in the middle domain, resulted in a wild-type phenotype (Fig 3A) in contrast to the inviability of strains expressing the same truncations at wild-type levels (Fig 2C) These data indicate that the truncated proteins have reduced function but this is compensated by increased expression so that the overall level of function is above the threshold level for cellular viability Overexpression of the shorter truncation mutants, Cdc37(1–190) and Cdc37(1–220), which lack the middle and C-terminal domains, resulted in growth comparable to expression of pREP1-cdc37 (Fig 3A) Expression of these truncation mutants at wild-type levels was previously shown (Fig 2C) to support limited growth, most likely due to their reduced protein levels This is confirmed by the complete restoration of viability by expression of these truncations at levels substantially greater than endogenous Cdc37 from pREP1 (Fig 3B) Proteolysis of Cdc37 was observed in these experiments, most notably for Cdc37(1–190) and Cdc37(1–220) (Fig 3B) Overexpression of the truncation Cdc37(1–155), which lacks part of the N-terminal domain, was unable to support cellular viability A Fig Overexpression of the N-terminal domain of Cdc37 sustains cellular viability in S pombe (A) Cdc37 truncation mutants were overexpressed from pREP1 in the S pombe plasmid shuffle strain ED1526 to determine ability to sustain cellular viability (B) The truncation mutants pREP1-cdc37155, pREP1-cdc37-190 and pREP1-cdc37220 were expressed in the S pombe wildtype strain ED1090 to compared to protein levels against endogenous Cdc37 by western blot of total cell protein extracts Protein levels were assayed by western blot with the anti-S pombe Cdc37 IgG Asterisk indicates proteolytic truncation of endogenous Cdc37 FEBS Journal 272 (2005) 4129–4140 ª 2005 FEBS B 4133 Hsp90 binding is not essential for S pombe Cdc37 (Fig 3A) Cdc37(1–155) protein was detected by western blot with the S pombe Cdc37 antibody and was found to be present at a greater level than the endogenous full length protein (Fig 3B) In summary, the data presented in Figs and show that expression of the full N-terminal domain of Cdc37 at levels greater than endogenous is sufficient for full cellular viability in S pombe There is a clear distinction between domains that are essential and those that are dispensable for Cdc37 function in vivo in S pombe The defining boundary appears to be between the N-terminal and middle domain The middle and C-terminal domains that contain the postulated Hsp90 and homodimerization domain are not essential for Cdc37 function in S pombe provided the level of expression is sufficient This points towards Cdc37 carrying out essential functions that are independent of Hsp90-binding and homodimerization A five amino acid in-frame insertion within the middle domain of S pombe Cdc37 abolishes function Mutants Cdc37-I120, Cdc37-I252, Cdc37-I386 and Cdc37-I422 generated by in vitro pentapeptide mutagenesis described in Experimental procedures contain five amino acid insertions commencing at residues 120, 252, 386 and 422, respectively (Fig 4A) The mutants were expressed from pREP81 in the plasmid shuffle assay (Fig 4B) Expression of Cdc37-I120, Cdc37-I386 and Cdc37-I422 resulted in a wild-type phenotype, supporting growth comparable to pREP81-cdc37 expression, showing that these insertions in the N- and A E L Turnbull et al C-terminal domains not dramatically affect Cdc37 function In contrast, the mutant Cdc37-I252 was unable to support cellular viability and no growth was observed (Fig 4B) Cells appeared sick, being heterogeneous in phenotype, characteristic of depletion of Cdc37 [23] According to the alignment of p50 and S pombe Cdc37 shown in Fig 2B, the Cdc37-I252 insertion is located at the edge of the postulated Hsp90 binding domain located in the six helix bundle of the middle domain [16] and may disrupt structure in this region A small fraction of Cdc37 occurs in a high molecular mass complex Hsp90 and Cdc37 from mammalian lysates were found by size exclusion chromatography to occur in a range of high molecular mass fractions consistent with observations that various proteins associate with these chaperones [6,9–11] We carried out size exclusion chromatography to determine whether S pombe Cdc37 occurred in a high molecular mass complex Recombinant Cdc37 was initially studied to establish the elution pattern of the chaperone alone The majority of recombinant Cdc37 was found to elute at around 200 kDa (Fig 5A) By analogy human Cdc37 is  50 kDa, but in its native state exists as a dimer [16] and is structurally elongated which might affect its apparent size Similar factors may be responsible for the unexpected elution profile of S pombe Cdc37 Size exclusion chromatography of S pombe whole cell protein extracts showed the majority of Cdc37 also eluted at around 200 kDa (Fig 5A) A small proportion of Cdc37 protein eluted as a high molecular mass complex(es) at  669 kDa, while no recombinant Cdc37 eluted at this position These data suggest that in vivo, a small fraction of Cdc37 interacts stably with other proteins to form a high molecular mass complex B Cdc37 and Hsp90 interact in high molecular mass complexes Fig A mutational insertion within the middle domain abolishes Cdc37 function (A) The location in Cdc37 of in-frame insertions of five codons generated by in vitro pentapeptide mutagenesis is shown schematically (B) Expression of in-frame insertion mutants at wild-type levels in the S pombe strain ED1526 by plasmid shuffle to determine the ability of these mutants to sustain cellular viability in a cdc37D 4134 We have been unable to identify any interaction between S pombe Hsp90 and Cdc37 by coimmunoprecipitation from unfractionated native cell extracts of S pombe, pull-down using recombinant proteins or yeast twohybrid assay A new technique was employed, using size exclusion chromatography to isolate, from cell extracts of S pombe, fractions containing the high molecular mass complex of Cdc37 and probing these to identify an interaction with Hsp90 The elution pattern of the Cdc37 high molecular mass complex and Hsp90-influenza hemagglutinin epitope (HA) from the size excluFEBS Journal 272 (2005) 4129–4140 ª 2005 FEBS E L Turnbull et al Hsp90 binding is not essential for S pombe Cdc37 A B C Fig A small proportion of total cellular Cdc37 occurs in a high molecular mass complex associated with Hsp90 (A) An extract prepared from ED1537 cells was fractionated by analytical size exclusion chromatography on a Superose column (lower panels) In a parallel experiment, recombinant S pombe Cdc37 expressed in E coli was run on an identical column (upper panel) The resulting fractions were western blotted and probed with antibodies specific for HA (to detect tagged Hsp90) or Cdc37 as indicated, to determine the distribution patterns of the two chaperones across the molecular mass range (B) Large scale preparative size exclusion chromatography of protein extracts from S pombe ED1537 cells was carried out on a Superose 12 column and western blot analysis with the anti-S pombe Cdc37 and anti-HA IgGs identified the elution patterns of Cdc37 and Hsp90 (C) Immunoprecipitation reactions were carried out using antirat (control), anti-S pombe Cdc37 and anti-HA IgGs on fractions from the Superose 12 column containing the Cdc37 high molecular mass complex sion chromatography overlapped We then asked whether Cdc37 and Hsp90-HA were stably associated in the high molecular complex To generate adequate material for analysis of the Cdc37 high molecular mass complex(es), preparative size exclusion chromatography was carried out on a Sephacryl S-300 column (Fig 5B) Fractions containing Cdc37 in high molecular mass complex(es) were pooled and used as a source for immunoprecipitations Immunoprecipitations using the S pombe Cdc37 antibody also precipitated Hsp90-HA (Fig 5C), indicating an interaction between the two chaperones in the high molecular mass complex However, the reverse immunoprecipitation using the HA antibody did not yield Cdc37, perhaps because the antibody may not have access to the HA epitope in the complex S pombe Cdc37 and Hsp90 interact genetically In the previous section we show biochemically that a small fraction of Cdc37 stably associates with Hsp90 FEBS Journal 272 (2005) 4129–4140 ª 2005 FEBS Further evidence for an interaction comes from genetic interactions between the genes that encode for Hsp90 and Cdc37 The Hsp90 temperature-sensitive mutant swo1-26 [26] was crossed to each of the four cdc37 temperature-sensitive mutants, and each double mutant was found to be synthetically lethal at temperatures permissive for the single mutants (data not shown) A different genetic interaction between these two chaperones is shown by suppression of the temperature-sensitive mutant cdc37-13 lethality by increased expression of Hsp90 (Fig 6A) In the converse experiment, increased expression of S pombe Cdc37 at low or high levels did not suppress the lethality of swo1-26 (data not shown) Double mutants of Hsp90 and Cdc37 temperature-sensitive genes in S pombe may be unable to sustain cellular viability at the permissive temperature either because Cdc37 and Hsp90 carry out important functions together (for instance, in a physical complex which does not form in the double mutant) or because they both have essential independent roles In the latter case, the synthetic lethal defect may arise because 4135 Hsp90 binding is not essential for S pombe Cdc37 A B Fig Hsp90 expression rescues a cdc37 temperature-sensitive mutant at the restrictive temperature (A) Serial dilutions of the temperature-sensitive mutant cdc37-13 with increased expression levels of Hsp90 incubated on yeast extract for four days at the permissive (28 °C) and nonpermissive (36 °C) temperatures (B) Cells of cdc37-13 with increased expression of Hsp90 plated on yeast extract as serial dilutions and incubated for days at 36 °C the cumulative effect of the loss of both chaperones results in chaperone activity falling below a critical threshold Hsp90 and Cdc37 may carry out the same or similar independent functions, being able to compensate for one another in some instances, shown by the ability of Hsp90 to partially rescue the temperature-sensitive mutant cdc37-13 Discussion Cdc37 sequence homology between different species is low, but our results show that human and S cerevisiae Cdc37 are functional homologues of Cdc37 in S pombe Human Cdc37 was less efficient than the S cerevisiae protein in sustaining cellular viability, whereas overexpression of the human homologue was required to rescue the S pombe cdc37D The structural domains of human Cdc37 have been defined [8] and were mapped onto S pombe Cdc37 to investigate the functional regions of this chaperone protein An interesting new result is that expression of the N-terminal domain of S pombe Cdc37 is sufficient for cellular viability These truncation mutants lack the postulated Hsp90-binding and homodimerization domains, indicating that these functions are not essential for Cdc37 activity in S pombe Interestingly, an interaction between Cdc37 and Hsp90 was detected both biochemically and genetically Size exclusion chromatography 4136 E L Turnbull et al showed that a small proportion of total cellular Cdc37 is found in a high molecular mass complex in association with Hsp90, indicating that these two chaperones interact in a nonessential manner in S pombe Our observations are consistent with those of Lee et al [27], showing that the C-terminal domain of Cdc37 is completely dispensable for function In S cerevisiae the truncation mutant Cdc37(1–355) which lacks the latter part of the middle domain and the entire C-terminal domain was unable to restore cellular viability in a cdc37Dstrain whether expressed at low or high level [27] However, similar S pombe mutants, Cdc37(1–428), Cdc37(1–412), Cdc37(1–385) and Cdc37(1–351), truncated around the putative Hsp90-binding and homodimerization domains, were able to sustain cellular viability when overexpressed Lethality at wild-type expression levels of these truncation mutants was most likely the result of reduced function, as protein levels were not compromised and there was no difference in the relative amounts of soluble and insoluble Cdc37 in the truncation mutants One explanation for this phenomenon is that the N-terminal domain may be titrated away from carrying out essential roles by the aberrant middle domain attempting (for example) to interact with Hsp90 or homodimerize Alternatively, folding may be disrupted, negatively affecting the protein structure, as these mutants are truncated in the six a-helix bundle identified in human Cdc37 [16] Disruption of the protein structure in this region might affect essential N-terminal interactions between Cdc37 and client proteins Whatever the reason, it appears that Cdc37 proteins with a defective middle domain are more compromised than mutants entirely lacking it Perhaps significantly, the mutant I252, in which five amino acid residues are inserted within this region, is not viable We have shown that truncation mutants, Cdc37(1–190) and Cdc37(1–220), lacking the majority of the middle and all of the C-terminal domains, supported limited growth at wild-type levels Overexpression of these truncations, increasing the protein abundance above endogenous Cdc37 levels, was able to sustain cellular viability This differs from the observations in S cerevisiae which showed that in a cdc37Dstrain overexpression of the truncation mutants Cdc37(1–148) and Cdc37(1–239), also truncated around the N-terminal and middle domain boundary, enabled slow growth in a temperature dependent manner [27] We have demonstrated that in S pombe, the middle and C-terminal domains are completely dispensable for cellular viability provided protein levels are not a limiting factor Truncations within the N-terminal domain were unable to sustain cellular viability FEBS Journal 272 (2005) 4129–4140 ª 2005 FEBS E L Turnbull et al at low or high expression levels Overexpression of Cdc37(1–155), truncated into the N-terminal domain, yielded protein at a level greater than endogenous Cdc37, suggesting loss of essential Cdc37 function was the limiting factor Although Cdc37 truncation mutants lacking the postulated Hsp90-binding domain can sustain cellular viability in S pombe, we have identified an interaction biochemically and genetically between these two molecular chaperones A small fraction of total S pombe Cdc37 forms a high molecular mass complex that was also found to contain Hsp90 Identification of an interaction between Hsp90 and Cdc37 has been problematic due to the instability of the interaction and the small amount of Cdc37 that is present in the high molecular mass complex(es) Biochemical interactions between Hsp90 and Cdc37 have been found to be very salt labile [28] and more unstable in yeast than in mammalian systems [20] Our data show that Cdc37 does interact with Hsp90 in S pombe, but the presence of the postulated Hsp90-binding domain is not essential for cellular viability It is possible that Cdc37 carries out essential functions independently of Hsp90 binding and that these roles not require homodimerization of Cdc37 The N-terminal domain of Cdc37 may be involved in chaperone activities independently of other co-chaperones as human Cdc37 has been shown to play an crucial role in promoting complex assembly between cyclin-dependent kinases and their cyclin partners [5] Alternatively, Cdc37 may interact with other co-chaperones possibly through its N-terminal domain as S cerevisiae Cdc37 has been found to maintain clients in an activation competent state in association with the co-chaperones Hsp70 and Hdj1 [15] Yet another possibility is that normally the Cdc37–client complex interacts with Hsp90, perhaps presenting the client to Hsp90 When only the N-terminal domain of Cdc37 is expressed, the Cdc37–client complex forms but is unable to interact in the usual way with Hsp90, but does so by random encounters in the cytoplasm This would be expected to be an inefficient process, and indeed truncation mutants expressing only the N-terminal domain grow poorly unless the expression level is increased Experimental procedures Alignment of Cdc37 protein sequence Cdc37 protein sequences were aligned with vector ntitm (Invitrogen Ltd., Paisley, UK) using BLOSUM62MT2 matrix and the output was displayed by genedoc (http:// www.psc.edu/biomed/genedoc/) FEBS Journal 272 (2005) 4129–4140 ª 2005 FEBS Hsp90 binding is not essential for S pombe Cdc37 Antibodies An anti-Cdc37 IgG was raised in rabbit against full length recombinant S pombe Cdc37 and affinity purified against recombinant GST-Cdc37 Anti-HA 12CA5 monoclonal antibodies (Roche Applied Science, Lewes, UK), antirabbit IgG HRP-linked antibody (Amersham Biosciences UK Ltd., Little Chalfont, UK), antimouse IgG HRP-linked antibody (Amersham) and antirat IgG HRP-linked antibody (Amersham) were used as appropriate Cloning and expression vectors S pombe cdc37 [23] was cloned into pREP vectors (pREP1, pREP81) for expression in S pombe pREP1 is a strong, thiamine-repressible promoter, while pREP81 retains the thiamine-repressibility but expression is 80–100-fold less [29] For expression in S pombe, the complete open reading frame of the human p50cdc37 cDNA was subcloned following PCR amplification from plasmid pET16b-p50 (kind gift from C Prodromou, Institute of Cancer Research, London, UK) into the Nde1-Xma1 sites of pREP1 and pREP81 vectors Similarly, S cerevisiae CDC37 was amplified from the plasmid E119 [30] by PCR and ligated into the Nde1-Xma1 sites of the vectors pREP1 and pREP81 S pombe cdc37+ was PCR amplified and cloned into the BamH1-EcoR1 sites of the vector pGEX1 (Amersham) for expression in Escherichia coli Truncations of S pombe cdc37 were generated by PCR mutagenesis introducing a stop codon followed by the restriction site Xma1 for cloning into the sites Nde1-Xma1 site of the pREP vectors S pombe swo1+ (the gene encoding Hsp90 in S pombe) was a kind gift from K Belaya (Department of Genetics, University of Cambridge, The Wellcome Trust ⁄ Cancer Research UK Gurdon Institute, UK) who PCR amplified it from the cosmid c926 (Wellcome Trust Sanger Institute, Cambridge, UK) and ligated it into Nde1-Xma1 sites of pREP vectors The DNA sequences of all cloned inserts were verified by DNA sequencing Random mutational analysis of S pombe cdc37 GPStm-LS Linker Scanning System (NEB Ltd., Hitchin, UK) (#E7102S) was used to randomly mutate cdc37 by in vitro pentapeptide transposition introducing 15 bp insertions TnsABC* Transposase was used to insert a transposon derived from GPS5 (Transprimer-5 donor plasmid) randomly into target cdc37 fragments previously excised from pPRE81 with Nde1-Xma1 and purified Fragments of cdc37 containing insertions were identified by gel electrophoresis, cut out of the gel and purified, then ligated into the restriction sites Nde1 and Xma1 of pREP81 The transposon transprimer was removed by restriction digestion with Pme1 followed by recircularization of plasmids by DNA ligase Resulting plasmids were sequenced to deter- 4137 Hsp90 binding is not essential for S pombe Cdc37 mine the identity and location of the nucleotide insertion Four mutants obtained contained in-frame insertions resulting in the insertion of five amino acids into the Cdc37 protein Stop codons were inserted by the five codon insertion in four mutants, each adding only 1–3 extra amino acids after the initial insertion site Six mutants, Cdc37(1– 412), Cdc37(1–385), Cdc37(1–321), Cdc37(1–273), Cdc37(1– 264) and Cdc37(1–252), contained insertions that generated a frameshift This resulted in an extra 10, 10, 8, 10, and amino acids, respectively, being added followed by a stop codon Site directed mutagenesis of S pombe cdc37 to generate truncation mutants Truncation mutants were generated by PCR amplification using a mutagenic oligonucleotide at the 3¢ end to introduce a stop codon followed by the restriction site Xma1 Truncations of cdc37 were cloned into Nde1-Xma1 of pREP vectors and sequenced Yeast strains S pombe strains used were ED1090 (ura4-D18 leu1-32), ED1560 (swo1-26 leu1-32) [26] was a kind gift from P Russell (The Scripps Research Institute, La Jolla, CA, USA) ED1537 (swo1 : 2HA-ura4+ ura4-D18 leu1-32), a gift from J Jimenez (Laboratorio Andaluz de Biologia, Universidad Pablo de Olavide, Sevilla, Spain), expresses HA-tagged Hsp90 from the endogenous swo1 (Hsp90) locus The strain was used in biochemical experiments to investigate interactions between Hsp90 and Cdc37 The S pombe strain ED1526 [23] used for plasmid shuffle is deleted for endogenous cdc37 and kept alive by pREP82-cdc37+ We also used temperature-sensitive mutants of cdc37; cdc37-681 [24], cdc37-184, cdc37-13 and cdc37-J (E L Turnbull and P A Fantes, unpublished observations) Medium (minimal medium and yeast extract medium) for standard growth conditions was used as described in [31] S pombe was grown at 32 °C except for temperature-sensitive mutants which were grown at the permissive temperature of 28 °C and the restrictive temperature of 36 °C Assay of mutant cdc37 genes in a cdc37D strain by plasmid shuffle in S pombe ED1526 cdc37::his1+ ade6 ura4-D18 leu1-32 his1-102 pREP82-cdc37+ was transformed with leu+ plasmids (pREP1 or pREP81) expressing wild-type or mutant cdc37 Cells were precultured overnight in MM containing adenine and uracil The attenuance at 600 nm was adjusted to 0.5 and serial dilutions were spotted onto MM + adenine + uracil plates with and without 5¢ fluoro-2¢-deoxyuridine (5FOA) Plates were incubated at 25, 28, 32 and 36 °C for 4138 E L Turnbull et al days 5FOA selects against ura4+ cells but allows ura4) cells to grow Strains carrying a functional cdc37 construct expressed from pREP1 or pREP81 are able to grow in the absence of pREP82-cdc37+ and are 5FOA resistant Strains with a nonfunctional cdc37 construct, cannot lose the pREP82-cdc37+ and are unable to grow on 5FOA medium Recombinant protein purification GST fusion proteins of S pombe Cdc37 were produced in E coli BL21 cells Cells were lysed by sonication in 200 mm Tris [pH 8], mm EDTA and mm EGTA Recombinant protein was absorbed onto Glutathione Sepharosetm 4B (Amersham) in buffer containing 500 mm NaCl, 0.5% (v ⁄ v) NP-40, 50 mm Tris pH 7.6, mm EDTA, mm EGTA and 1· Complete Inhibitors (Roche) Recombinant protein was eluted in 200 mm Tris (pH 8), mm EDTA, mm EGTA and 50 mm glutathione Samples were dialysed against 150 mm NaCl, 20 mm Tris pH 7.6, mm EDTA and mm EGTA Protein concentration was then determined by Bradford Assay (Bio-Rad Laboratories Ltd., Hemel Hempstead, UK) Size exclusion chromatography A culture of S pombe strain ED1537 at A600 of 0.5 was harvested and whole cell lysate extracted using glass beads and vortexing in lysis buffer [150 mm NaCl, 0.5% (v ⁄ v) NP-40, 50 mm Tris (pH 7.5), 10% (w ⁄ v) glycerol, 10· Complete protease inhibitors, 20 mm molybdate] The insoluble debris was removed by centrifugation at °C for 15 at 20 000 g Protein concentration was determined by Bradford Protein Assay (Bio-Rad) Size exclusion chromatography was carried out on either a Superose column (for analytical preparation) or a Sephacryl S-300 HR 26 ⁄ 60 column (for preparative analysis) in SEC buffer [20 mm Hepes (pH 7.9), mm MgCl2, 150 mm KCl, 10% (w ⁄ v) glycerol, mm dithiothreitol] and maintained at °C Fraction samples were then run using SDS ⁄ PAGE for analysis by western blot Immunoprecipitation The high molecular mass fractions containing Cdc37 from the Sephacryl S-300 column were pooled and quantified by Bradford Protein Assay (Bio-Rad) Equal amounts of protein were used in each immunoprecipitation experiment Protein A Sepharosetm beads CL-4B (Amersham) were incubated with anti-(S pombe Cdc37) IgG, anti-HA IgG or anti-rat IgG (Amersham) for 30 at °C Immunoprecipitations were carried out at °C for h Immunoprecipitates were washed four times with mL of lysis buffer and resuspended in 2· SDS gel loading buffer Samples were run on polyacrylamide gels and western blotted using antiS pombe Cdc37 and anti-HA IgGs FEBS Journal 272 (2005) 4129–4140 ª 2005 FEBS E L Turnbull et al Acknowledgements We wish to thank Paul Russell, Juan Jimenez, Chris Prodromou and Kazuhiro Shiozaki for reagents Thank you to Kate Belaya and Paul Westwood for their valuable contributions to this work, Adrian Bird and Robert Klose for use of their equipment and to the University of Edinburgh and BBSRC for funding of this project References Reed SI (1980) The selection of S cerevisiae mutants defective in the start event of cell division Genetics 95, 561–577 Hunter T & Sefton BM (1980) Transforming gene product of Rous sarcoma virus phosphorylates tyrosine Proc Natl Acad Sci USA 77, 1311–1315 MacLean M & Picard D (2003) Cdc37 goes beyond Hsp90 and kinases Cell Stress Chaperones 8, 114–119 Dai K, Kobayashi R & Beach D (1996) Physical interaction of mammalian CDC37 with CDK4 J Biol Chem 271, 22030–22034 Lamphere L, Fiore F, Xu X, Brizuela L, Keezer S, Sardet C, Draetta GF & Gyuris J (1997) Interaction between Cdc37 and Cdk4 in human cells Oncogene 14, 1999–2004 Stepanova L, Leng X, Parker SB & Harper JW (1996) Mammalian p50Cdc37 is a protein kinase-targeting subunit of Hsp90 that binds and stabilizes Cdk4 Genes Dev 10, 1491–1502 Grammatikakis N, Lin JH, Grammatikakis A, Tsichlis PN & Cochran BH (1999) p50 (cdc37) acting in concert with Hsp90 is required for Raf-1 function Mol Cell Biol 19, 1661–1672 Shao J, Irwin A, Hartson SD & Matts RL (2003) Functional dissection of cdc37: characterization of domain structure and amino acid residues critical for protein kinase binding Biochemistry 42, 12577–12588 Boudeau J, Deak M, Lawlor MA, Morrice NA & Alessi DR (2003) Heat-shock protein 90 and Cdc37 interact with LKB1 and regulate its stability Biochem J 370, 849–857 10 Mahony D, Parry DA & Lees E (1998) Active cdk6 complexes are predominantly nuclear and represent only a minority of the cdk6 in T cells Oncogene 16, 603–611 11 Wang H, Goode T, Iakova P, Albrecht JH & Timchenko NA (2002) C ⁄ EBPalpha triggers proteasome-dependent degradation of cdk4 during growth arrest Embo J 21, 930–941 12 Bandhakavi S, McCann RO, Hanna DE & Glover CV (2003) A positive feedback loop between protein kinase CKII and Cdc37 promotes the activity of multiple protein kinases J Biol Chem 278, 2829–2836 FEBS Journal 272 (2005) 4129–4140 ª 2005 FEBS Hsp90 binding is not essential for S pombe Cdc37 13 Miyata Y & Nishida E (2004) CK2 controls multiple protein kinases by phosphorylating a kinase-targeting molecular chaperone, Cdc37 Mol Cell Biol 24, 4065– 4074 14 Shao J, Prince T, Hartson SD & Matts RL (2003) Phosphorylation of serine 13 is required for the proper function of the Hsp90 co-chaperone, Cdc37 J Biol Chem 278, 38117–38120 15 Kimura Y, Rutherford SL, Miyata Y, Yahara I, Freeman BC, Yue L, Morimoto RI & Lindquist S (1997) Cdc37 is a molecular chaperone with specific functions in signal transduction Genes Dev 11, 1775–1785 16 Roe SM, Ali MM, Meyer P, Vaughan CK, Panaretou B, Piper PW, Prodromou C & Pearl LH (2004) The mechanism of Hsp90 regulation by the protein kinasespecific cochaperone p50 (cdc37) Cell 116, 87–98 17 Zhang W, Hirshberg M, McLaughlin SH, Lazar GA, Grossmann JG, Nielsen PR, Sobott F, Robinson CV, Jackson SE & Laue ED (2004) Biochemical and structural studies of the interaction of Cdc37 with Hsp90 J Mol Biol 340, 891–907 18 Siligardi G, Panaretou B, Meyer P, Singh S, Woolfson DN, Piper PW, Pearl LH & Prodromou C (2002) Regulation of Hsp90 ATPase activity by the co-chaperone Cdc37p ⁄ p50cdc37 J Biol Chem 277, 20151–20159 19 McLaughlin SH, Ventouras LA, Lobbezoo B & Jackson SE (2004) Independent ATPase activity of Hsp90 subunits creates a flexible assembly platform J Mol Biol 344, 813–826 20 Farrell A & Morgan DO (2000) Cdc37 promotes the stability of protein kinases Cdc28 and Cak1 Mol Cell Biol 20, 749–754 21 Abbas-Terki T, Donze O & Picard D (2000) The molecular chaperone Cdc37 is required for Ste11 function and pheromone-induced cell cycle arrest FEBS Lett 467, 111–116 22 Millson SH, Truman AW, Wolfram F, King V, Panaretou B, Prodromou C, Pearl LH & Piper PW (2004) Investigating the protein–protein interactions of the yeast Hsp90 chaperone system by two-hybrid analysis: potential uses and limitations of this approach Cell Stress Chaperones 9, 359–368 23 Westwood PK, Martin IV & Fantes PA (2004) Fission yeast Cdc37 is required for multiple cell cycle functions Mol Genet Genomics 271, 82–90 24 Tatebe H & Shiozaki K (2003) Identification of Cdc37 as a novel regulator of the stress-responsive mitogenactivated protein kinase Mol Cell Biol 23, 5132–5142 25 Gerber MR, Farrell A, Deshaies RJ, Herskowitz I & Morgan DO (1995) Cdc37 is required for association of the protein kinase Cdc28 with G1 and mitotic cyclins Proc Natl Acad Sci USA 92, 4651–4655 26 Aligue R, Akhavan-Niak H & Russell P (1994) A role for Hsp90 in cell cycle control: Wee1 tyrosine kinase 4139 Hsp90 binding is not essential for S pombe Cdc37 activity requires interaction with Hsp90 Embo J 13, 6099–6106 27 Lee P, Rao J, Fliss A, Yang E, Garrett S & Caplan AJ (2002) The Cdc37 protein kinase-binding domain is sufficient for protein kinase activity and cell viability J Cell Biol 159, 1051–1059 28 Hartson SD, Irwin AD, Shao J, Scroggins BT, Volk L, Huang W & Matts RL (2000) p50 (cdc37) is a nonexclusive Hsp90 cohort which participates intimately in Hsp90-mediated folding of immature kinase molecules Biochemistry 39, 7631–7644 29 Basi G, Schmid E & Maundrell K (1993) TATA box mutations in the Schizosaccharomyces pombe nmt1 pro- 4140 E L Turnbull et al moter affect transcription efficiency but not the transcription start point or thiamine repressibility Gene 123, 131–136 30 Schutz AR, Giddings TH Jr, Steiner E & Winey M (1997) The yeast CDC37 gene interacts with MPS1 and is required for proper execution of spindle pole body duplication J Cell Biol 136, 969–982 31 Gallagher IM, Alfa CE & Hyams JS (1993) p63cdc13, a B-type cyclin, is associated with both the nucleolar and chromatin domains of the fission yeast nucleus Mol Biol Cell 4, 1087–1096 FEBS Journal 272 (2005) 4129–4140 ª 2005 FEBS ... sustain cellular viability These truncated proteins not contain the postulated Hsp90-binding domain, suggesting that binding of the cochaperone Hsp90 by Cdc37 is not required for cellular viability. .. of S pombe Cdc37, although the human protein may act inefficiently in S pombe blot analysis on GST, S pombe protein extracts and GST -Cdc37 using depleted serum results in a loss of signal against... S pombe Cdc37 to investigate the functional regions of this chaperone protein An interesting new result is that expression of the N-terminal domain of S pombe Cdc37 is sufficient for cellular viability