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Trypanosoma cruzi heat-shock protein 90 can functionally complement yeast
MOLECULAR
Molecular
and Biochemical
Parasitology
70 (1995) 199-202
i%bEMICAL PARASITOLOGY
Short communication
Trypanosoma cruzi heat-shock protein 90 can functionally complement yeast Gaby Palmer a, Jean-Frangois
Louvion a, Randal S. Tibbetts b, David M. Engman b, Didier Picard a,*
dDtpartement de Biologic Cellulaire UniuersitC de Get&e Sciences III, CH-1211 Gen&te 4, Switzerland h Departments of Pathology and Microbiology-Immunology, Northwestern UniL,ersityMedical School, 303 East Chicago AL)enue, Chicago, IL 60611. USA Received 1 November
Keywords: Trypanosoma cruzi; Heat-shock
1994; accepted 6 January
protein 90; Saccharomyces cereoisiae; Genetic complementation
The molecular analysis of parasite proteins would be greatly facilitated if they could be expressed and characterized in the budding yeast Saccharomyces cereuisiae. This would allow yeast genetics to be applied to study a parasite protein of interest, particularly if the protein complemented a deficient yeast strain. We chose heat shock protein 90 (HSP90) as a model protein. Expression of at least one of the two yeast genes, HSP82 and HSC82, which encode two isoforms of this cytosolic protein, is essential for viability [l]. We show that the HSP90 homologue of Trypanosoma cruzi, HSP83 [2], can functionally replace the yeast protein. While the HSP90a and HSP90/? isoforms from the parasite’s human host share this ability [3,4], we have found that the Escherichia coli HSPBO homologue, htpG, is apparently too divergent to complement. HSP90 is an extremely abundant and highly conserved cytosolic protein which has many characteristics of a molecular chaperone [5-71. Its functions are
* Corresponding author. Tel. (41-22) 781-1747; c-mail: PicardQsc2a.unige.ch
1995
702-6813;
Fax (41-22)
0166-6851/95/$09.50 0 1995 Elscvier Science B.V. All rights reserved SSDI 0166-6851(95)00007-O
poorly understood both in parasites and in other organisms. In trypanosomatid parasites, HSP90 is strongly induced when the parasite is subjected to a ‘heat-shock’ during passage from the insect vector to the warm-blooded vertebrate host. High heat inducibility of the HSP83 gene appears to correlate with vertebrate infectivity and virulence [8,9]. Moreover, immunological screening of serum from patients suffering from trypanosomatid-induced Chagas’ disease and leishmaniasis has demonstrated that HSP83 is an immunodominant antigen [lo-121. For genetic complementation experiments we took advantage of a haploid yeast strain whose two HSP90 genes, HSP82 and HSC82, had been disrupted by insertion of the LEU2 gene [l]. Essential HSP90 function was provided by an episomal expression vector. In this particular strain, HHl-KAT6, viability is ensured by the galactose-inducible expression of human HSP90P. Thus, human HSP90/3, as previously mentioned [3], and human HSP90a [4] are able to complement yeast. To test other HSP90 proteins, appropriate coding sequences were placed under the control of a constitutive promoter on an episomal vector and introduced into yeast. The T.
200
G. Palmer et al. /Molecular
and Biochemical Parasitology 70 (1995) 199-202
cruzi and E. coli HSP90 homologues were expressed both as wild-type proteins and as proteins containing an N-terminal flu epitope tag for immunologic detection. As long as such transformants are cultured on galactose as a carbon source both the human HSP90P and the additional exogenous HSP90 protein are expressed. Complementation by the constitutively expressed exogenous HSP90 homologue can be evaluated by turning off expression of the human HSP90P by switching to glucose as a carbon source.
I I
Table 1 Genetic complementation
of a HSP90-deficient
yeast strain ’
Protein expressed b
Flu tag ’
Complementation d
None S. cereuisiae HSP82
NA no
no
NA
T. cruzi HSP83
yes no
yes yes yes yes yes no no
yes yes ND
Human HSP90P E. coli htpG
yes no yes no
Expression confirmed ’
yes yes yes ND
NA, not applicable; ND, not done. a In haploid yeast strain HID-KAT6 (provided by S.L. Lindquist), the HSP82 and HSC82 genes are disrupted by insertion of the LEU2 marker [l] resulting in the following genotype: Mata ade2 leu2 ura3 his3 trpl hsc82::LEU2 hsp82::LEU2. Essential HSP90 function is provided by the galactose-inducible human HSP90p expression vector pGall-hhsp90. pGall-hhsp90 was constructed by inserting the GALI-GAL10 promoter region as a BamHIEcoRI fragment and HSP90P cDNA sequences from plasmed pKNl-3 (a gift from N.F. Rebbe) into yeast shuttle vector pUN90
[ml. b Constitutive
expression of all but the human HSP90P homologue was achieved with plasmid p2IJ. It contains the 2~ yeast replicon and the glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter for high level expression in the URA3-containing plasmid pRS306 [17]. p2U is a high-copy number yeast plasmid and was constructed analogously to the previously described plasmid p2HG [3]. The coding regions for S. cereuisiae HSP82, T. cruzi HSP83 and E. coli htpG were excised from plasmids p’IT8 (a gift from S.L. Lindquistl, pGEX-hsp83 [18] and pBJ2 1191, respectively. ’ The influenza haemagglutinin epitope (amino-acid sequence -YPYDVPDYA-1 was used as an N-terminal tag for recognition ,“y mouse monoclonal antibody 12CAS (Berkeley Antibody). Genetic complementation of strain HHl-KAT6. ‘yes’ indicates growth on glucose as a carbon source in the absence of the galactose-induced human HSP90P. e ‘yes’ indicates that expression of HSP90 homologues was confirmed by immunoblot analysis (see Fig. 1; and data not shown).
anti-f/u
I
Fig. 1 Immunoblot analysis of yeast strains expressing HSPBO homologues. (A) Analysis of control strains which are isogenic except for the presence of an expression vector for either yeast HSP82 or human HSP90/3 (strain background is HHl-KAT4). The immunoblotting experiment shows the total absence of yeast HSP82/HSC82 in strain HHl-KAT6. Yeast HSP82 was revealed with the polyclonal rabbit antiserum 4-l-8 raised against a yeastspecific C-terminal peptide (anti-HSP82); this antiserum recognizes both HSP82 and HSC82. Note that HSP90P is expressed from a galactose-inducible promoter; it is only expressed in cells grown on galactose (gal) but not on glucose @cl (see, for example, the second and third lane in panel Bl. (B) Top panel: Immunoblotting for human HSP90P. Its presence was revealed by probing with a polyclonal rabbit antiserum which was raised against murine HSP90 (anti-HSP90) and is specific for mammalian HSP90. Bottom panel: T. cruzi HSP83 and E. coli htpG, which were tagged with the flu epitope, were probed with a monoclonal antibody against the flu epitope tag (anti-flu). Note that the latter two proteins are constitutively expressed and that the strain expressing E. coli htpG can only grow when the human HSP90/3 is coexpressed (i.e., on galactosel. Arrows indicate the major protein species (E. coli htpG is about 9-kDa smaller than T. cnui HSP83). Yeast cells were grown in appropriate selection medium containing either 2% glucose or 2% galactose. Extracts were prepared at 4°C by breaking cells with glass beads in a low-salt buffer. Equal amounts of total protein were loaded for a given set of lanes.
Table 1 summarizes the results from this type of experiment. It shows that the T. crwi HSP83, with or without the flu tag, is able to substitute for the yeast HSP90 proteins. Over a wide range of temperatures (25--37°C) growth of these strains is indistinguishable from that of control wild-type strains or of strains expressing yeast HSP82 from a 2~ plasmid. In contrast, the E. coli HSP90 homologue htpG cannot support the growth of a HSP82/HSC82-deficient yeast strain. Using specific antibodies in immunoblotting experiments we confirmed the expression of the various HSP90 proteins in the different
G. Palmer et al. /Molecular
and Biochemical Parasitology
strains (Fig. 1). Upon shifting the cells to glucose, human HSP90P was indeed replaced by T. cruzi HSP83, thus confirming that the T. cruzi protein can function in yeast in the absence of any other HSP90 homologue, including the yeast HSP82 and HSC82 proteins. Sequence conservation of the T. cruzi, human and yeast HSP90 proteins is 63% (identity) for all three pair-wise combinations whereas E. coli htpG, which is unable to complement yeast, is only about 42% identical to the three eukaryotic proteins. HSP90 sequence conservation is considerably higher ( 2 84% identity) within the trypanosomatid family. These proteins also form a separate group with respect to the extremely conserved C-terminal HSP90 pentapeptide motif MEEVD. Three of the four known trypanosomatid HSP90 sequences (T. cruzi, Leishmania amazonensis and L. donovani, but not that of T. brucei), are the only known eukaryotic HSP90 proteins which have either Q or L instead of E in the third position. While the significance of this divergence, as well as the function of this conserved tail have yet to be determined, our results clearly indicate that the highly conserved E in the third position is not required in yeast. Thus, we confirm and extend previous reports on expression of trypanosomatid proteins in yeast. Yeast has been used to overexpress the bifunctional thymidylate synthase-dihydrofolate reductase from L. major [13]. Furthermore, it has been demonstrated that expression site associated genes encoding adenylate cyclase from T. brucei and T. equiperdum can functionally replace the S. cerevisiae homologue CH’U [14,15]. Yeast genetics can now be applied to the functional dissection of several proteins including HSP83 from T. cruzi, and it may well be more generally useful to study proteins/genes from parasites which have more limited genetics.
Acknowledgements We are greatly indebted to S.L. Lindquist for providing several yeast strains including HHl-KAT6, plasmids and antiserum against yeast HSP90. We are also grateful to N.F. Rebbe and B. Craig for providing the human HSP90/3 and E. coli htpG plasmids,
70 (1995) 199-202
201
respectively. This work was supported by the Swiss National Science Foundation and the Canton de Genbve.
References [l] Borkovich, K.A., Farrelly, F.W., Finkelstein, D.B., Taulien, J. and Lindquist, S. (1989) hsp82 is an essential protein that is required in higher concentrations for growth of cells at higher temperatures. Mol. Cell. Biol. 9, 3919-3930. [2] Dragon, E., Sias, S.R., Kato, E.A. and Gabe, J.D. (1987) The genome of Trypanosoma cruzi contains a constitutively expressed, tandemly arranged multicopy gene homologous to a major heat shock protein. Mol. Cell. Biol. 7, 1271-1275. [3] Picard, D., Khursheed, B., Garabedian, M.J., Fortin, M.G., Lindquist, S. and Yamamoto, K.R. (1990) Reduced levels of hsp90 compromise steroid receptor action in rlioo. Nature 348, 166-168. [4] Minami, Y., Kimura, Y., Kawasaki, H., Suzuki, K. and Yahara, I. (1994) The carboxy-terminal region of mammalian HSP90 is required for its dimerization and function in ko. Mol. Cell. Biol. 14, 1459-1464. [5] Georgopoulos, C. and Welch, W.J. (1993) Role of the major heat shock proteins as molecular chaperones. Annu. Rev. Cell Biol. 9, 601-634. [6] Picard, D. (1993) Steroid-binding domains for regulating the functions of heterologous proteins in cis. Trends Cell Biol. 3, 278-280. [7] Jakob, U. and Buchner, J. (1994) Assisting spontaneity: the role of Hsp90 and small Hsps as molecular chaperones. Trends Biochem. Sci. 19, 205-211. [8] Van der Ploeg, L.H., Giannini, S.H. and Cantor, C.R. (1985) Heat shock genes: regulatory role for differentiation in parasitic protozoa. Science 228, 1443-1446. [9] Shapira, M., McEwen, J.G. and Jaffe, C.L. (1988) Temperature effects on molecular processes which lead to stage differentiation on Leishmania. EMBO J. 7, 289.5-2901. [lo] Engman, D.M., Dragon, E.A. and Donelson, J.E. (1990) Human humoral immunity to hsp70 during T. cruzi infection. J. Immunol. 144, 3987-3991. [ll] Nafziger, D.A., Recinos, R.F., Hunter, C.A. and Donelson, J.E. (1991) Patients infected with Leishmania donovani chagasi can have antibodies that recognize heat shock and acidic ribosomal proteins of Trypanosoma cruzi. Mol. Biochem. Parasitol. 49, 325-328. [12] de Andrade, CR., Kirchhoff, L.V., Donelson, J.E. and Otsu. K. (1992) Recombinant Leishmania Hsp90 and Hsp70 are recognized by sera from visceral leishmaniasis patients but not Chagas’ disease patients. J. Clin. Microbial. 30, 330-335. 1131 Grumont, R., Sirawaraporn, W. and Santi, D.V. (1988) Heterologous expression of the bifunctional thymidylate synthase-dihydrofolate reductase from Leishmania major. Biochemistry 27, 3776-3784. [14] Ross, D.T., Raibaud, A., Florent, I.C., Sather, S., Gross, M.K., Storm, D.R. and Eisen, H. (1991) The trypanosome VSG expression site encodes adenylate cyclase and a
202
G. Palmer et al. /Molecular
and Biochemical
leucine-rich putative regulatory gene. EMBO J. 10, 20472053. [15] Paindavoine, P., Rolin, S., Van Assel, S.. Geuskens, M., Jauniaux, J.C., Dinsart, C., Huet, G. and Pays, E. (1992) A gene from the variant surface glycoprotein expression site encodes one of several transmembrane adenylate cyclases located on the flagellum of Trypanosoma brucei. Mol. Cell. Biol. 12, 1218-1225. [16] Elledge, S.J. and Davis, R.W. (1988) A family of versatile centromeric vectors designed for use in the sectoring-shuffle mutagenesis assay in Saccharomyces cerevisiae. Gene 70. 303-312.
Parasitology
70 (1995) 199-202
[17] Sikorski, R.S. and Hieter, P. (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cereuisiae. Genetics 122, 19-27. [18] Nadeau, K., Sullivan, M.A., Bradley, M., Engman, D.M. and Walsh, C.T. (1992) 83-kilodalton heat shock proteins of trypanosomes are potent peptide-stimulated ATPases. Protein Sci. 1, 970-979. [19] Bardwell, J.C. and Craig, E.A. (1987) Eukaryotic Mr 83.000 heat shock protein has a homologue in Escherichia coli. Proc. Natl. Acad. Sci. USA 84. 5177-5181.
Molecular
and Biochemical
Parasitology
70 (1995) 199-202
i%bEMICAL PARASITOLOGY
Short communication
Trypanosoma cruzi heat-shock protein 90 can functionally complement yeast Gaby Palmer a, Jean-Frangois
Louvion a, Randal S. Tibbetts b, David M. Engman b, Didier Picard a,*
dDtpartement de Biologic Cellulaire UniuersitC de Get&e Sciences III, CH-1211 Gen&te 4, Switzerland h Departments of Pathology and Microbiology-Immunology, Northwestern UniL,ersityMedical School, 303 East Chicago AL)enue, Chicago, IL 60611. USA Received 1 November
Keywords: Trypanosoma cruzi; Heat-shock
1994; accepted 6 January
protein 90; Saccharomyces cereoisiae; Genetic complementation
The molecular analysis of parasite proteins would be greatly facilitated if they could be expressed and characterized in the budding yeast Saccharomyces cereuisiae. This would allow yeast genetics to be applied to study a parasite protein of interest, particularly if the protein complemented a deficient yeast strain. We chose heat shock protein 90 (HSP90) as a model protein. Expression of at least one of the two yeast genes, HSP82 and HSC82, which encode two isoforms of this cytosolic protein, is essential for viability [l]. We show that the HSP90 homologue of Trypanosoma cruzi, HSP83 [2], can functionally replace the yeast protein. While the HSP90a and HSP90/? isoforms from the parasite’s human host share this ability [3,4], we have found that the Escherichia coli HSPBO homologue, htpG, is apparently too divergent to complement. HSP90 is an extremely abundant and highly conserved cytosolic protein which has many characteristics of a molecular chaperone [5-71. Its functions are
* Corresponding author. Tel. (41-22) 781-1747; c-mail: PicardQsc2a.unige.ch
1995
702-6813;
Fax (41-22)
0166-6851/95/$09.50 0 1995 Elscvier Science B.V. All rights reserved SSDI 0166-6851(95)00007-O
poorly understood both in parasites and in other organisms. In trypanosomatid parasites, HSP90 is strongly induced when the parasite is subjected to a ‘heat-shock’ during passage from the insect vector to the warm-blooded vertebrate host. High heat inducibility of the HSP83 gene appears to correlate with vertebrate infectivity and virulence [8,9]. Moreover, immunological screening of serum from patients suffering from trypanosomatid-induced Chagas’ disease and leishmaniasis has demonstrated that HSP83 is an immunodominant antigen [lo-121. For genetic complementation experiments we took advantage of a haploid yeast strain whose two HSP90 genes, HSP82 and HSC82, had been disrupted by insertion of the LEU2 gene [l]. Essential HSP90 function was provided by an episomal expression vector. In this particular strain, HHl-KAT6, viability is ensured by the galactose-inducible expression of human HSP90P. Thus, human HSP90/3, as previously mentioned [3], and human HSP90a [4] are able to complement yeast. To test other HSP90 proteins, appropriate coding sequences were placed under the control of a constitutive promoter on an episomal vector and introduced into yeast. The T.
200
G. Palmer et al. /Molecular
and Biochemical Parasitology 70 (1995) 199-202
cruzi and E. coli HSP90 homologues were expressed both as wild-type proteins and as proteins containing an N-terminal flu epitope tag for immunologic detection. As long as such transformants are cultured on galactose as a carbon source both the human HSP90P and the additional exogenous HSP90 protein are expressed. Complementation by the constitutively expressed exogenous HSP90 homologue can be evaluated by turning off expression of the human HSP90P by switching to glucose as a carbon source.
I I
Table 1 Genetic complementation
of a HSP90-deficient
yeast strain ’
Protein expressed b
Flu tag ’
Complementation d
None S. cereuisiae HSP82
NA no
no
NA
T. cruzi HSP83
yes no
yes yes yes yes yes no no
yes yes ND
Human HSP90P E. coli htpG
yes no yes no
Expression confirmed ’
yes yes yes ND
NA, not applicable; ND, not done. a In haploid yeast strain HID-KAT6 (provided by S.L. Lindquist), the HSP82 and HSC82 genes are disrupted by insertion of the LEU2 marker [l] resulting in the following genotype: Mata ade2 leu2 ura3 his3 trpl hsc82::LEU2 hsp82::LEU2. Essential HSP90 function is provided by the galactose-inducible human HSP90p expression vector pGall-hhsp90. pGall-hhsp90 was constructed by inserting the GALI-GAL10 promoter region as a BamHIEcoRI fragment and HSP90P cDNA sequences from plasmed pKNl-3 (a gift from N.F. Rebbe) into yeast shuttle vector pUN90
[ml. b Constitutive
expression of all but the human HSP90P homologue was achieved with plasmid p2IJ. It contains the 2~ yeast replicon and the glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter for high level expression in the URA3-containing plasmid pRS306 [17]. p2U is a high-copy number yeast plasmid and was constructed analogously to the previously described plasmid p2HG [3]. The coding regions for S. cereuisiae HSP82, T. cruzi HSP83 and E. coli htpG were excised from plasmids p’IT8 (a gift from S.L. Lindquistl, pGEX-hsp83 [18] and pBJ2 1191, respectively. ’ The influenza haemagglutinin epitope (amino-acid sequence -YPYDVPDYA-1 was used as an N-terminal tag for recognition ,“y mouse monoclonal antibody 12CAS (Berkeley Antibody). Genetic complementation of strain HHl-KAT6. ‘yes’ indicates growth on glucose as a carbon source in the absence of the galactose-induced human HSP90P. e ‘yes’ indicates that expression of HSP90 homologues was confirmed by immunoblot analysis (see Fig. 1; and data not shown).
anti-f/u
I
Fig. 1 Immunoblot analysis of yeast strains expressing HSPBO homologues. (A) Analysis of control strains which are isogenic except for the presence of an expression vector for either yeast HSP82 or human HSP90/3 (strain background is HHl-KAT4). The immunoblotting experiment shows the total absence of yeast HSP82/HSC82 in strain HHl-KAT6. Yeast HSP82 was revealed with the polyclonal rabbit antiserum 4-l-8 raised against a yeastspecific C-terminal peptide (anti-HSP82); this antiserum recognizes both HSP82 and HSC82. Note that HSP90P is expressed from a galactose-inducible promoter; it is only expressed in cells grown on galactose (gal) but not on glucose @cl (see, for example, the second and third lane in panel Bl. (B) Top panel: Immunoblotting for human HSP90P. Its presence was revealed by probing with a polyclonal rabbit antiserum which was raised against murine HSP90 (anti-HSP90) and is specific for mammalian HSP90. Bottom panel: T. cruzi HSP83 and E. coli htpG, which were tagged with the flu epitope, were probed with a monoclonal antibody against the flu epitope tag (anti-flu). Note that the latter two proteins are constitutively expressed and that the strain expressing E. coli htpG can only grow when the human HSP90/3 is coexpressed (i.e., on galactosel. Arrows indicate the major protein species (E. coli htpG is about 9-kDa smaller than T. cnui HSP83). Yeast cells were grown in appropriate selection medium containing either 2% glucose or 2% galactose. Extracts were prepared at 4°C by breaking cells with glass beads in a low-salt buffer. Equal amounts of total protein were loaded for a given set of lanes.
Table 1 summarizes the results from this type of experiment. It shows that the T. crwi HSP83, with or without the flu tag, is able to substitute for the yeast HSP90 proteins. Over a wide range of temperatures (25--37°C) growth of these strains is indistinguishable from that of control wild-type strains or of strains expressing yeast HSP82 from a 2~ plasmid. In contrast, the E. coli HSP90 homologue htpG cannot support the growth of a HSP82/HSC82-deficient yeast strain. Using specific antibodies in immunoblotting experiments we confirmed the expression of the various HSP90 proteins in the different
G. Palmer et al. /Molecular
and Biochemical Parasitology
strains (Fig. 1). Upon shifting the cells to glucose, human HSP90P was indeed replaced by T. cruzi HSP83, thus confirming that the T. cruzi protein can function in yeast in the absence of any other HSP90 homologue, including the yeast HSP82 and HSC82 proteins. Sequence conservation of the T. cruzi, human and yeast HSP90 proteins is 63% (identity) for all three pair-wise combinations whereas E. coli htpG, which is unable to complement yeast, is only about 42% identical to the three eukaryotic proteins. HSP90 sequence conservation is considerably higher ( 2 84% identity) within the trypanosomatid family. These proteins also form a separate group with respect to the extremely conserved C-terminal HSP90 pentapeptide motif MEEVD. Three of the four known trypanosomatid HSP90 sequences (T. cruzi, Leishmania amazonensis and L. donovani, but not that of T. brucei), are the only known eukaryotic HSP90 proteins which have either Q or L instead of E in the third position. While the significance of this divergence, as well as the function of this conserved tail have yet to be determined, our results clearly indicate that the highly conserved E in the third position is not required in yeast. Thus, we confirm and extend previous reports on expression of trypanosomatid proteins in yeast. Yeast has been used to overexpress the bifunctional thymidylate synthase-dihydrofolate reductase from L. major [13]. Furthermore, it has been demonstrated that expression site associated genes encoding adenylate cyclase from T. brucei and T. equiperdum can functionally replace the S. cerevisiae homologue CH’U [14,15]. Yeast genetics can now be applied to the functional dissection of several proteins including HSP83 from T. cruzi, and it may well be more generally useful to study proteins/genes from parasites which have more limited genetics.
Acknowledgements We are greatly indebted to S.L. Lindquist for providing several yeast strains including HHl-KAT6, plasmids and antiserum against yeast HSP90. We are also grateful to N.F. Rebbe and B. Craig for providing the human HSP90/3 and E. coli htpG plasmids,
70 (1995) 199-202
201
respectively. This work was supported by the Swiss National Science Foundation and the Canton de Genbve.
References [l] Borkovich, K.A., Farrelly, F.W., Finkelstein, D.B., Taulien, J. and Lindquist, S. (1989) hsp82 is an essential protein that is required in higher concentrations for growth of cells at higher temperatures. Mol. Cell. Biol. 9, 3919-3930. [2] Dragon, E., Sias, S.R., Kato, E.A. and Gabe, J.D. (1987) The genome of Trypanosoma cruzi contains a constitutively expressed, tandemly arranged multicopy gene homologous to a major heat shock protein. Mol. Cell. Biol. 7, 1271-1275. [3] Picard, D., Khursheed, B., Garabedian, M.J., Fortin, M.G., Lindquist, S. and Yamamoto, K.R. (1990) Reduced levels of hsp90 compromise steroid receptor action in rlioo. Nature 348, 166-168. [4] Minami, Y., Kimura, Y., Kawasaki, H., Suzuki, K. and Yahara, I. (1994) The carboxy-terminal region of mammalian HSP90 is required for its dimerization and function in ko. Mol. Cell. Biol. 14, 1459-1464. [5] Georgopoulos, C. and Welch, W.J. (1993) Role of the major heat shock proteins as molecular chaperones. Annu. Rev. Cell Biol. 9, 601-634. [6] Picard, D. (1993) Steroid-binding domains for regulating the functions of heterologous proteins in cis. Trends Cell Biol. 3, 278-280. [7] Jakob, U. and Buchner, J. (1994) Assisting spontaneity: the role of Hsp90 and small Hsps as molecular chaperones. Trends Biochem. Sci. 19, 205-211. [8] Van der Ploeg, L.H., Giannini, S.H. and Cantor, C.R. (1985) Heat shock genes: regulatory role for differentiation in parasitic protozoa. Science 228, 1443-1446. [9] Shapira, M., McEwen, J.G. and Jaffe, C.L. (1988) Temperature effects on molecular processes which lead to stage differentiation on Leishmania. EMBO J. 7, 289.5-2901. [lo] Engman, D.M., Dragon, E.A. and Donelson, J.E. (1990) Human humoral immunity to hsp70 during T. cruzi infection. J. Immunol. 144, 3987-3991. [ll] Nafziger, D.A., Recinos, R.F., Hunter, C.A. and Donelson, J.E. (1991) Patients infected with Leishmania donovani chagasi can have antibodies that recognize heat shock and acidic ribosomal proteins of Trypanosoma cruzi. Mol. Biochem. Parasitol. 49, 325-328. [12] de Andrade, CR., Kirchhoff, L.V., Donelson, J.E. and Otsu. K. (1992) Recombinant Leishmania Hsp90 and Hsp70 are recognized by sera from visceral leishmaniasis patients but not Chagas’ disease patients. J. Clin. Microbial. 30, 330-335. 1131 Grumont, R., Sirawaraporn, W. and Santi, D.V. (1988) Heterologous expression of the bifunctional thymidylate synthase-dihydrofolate reductase from Leishmania major. Biochemistry 27, 3776-3784. [14] Ross, D.T., Raibaud, A., Florent, I.C., Sather, S., Gross, M.K., Storm, D.R. and Eisen, H. (1991) The trypanosome VSG expression site encodes adenylate cyclase and a
202
G. Palmer et al. /Molecular
and Biochemical
leucine-rich putative regulatory gene. EMBO J. 10, 20472053. [15] Paindavoine, P., Rolin, S., Van Assel, S.. Geuskens, M., Jauniaux, J.C., Dinsart, C., Huet, G. and Pays, E. (1992) A gene from the variant surface glycoprotein expression site encodes one of several transmembrane adenylate cyclases located on the flagellum of Trypanosoma brucei. Mol. Cell. Biol. 12, 1218-1225. [16] Elledge, S.J. and Davis, R.W. (1988) A family of versatile centromeric vectors designed for use in the sectoring-shuffle mutagenesis assay in Saccharomyces cerevisiae. Gene 70. 303-312.
Parasitology
70 (1995) 199-202
[17] Sikorski, R.S. and Hieter, P. (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cereuisiae. Genetics 122, 19-27. [18] Nadeau, K., Sullivan, M.A., Bradley, M., Engman, D.M. and Walsh, C.T. (1992) 83-kilodalton heat shock proteins of trypanosomes are potent peptide-stimulated ATPases. Protein Sci. 1, 970-979. [19] Bardwell, J.C. and Craig, E.A. (1987) Eukaryotic Mr 83.000 heat shock protein has a homologue in Escherichia coli. Proc. Natl. Acad. Sci. USA 84. 5177-5181.