Hmo1p, a High Mobility Group 1/2 Homolog, Genetically and Physically Interacts With the Yeast FKBP12 Prolyl Isomerase

Corresponding author: Joseph Heitman, 322 Carl Bldg., Research Dr., Box 3546 Duke University Medical Center, Durham, NC 27710., heitm001{at}mc.duke.edu (E-mail)

Communicating editor: M. D. ROSE

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The immunosuppressive drugs FK506 and rapamycin bind to the cellular protein FKBP12, and the resulting FKBP12-drug complexes inhibit signal transduction. FKBP12 is a ubiquitous, highly conserved, abundant enzyme that catalyzes a rate-limiting step in protein folding: peptidyl-prolyl cis-trans isomerization. However, FKBP12 is dispensible for viability in both yeast and mice, and therefore does not play an essential role in protein folding. The functions of FKBP12 may involve interactions with a number of partner proteins, and a few proteins that interact with FKBP12 in the absence of FK506 or rapamycin have been identified, including the ryanodine receptor, aspartokinase, and the type II TGF-ß receptor; however, none of these are conserved from yeast to humans. To identify other targets and functions of FKBP12, we have screened for mutations that are synthetically lethal with an FKBP12 mutation in yeast. We find that mutations in HMO1, which encodes a high mobility group 1/2 homolog, are synthetically lethal with mutations in the yeast FPR1 gene encoding FKBP12. hmo1 and fpr1 mutants share two phenotypes: an increased rate of plasmid loss and slow growth. In addition, Hmo1p and FKBP12 physically interact in FKBP12 affinity chromatography experiments, and two-hybrid experiments suggest that FKBP12 regulates Hmo1p-Hmo1p or Hmo1p-DNA interactions. Because HMG1/2 proteins are conserved from yeast to humans, our findings suggest that FKBP12-HMG1/2 interactions could represent the first conserved function of FKBP12 other than mediating FK506 and rapamycin actions.

FKBP12 is a highly conserved protein that serves as the intracellular receptor for the immunosuppressive drugs FK506 and rapamycin. Each drug-FKBP12 complex functions in a unique manner; the FKBP12-FK506 complex inhibits the calcium-calmodulin-dependent serine-threonine phosphatase calcineurin, while the FKBP12-rapamycin complex inhibits mTOR (RAFT/FRAP; HEITMAN et al. 1991A , HEITMAN et al. 1991B ; LIU et al. 1991 ; SABATINI et al. 1994 ; SABERS et al. 1995 ). FKBP12, calcineurin, and TOR are all conserved from yeast to man, and the mechanisms of FK506 and rapamycin immunosuppression and antimicrobial action are essentially identical. FKBP12 also exhibits peptidyl-prolyl isomerase activity in vitro, a rate-limiting step in protein folding (KOLTIN et al. 1991 ; WIEDERRECHT et al. 1991 ).

Several proteins that interact with mammalian FKBP12 have now been identified. FKBP12 associates with several large protein complexes, including the multi-drug resistance pump and the ryanodine, inositol trisphosphate (IP₃), and the type I TGF-ß receptor (JAYARAMAN et al. 1992 ; TIMERMAN et al. 1993 ; BRILLANTES et al. 1994 ; WANG et al. 1994 ; CAMERON et al. 1995A , CAMERON et al. 1995B ; HEMENWAY and HEITMAN 1996 ; WANG et al. 1996B ; BASSING et al. 1998 ). In the case of the ryanodine and IP₃ receptors, it is thought that FKBP12 may anchor calcineurin to these substrates, resulting in calcium-regulated dephosphorylation (CAMERON et al. 1995A , CAMERON et al. 1995B ). FKBP12-deficient mice have recently been described (SHOU et al. 1998 ). These knock-out mice have very poor survival rates and suffer from several heart defects that mimic a human congenital heart disorder. In vitro studies with FKBP12-deficient cells indicated that FKBP12 is important in the modulation of calcium release activity of both the cardiac and skeletal ryanodine receptors, in accord with the previous biochemical studies described above. These FKBP12-deficient cells, however, had no defects in TGF-ß signaling; thus, the physiological relevance of the FKBP12-TGF-ß receptor interaction is still unclear (BASSING et al. 1998 ). In addition, FKBP12 interacts with FAP48, a protein of unknown function, and the transcription factor YY1, though the physiological role of these interactions has not yet been determined (YANG et al. 1995 ; CHAMBRAUD et al. 1996 ).

Mammalian and yeast FKBP12 are remarkably conserved; they share 54% identity and have superimposable crystal structures (HEITMAN et al. 1991A , HEITMAN et al. 1991B ; ROTONDA et al. 1993 ). Interestingly, none of the targets of mFKBP12 identified thus far are found in Saccharomyces cerevisiae. Other proteins that physically associate with yeast FKBP12 have been identified. First, a weak physical interaction between FKBP12 and calcineurin in the absence of FK506 has been demonstrated in yeast, and mutations in fpr1, the gene encoding FKBP12, affect processes that involve calcineurin (CARDENAS et al. 1994 ). In addition, aspartokinase was identified in a two-hybrid screen for yFKBP12-interacting proteins (ALARCON and HEITMAN 1997 ). Aspartokinase is an enzyme in the biosynthetic pathway of methionine and threonine. The activity of aspartokinase is feedback-inhibited by products of the pathway, and FKBP12 regulates this feedback inhibition.

Here we identify another target of yeast FKBP12, the high mobility group 1/2 homolog HMO1, by screening for mutations that are synthetically lethal with fpr1 mutations. High mobility group (HMG) proteins are nonhistone, chromatin-binding proteins. HMG proteins are classified into three types [HMG1/2, HMG14/17, HMG-I(Y)] on the basis of molecular weight, DNA binding specificity, and sequence motif (LANDSMAN and BUSTIN 1993 ). HMG1/2 proteins have two copies of an HMG box, an 80-amino-acid motif that has DNA-binding activity. There are two types of HMG box-containing proteins: transcription factors and chromatin-associated proteins. The chromatin-associated class binds DNA with little sequence specificity, instead recognizing DNA structure such as kinks, cruciforms, or single-stranded DNA. These chromatin-associated HMG proteins contain two HMG boxes and an acidic carboxy terminus. HMG1/2 proteins have been implicated in the regulation of chromatin structure, replication, and transcription, but the in vivo functions of these proteins are still unclear (TREMETHICK and MOLLOY 1986 , TREMETHICK and MOLLOY 1988 ).

In yeast, there are three proteins that contain two HMG boxes, one of which is mitochondrial. An HMG-1/2 protein in yeast, Hmo1p, was recently identified as a predominantly nuclear protein that can bind DNA (LU et al. 1996 ). hmo1 mutants exhibit a slow growth defect and an increased rate of plasmid loss, and chromatin isolated from these mutants is hypersensitive to nuclease digestion. In addition to Hmo1p, yeast express a related protein, Hmo2p (LU et al. 1996 ).

In this study, we conducted a genetic screen for mutations that exhibit synthetic lethality with mutations in FPR1, which encodes yeast FKBP12. A single FKBP12 synthetic lethal mutation was identified and shown to be allelic with HMO1, which encodes a yeast HMG1/2 protein. We confirm that null mutations in fpr1 and hmo1 are synthetically lethal. In addition, we show that Hmo1p and FKBP12 physically interact in vitro, and hmo1 and fpr1 mutants share common phenotypes. Finally, by two-hybrid analysis, we find that Hmo1p can dimerize, and dimerization is increased in the absence of FKBP12. Taken together, our findings suggest that FKBP12 regulates assembly of Hmo1p-Hmo1p complexes or Hmo1p-DNA interactions. Because HMG1/2 proteins are the first targets of FKBP12 that are conserved from yeast to humans, a conserved role for FKBP12 in regulating HMG1/2 protein function could explain the remarkable conservation of FKBP12.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Media and strains:
Media were prepared as described (SHERMAN 1991 ). Strains are described in Table 1.

Strain construction:
Yeast transformation and one-step gene disruption were as described (ROTHSTEIN 1991 ; GIETZ et al. 1995 ). The fpr1::hisG strains (KDY99 and KDY100) were engineered as follows. Plasmid pNKY51 (ALANI et al. 1987 ) was cleaved with BglII to release the URA3 gene flanked by hisG repeats. This fragment was ligated into the corresponding site of plasmid pYJH33 (HEITMAN et al. 1991B ), which contains a BglII site in the middle of the FPR1 open reading frame, to yield plasmid pKDd5. To disrupt fpr1 in the genome, pKDd5 was digested with EcoRI, which releases the fpr1::hisG-URA3-hisG fragment that was used to transform strains Y382 and Y388 (BENDER and PRINGLE 1991 ). Ura⁺, rapamycin-resistant transformants were selected, grown nonselectively, and then plated on 5-FOA media to select for loss of the URA3 gene. 5-FOA-resistant colonies were checked again for rapamycin resistance, indicating an fpr1::hisG genotype.

The HMO1 open reading frame was disrupted with the G418-resistance gene by PCR as described (WACH et al. 1994 ; LORENZ et al. 1995 ), using the following primers: (5'-CCTTCTGTCAAATTGAAGTCCGCCAAAGACTCCCTCGTCTCCCAGCTGAAGCTTCGTACTG-3') and (5'-GTTTTGCTTCCTTCTTTTTCTCTAAAATGTACATAGTTTTAGGCATAGGGCCACTAGTGGATCTG-3'). The resulting PCR product was used to transform a JK9-3da/ diploid strain. G418-resistant transformants were selected, and HMO1/hmo1 heterozygous mutants were confirmed by PCR. These heterozygous mutants were sporulated and dissected. Tetrads exhibited 2 G418-resistant:2 G418-sensitive segregation, and slow growth segregated with the G418-resistance phenotype, as expected.

Mutagenesis and synthetic lethal screen:
The colony sectoring assay has been previously described (KOSHLAND et al. 1985 ; BENDER and PRINGLE 1991 ). The ADE3 URA3 FPR1 plasmid (pKDw7) used for the screen was constructed by ligating a SmaI/SalI ADE3 fragment from plasmid LDB48 (T. Davis; KOSHLAND et al. 1985 ) into the corresponding sites of pYJH23, a 2µ URA3 FPR1 plasmid (HEITMAN et al. 1991B ).

Strains KDY99 and KDY100, both containing the ADE3 URA3 FPR1 plasmid pKDw7, were mutagenized with UV to 40% survival using 150 mJ of energy from a Stratalinker (Stratagene, La Jolla, CA). About 1000 cells were plated on each YPD plate; 28,170 total colonies were screened. After 5 days of incubation at 30° in the dark to prevent light-dependent repair of UV lesions, colonies were replica-plated to YPD + rapamycin (0.1 µg/ml), 5-FOA, and YPD medium. Eighteen nonsectoring, rapamycin- and 5-FOA-sensitive colonies were chosen. These candidate mutants were streak purified and tested again for the ability to sector. From this secondary screen, eight candidates remained nonsectoring. These nonsectoring mutants were transformed with a plasmid containing a wild-type copy of FKBP12 on a LEU2 vector (pYJH26; HEITMAN et al. 1991B ). One mutant, dead without FKBP12 (dwf11-2), readily lost the URA3 vector when transformed with the FKBP12 LEU2 plasmid (pYJH26). Other mutants remained nonsectoring, suggesting that the URA3 plasmid had integrated into the genome and that integration resulted in the original nonsectoring phenotype.

Cloning of DWF11/HMO1:
A 2µ LEU2 yeast genomic library (provided by C. Alarcon and S. Muir) was used to transform the dwf11-2 strain. From 8800 transformants, 17 5-FOA-resistant colonies were selected. Plasmids were isolated from yeast as described (HOFFMAN and WINSTON 1987 ) and amplified in Escherichia coli. Of the 17 plasmids, 6 contained the FPR1 gene, 5 contained RPL25, and 2 contained HMO1. The remaining 4 plasmids did not have an insert.

Targeted integration of HMO1 and RPL25 was performed as described (ROSE and BROACH 1991 ). The RPL25 gene contained within a HindIII fragment of the genomic clone was ligated into the corresponding sites of the integrating LEU2 vector YIplac128 (GIETZ and SUGINO 1988 ) to yield plasmid pIRPL25. pIRPL25 was cleaved with SpeI; SpeI cuts once in the RPL25 open reading frame and thus stimulates targeted integration at the RPL25 genomic locus. The linearized plasmid was used to transform the dwf11-2 mutant strain (dwf11-2 fpr1::hisG [URA3 ADE3 FPR1]). An RPL25 integrant was then crossed to the MATa wild-type strain (KDY100: fpr1::hisG DWF11). The resulting diploid was sporulated, and 30 tetrads were dissected. The segregation pattern showed independent segregation of the RPL25-LEU2 integration locus and the dwf11-2 locus, with 2 Leu⁺:2 Leu^- segregation, indicating that RPL25 is a suppressor of dwf11-2.

The same type of integration experiment was performed with HMO1; an EcoRI-SphI genomic fragment containing HMO1 was cloned into YIplac128, linearized with ApaI, and used to transform the dwf11-2 mutant strain. The HMO1-LEU2 integrant strain was crossed to both wild-type (KDY100: MATa fpr1::hisG DWF11) and mutant (KDY101-1b:MATa fpr1::hisG dwf11-2 [URA3 ADE3 URA3]) strains, and resulting diploids were sporulated and dissected. In the cross to wild type, all 20 tetrads exhibited a 4 FOA- and rapamycin-resistant:0 FOA- and rapamycin-sensitive, 2 Leu⁺:2 Leu^- segregation pattern. In the cross to the mutant strain, 14 of 15 tetrads showed the expected 2 FOA- and rapamycin-resistant:2 FOA- and rapamycin-sensitive segregation. Thus, HMO1 is allelic with DWF11.

Determination of plasmid loss:
Plasmid loss was determined as described (LU et al. 1996 ). Briefly, the isogenic strains JK9-3da (wild type), KDY69 (fpr1::hisG), and KDY103-1d (hmo1::G418) were transformed with the CEN LEU2 plasmid pRS315 (SIKORSKI and HIETER 1989 ) and grown to mid-log phase in medium lacking leucine. Dilutions of the cultures were plated on synthetic complete medium and then replica-plated to medium lacking leucine to determine the initial fraction (F_i) of colonies containing the plasmid. Cultures were then collected by centrifugation and resuspended in nonselective YPD medium. After several doublings, the final fraction (F_f) of colonies containing the plasmid was again determined by diluting on nonselective medium and then replica-plating on selective medium. Plasmid loss rate was calculated as 1–10^m, where m = .

FKBP12-Hmo1p-binding assays:
Purification of FKBP12 was previously described (ALARCON and HEITMAN 1997 ). Preparation of yeast extracts, FKBP12 affinity chromatography, and Western blotting was as described (CARDENAS et al. 1994 ). The Hmo1p polyclonal antibodies were kindly provided by Steve Brill (LU et al. 1996 ).

Two-hybrid experiments:
Two-hybrid vectors containing FKBP12 were previously described (CARDENAS et al. 1994 ). HMO1 expression plasmids were engineered by amplifying the HMO1 open reading frame via PCR with the following primers: (5'-ACTGAATTCAGACTACAGATCCTTCTGTC-3') and (5'-ACTGGATCCACGATTTTAGCGATGTTCCCC-3'). The resulting PCR product was cleaved with EcoRI and BamHI and cloned into the corresponding sites of pGAD424 and pGBT9 [plasmids described in FIELDS and SONG 1989 ] to yield plasmids pKDw35 and pKDw36. Both pKDw35 and pKDw36 complement the growth defect of an hmo1::G418 mutant strain, and thus both GAL4(BD)-Hmo1 and GAL4(AD)-Hmo1 hybrid proteins are functional. The yeast reporter strains Y190, SMY1, PJ69-4a, and SMY87 were cotransformed with the plasmids described in Table 2 and were selected on medium lacking leucine and tryptophan. ß-Galactosidase assays were as described (CARDENAS et al. 1994 ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation of mutations that are synthetically lethal with fpr1 mutations:
We used an ade2 ade3 colony-sectoring assay (KOSHLAND et al. 1985 ; BENDER and PRINGLE 1991 ) to identify genes that genetically interact with FKBP12 (Figure 1). We started with two ade2 ade3 strains of opposite mating type and with additional complementing auxotrophic mutations lys2 and trp1. While ade2 mutants form red colonies because they accumulate an intermediate in adenine biosynthesis that fluoresces red, ade2 ade3 mutants are white because the Ade3 enzyme lies upstream of Ade2 in the pathway. The initial strain set is also Ura^- and thus FOA-resistant.

View larger version (15K): In this window In a new window Download PPT slide   Figure 1. Strategy for an FKBP12 synthetic lethal screen. The starting strains harbor ura3, fpr1, ade2, and ade3 mutations and thus they are phenotypically FOA resistant, rapamycin resistant, and white. The strains were transformed with a plasmid carrying wild-type copies of the FPR1, URA3, and ADE3 genes and then UV mutagenized. Strains that retain the plasmid are Ura+, 5-FOA sensitive, rapamycin sensitive, and red. Strains that have lost the plasmid are Ura-, 5-FOA resistant, rapamycin resistant, and white. Mutations that require FPR1 for viability produce nonsectoring red colonies that remain sensitive to 5-FOA and rapamycin.

The fpr1 gene was disrupted with the hisG cassette (ALANI et al. 1987 ) in the ade2 ade3 ura3 backgrounds, rendering the strains rapamycin-resistant, because FKBP12 is the intracellular receptor for rapamycin. The resulting fpr1::hisG mutant strains were transformed with a 2µ plasmid carrying wild-type copies of the URA3, FPR1, and ADE3 genes. Initial transformants were Ura⁺, FOA, and rapamycin sensitive and red; however, the transformed cells can lose the plasmid because none of the genes contained on the plasmid are essential. These transformed cells thus give rise to red (cells retaining the plasmid)- and white (cells that lost the plasmid)-sectored colonies. Mutations that are lethal in combination with mutations in fpr1 are no longer able to lose the plasmid and cause cells to give rise to nonsectoring, red, and FOA- and rapamycin-sensitive colonies. Thus, we mutagenized the fpr1 ura3 ade2 ade3 strains containing the URA3/FPR1/ADE3 plasmid and screened for nonsectoring, red, FOA-sensitive colonies.

Eight nonsectoring mutants were isolated. In addition to colonies containing mutations synthetically lethal with fpr1, colonies may also be nonsectoring if the URA3/FPR1/ADE3 plasmid integrated into the cellular genomic DNA. To distinguish between fpr1 synthetic lethal mutants and integrants, candidates were transformed with a LEU2 plasmid bearing the FPR1 gene. Integrants would remain 5-FOA sensitive, while fpr1 synthetic lethal mutants would be able to shuffle the original FPR1 URA3 plasmid and become 5-FOA resistant. In addition, integrants would confer dominant 5-FOA and rapamycin sensitivity, while synthetic lethal mutants are often recessive. As illustrated in Figure 2, one candidate, which we designated dwf11-2, was recessive when crossed to its wild-type parent (KDY100: fpr1::hisG DWF11; sectoring was restored) and also became 5-FOA resistant when transformed with an FPR1 LEU2 plasmid, indicating loss of the FPR1 URA3 plasmid. As expected, the dwf11-2 strain containing the FPR1/LEU2 plasmid remained rapamycin sensitive because the presence of FKBP12 was still required for its viability. The dwf11-2 mutant exhibited a slow growth defect; while wild-type FKBP12 complemented the 5-FOA-sensitive nonsectoring phenotypes of the fpr1::hisG dwf11-2 [FPR1 URA3 ADE3] strain, it did not complement the slow growth defect conferred by dwf11-2 (Figure 2).

View larger version (108K): In this window In a new window Download PPT slide   Figure 2. The synthetic lethality between dwf11-2 and fpr1 mutations is recessive and complemented by either the wild-type FPR1 or HMO1 gene. The dwf11-2 fpr1::hisG [FPR1 URA3 ADE3] strain was crossed to the wild-type parental strain KDY100 (fpr1 DWF11), and the resulting diploid was grown on YPD, 5-FOA, and YPD + rapamycin medium (top). Similarly, the dwf11-2 strain was transformed with either an empty LEU2 plasmid [LEU2] (YEplac181) or a LEU2 plasmid containing the wild-type FPR1 [FPR1 LEU2] or HMO1 gene [HMO1 LEU2]. Transformants were selected on medium lacking leucine and then grown on YPD, 5-FOA, and YPD + rapamycin medium (bottom), demonstrating that the resident FPR1 URA3 ADE3 plasmid could be lost following transformation with the FPR1 LEU2 or HMO1 LEU2 plasmids but not with the control LEU2 plasmid.

dwf11-2 is allelic with HMO1:
We cloned the wild-type DWF11 gene by complementation of the 5-FOA sensitivity exhibited by the dwf11-2 fpr1::hisG [FPR1 URA3 ADE3] mutant strain. A genomic library constructed in a 2µ LEU2 plasmid was introduced, and we screened for the ability to lose the resident URA3 FPR1 plasmid and form 5-FOA-resistant colonies. Presumptive complementing clones were rescued in E. coli, confirmed by retransforming into the dwf11-2 mutant strain, and identified by restriction mapping and sequencing. As expected, several clones containing the FPR1 gene encoding FKBP12 were obtained; these complementing clones rendered colonies 5-FOA resistant, but rapamycin sensitive. In addition, two other classes of complementing clones were identified. In these cases, sectoring of the FPR1 URA3 ADE3 resident plasmid occurred to result in white, 5-FOA- and rapamycin-resistant colonies, indicating no functional FKBP12 was present. One set carried the RPL25 gene, which encodes an essential ribosomal protein, while the other carried the HMO1 gene, which encodes a high mobility group 1/2 chromatin-associated protein (LU et al. 1996 ; PERNAMBUCO et al. 1996 ).

To resolve whether RPL25 or HMO1 encoded the wild-type copy of DWF11 (vs. functioning as an extragenic suppressor), targeted integration was performed with both genes (see MATERIALS AND METHODS). These genetic analyses indicated that HMO1 was allelic with DWF11. We next tested whether the wild-type HMO1 gene could complement in low copy number the slow growth defect and fpr1 synthetic lethality of dwf11-2 mutants. When expressed from a CEN LEU2 plasmid, the HMO1 gene complemented both of these dwf 11-2 mutant phenotypes (Figure 2). RPL25 also complemented the fpr1 synthetic lethality of dwf11-2 mutants and the slow growth phenotype of dwf11-2 but not of fpr1, indicating that RPL25 is a suppressor of dwf11-2/hmo1 and not of FKBP12 (data not shown).

We next tested whether knock-out alleles of fpr1 and hmo1 were also synthetically lethal in a second strain background. The HMO1 open reading frame was replaced with the G418-resistance gene, and the resulting hmo1::G418 strain was crossed to an fpr1::hisG null mutant strain. The HMO1/hmo1 FPR1/fpr1 diploid was sporulated, and 30 tetrads were dissected. The fpr1::hisG and hmo1::G418 alleles were followed by rapamycin and G418 resistance, respectively. Of the 30 tetrads, no rapamycin-resistant, G418-resistant segregants were isolated, and viability segregated as expected for two independent genes, indicating synthetic lethality between fpr1 and hmo1 mutations (a representative sample of tetrads is illustrated in Figure 3). Null mutations in FKBP12 were not synthetically lethal with null mutations in hmo2, the other HMG1/2 homolog in yeast (data not shown).

View larger version (48K): In this window In a new window Download PPT slide   Figure 3. fpr1 and hmo1 mutations are synthetically lethal. Haploid fpr1::hisG and hmo1::G418 null mutants were mated, and the resulting diploids were sporulated and dissected. A representative sample of tetrads is illustrated here in which tetrads are numbered 1 to 10, and the four meiotic segregants of each individual tetrad are lettered A to D. Segregants were replica plated to medium containing either rapamycin or G418. Segregation of the fpr1 and hmo1 mutations was followed by rapamycin-resistance and G418-resistance, respectively. Inviable segregants and their predicted phenotypes and genotypes are indicated in parentheses.

fpr1 mutants have an increased rate of plasmid loss:
HMO1 was originally identified biochemically by an associated helicase activity, though Hmo1 does not have intrinsic helicase activity (LU et al. 1996 ). The Hmo1 protein is localized primarily to the nucleus, but a significant portion is also found in the cytoplasm (LU et al. 1996 ). This study also showed that hmo1 mutants have a slow growth phenotype and also exhibit an increased rate of plasmid loss (3.5-fold; LU et al. 1996 ). Strains lacking fpr1 also lose plasmids more readily (1.9-fold) than isogenic FPR1 wild-type strains (data not shown).

Both hmo1 and fpr1 mutants exhibit slow growth (HEITMAN et al. 1991B ; LU et al. 1996 ). We tested whether overexpression of wild-type Hmo1 can suppress the fpr1 mutant growth defect, and, similarly, whether overexpression of FKBP12 can suppress the hmo1 mutant growth defect. There was no suppression in either case (data not shown).

Hmo1p and FKBP12 proteins physically interact:
To examine whether Hmo1p and FKBP12 physically interact, we tested the ability of Hmo1p to bind to an FKBP12 affinity matrix. Yeast FKBP12 was tagged at its amino terminus with a hexahistidine tag, overexpressed in bacteria, purified by nickel affinity chromatography, and coupled to affigel beads as described (CARDENAS et al. 1994 ). The FKBP12 affinity matrix (or affigel beads alone as a control) was then incubated with total cellular extracts prepared from either wild-type or hmo1 mutant strains; bound proteins were eluted, fractionated by SDS-PAGE, and transferred to nitrocellulose; and the Hmo1 protein was detected by Western blot with anti-Hmo1 antibodies (LU et al. 1996 ). Hmo1 specifically interacted with FKBP12, and addition of FK506 disrupted the Hmo1p-FKBP12 complex (Figure 4), suggesting that Hmo1p may bind to the FKBP12-drug-binding/active site. In contrast, no Hmo1p interacted with affigel beads alone, indicating a specific interaction between FKBP12 and Hmo1p. Note that because this experiment involves incubating total yeast extracts with an in vitro FKBP12 affinity matrix, other proteins present in the extract in addition to Hmo1p may be components of the Hmo1p-FKBP12 complex.

View larger version (21K): In this window In a new window Download PPT slide   Figure 4. Hmo1p and FKBP12 proteins physically associate. Wild-type (WT) or hmo1 mutant () extracts were incubated with either FKBP12-coupled affigel beads in the absence (lane 3) or presence (lane 4) of FK506 (20 µM), or affigel beads alone (lane 6). The affigel beads were washed four times with lysis buffer then eluted with sample buffer and analyzed by Western blot using anti-Hmo1p antibodies. Lane 1 is untreated total cell extract from a wild-type strain and lane 2 is untreated total cell extract from the isogenic hmo1 mutant strain.

Hmo1p homodimerizes in the two-hybrid system:
To further analyze FKBP12-Hmo1p interactions, a series of two-hybrid experiments were performed. For this purpose, we fused Hmo1p to the GAL4-DNA-binding domain (GAL4 BD-Hmo1p) and activation domain (GAL4 AD-Hmo1p) and tested their ability to interact, both with each other and with fusions of FKBP12 to GAL4 BD and GAL4 AD that were previously described (CARDENAS et al. 1994 ; LORENZ and HEITMAN 1995 ). All fusion proteins were able to complement fpr1 or hmo1 mutant phenotypes (CARDENAS et al. 1994 ; data not shown).

The first set of two-hybrid experiments was performed in the PJ69-4a strain background (JAMES et al. 1996 ). Only a quite modest interaction between FKBP12 and Hmo1p was detected in the two-hybrid system above background levels of the two fusion proteins alone (10.4 ß-galactosidase units and 9.3 units vs. 22.1 units; Table 2, top). On the other hand, GAL4 BD-Hmo1p and GAL4 AD-Hmo1p interacted to yield a signal that was greater than that produced by either single fusion protein alone, consistent with Hmo1p-Hmo1p homodimerization (Table 2, top). This is in accord with results from the initial report on the identification of Hmo1p in which the purification of Hmo1p indicated that Hmo1p forms an oligomeric complex in solution (LU et al. 1996 ). Interestingly, we found that a high level of expression of the GAL4 AD-Hmo1 fusion protein alone was toxic in strains lacking FKBP12 (SMY87), but tolerated in an isogenic strain expressing FKBP12 (PJ69-4a). Because Hmo1p is a DNA-binding protein that may globally associate with chromatin, tethering the GAL4 transcriptional activation domain to chromatin may be toxic to the cell by perturbing gene expression. That expression of FKBP12 relieves this toxic effect again supports an Hmo1p-FKBP12 interaction and suggests that FKBP12 could, for example, regulate Hmo1p interactions with either itself or DNA.

We also performed a second set of two-hybrid experiments in a different two-hybrid host strain background. In either the FKBP12 wild-type strain (Y190) or in an isogenic fpr1 mutant (SMY1), expression of the GAL4 AD-Hmo1 hybrid protein was tolerated (Table 2, bottom; HARPER et al. 1993 ). In the Y190 FKBP12 wild-type strain, modest interactions were detected between the GAL4 AD-Hmo1 and the GAL4 BD-Hmo1 fusion proteins (1.3 units background vs. 3.1 units) and also between the GAL4 DB-FKBP12 and GAL4 AD-Hmo1 fusion proteins (0.1 units background vs. 3.0 units). Moreover, the level of Hmo1p dimerization was increased by ~3.5-fold in the presence of FK506 (3.1 units to 10.9 units). The Hmo1p-Hmo1p interaction was also dramatically increased in the two-hybrid host strain lacking FKBP12 (3.1 units in the FKBP12 wild-type strain compared to 207 units in the strain lacking FKBP12). The Hmo1p-Hmo1p interaction was only very modestly affected by FK506 in the FKBP12 mutant strain, indicating that the effects of FK506 are largely mediated by FKBP12. Taken together, these observations support the conclusions that Hmo1p interacts with itself and with FKBP12, and that FKBP12 inhibits Hmo1p-Hmo1p interactions.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We have addressed the normal cellular functions of the peptidyl-prolyl isomerase FKBP12, the cellular target of the immunosuppressive drugs FK506 and rapamycin. FKBP12 is highly conserved from yeast to man, yet is not essential for viability in either yeast or mice. A relatively limited number of FKBP12 target proteins have been identified, but none are conserved between yeast and multicellular eukaryotes. We have therefore taken genetic approaches in yeast to identify other proteins whose function either overlaps with, or is dependent upon, FKBP12.

Here we report the isolation of mutations that are synthetically lethal with FKBP12 mutations. By this approach, we identified a mutation in the HMO1 gene as synthetically lethal with mutations in the FPR1 gene encoding FKBP12. Generation and analysis of an hmo1::G418 mutation confirmed that hmo1 and fpr1 mutations are synthetically lethal, even in a different yeast strain background. The HMO1 gene was previously identified and characterized by Brill and colleagues, who found that it encodes a yeast HMG1/2 protein homolog that is associated with chromatin and a helicase (LU et al. 1996 ). In addition to synthetic lethal interactions, we also found that fpr1 and hmo1 mutations confer two common phenotypes: slow growth and an increased rate of plasmid loss, providing additional evidence that the functions of the FKBP12 and Hmo1 proteins are related.

Additional findings indicate that FKBP12 and Hmo1p physically and functionally interact. Importantly, we demonstrate that FKBP12 and Hmo1p physically interact in vitro by chromatography with an FKBP12 affinity matrix. This interaction may occur via the FKBP12 active site/ligand-binding pocket because FK506 potently disrupted the FKBP12-Hmo1 complex. In addition, in vivo binding assays with the two-hybrid system revealed that Hmo1p can form a homodimer or homooligomer, in accord with previous observations of Brill and colleagues who found that the 35-kD Hmo1 protein fractionated as an ~330-kD multimer on a Superdex 200 sizing column (LU et al. 1996 ). Hmo1p-Hmo1p interactions in the two-hybrid assay were increased by either the FKBP12 ligand FK506 or by mutation of the FPR1 gene that encodes FKBP12. Moreover, expression of a GAL4 AD-Hmo1 fusion protein was tolerated in a two-hybrid host strain expressing FKBP12, but was toxic in an isogenic strain lacking FKBP12. Taken together, our findings reveal that FKBP12 and the HMG protein Hmo1 physically and genetically interact and provide evidence that FKBP12 may function to regulate oligomerization and function of an Hmo1-chromatin complex.

Our evidence supports the hypothesis that FKBP12 regulates the interaction and/or function of Hmo1 protein complexes and suggests that both the active site and other regions of FKBP12 interact with Hmo1p. FK506 potently disrupted the FKBP12-Hmo1p complexes detected in vitro, suggesting that Hmo1p interacts, at least in part, via the FKBP12 active site. On the other hand, FK506 had relatively modest effects on Hmo1p-Hmo1p interactions detected in the two-hybrid assay, whereas deletion of FKBP12 resulted in a much more marked increase in Hmo1p-Hmo1p dimerization. These observations suggest that Hmo1p may interact with regions of FKBP12 in addition to the active site, as is also the case in FKBP12 interactions with the yeast aspartokinase protein (ALARCON and HEITMAN 1997 ). Several FKBP12 active site mutants (F43Y, D44V, and F106Y) could complement the synthetic lethality of the fpr1::hisG dwf11-2 double mutant strain (data not shown). Previous studies revealed that D44 and F106 are involved in FK506 binding, whereas F43 mutant proteins bind FK506 (DECENZO et al. 1996 ; DOLINSKI et al. 1997 ). Thus, these active site/ligand-binding pocket residues are clearly not required for FKBP12-dependent viability of hmo1 mutant strains. Taken together, our findings indicate that FKBP12 and Hmo1p genetically and physically interact.

One finding from our studies was that null mutations in the FPR1 and HMO1 genes exhibited synthetic lethality. What is the molecular basis for synthetic lethality when the HMO1 gene is completely deleted, rather than more subtly mutated, in a strain lacking FKBP12? Biochemical evidence indicates that the Hmo1 and FKBP12 proteins physically interact, suggesting this is a direct effect. One plausible hypothesis is that the other HMG1/2 homolog, Hmo2p, might also be a target of FKBP12. In this model, when both Hmo1p and FKBP12 are absent, Hmo2p function would be more compromised than when either Hmo1p or FKBP12 are mutated individually. However, this model can be excluded because we find that mutations in HMO2 and FPR1 are not synthetically lethal (data not shown). An alternative hypothesis is that there may exist protein components of the FKBP12-Hmo1p complex that, in the absence of Hmo1p, are dependent on FKBP12 for function. Further studies are required to address the structure and function of Hmo1p-DNA chromatin complexes that might provide further insight.

Thus far, several mammalian targets of FKBP12 have been identified and studied in some detail, including the multidrug resistance pump and the ryanodine, IP₃, and type I TGF-ß receptors (JAYARAMAN et al. 1992 ; TIMERMAN et al. 1993 ; BRILLANTES et al. 1994 ; WANG et al. 1994 , WANG et al. 1996A ; CAMERON et al. 1995A , CAMERON et al. 1995B ; HEMENWAY and HEITMAN 1996 ; BASSING et al. 1998 ). With the exception of the MDR family of proteins, none of these proteins, however, are conserved in yeast, and the mammalian Mdr3 protein is quite divergent from its yeast counterparts. On the other hand, because HMG proteins are highly conserved, our identification of physical and functional interactions between the yeast HMG protein Hmo1 and FKBP12 reveals an FKBP12 target that is conserved from yeast to humans and could, at least in part, account for the remarkable conservation of FKBP12 during evolution. Finally, the finding that FKBP12 interacts with not only the yeast HMG protein Hmo1p but also the mammalian YY1 transcription factor (YANG et al. 1995 ) suggests a possible novel nuclear role for FKBP12 in the regulation of transcription and chromatin structure, which remains to be explored.

	ACKNOWLEDGMENTS

We thank Steve Brill, Alan Bender, Trish Davis and Nancy Kleckner for providing antisera, strains, and plasmids; Lora Cavallo and Scott Muir for technical assistance; and Clara Alarcon, Maria Cardenas, Mike Lorenz and Felix Persi for helpful discussions. We thank Fujisawa Pharmaceuticals for providing FK506. Joseph Heitman is an associate investigator of the Howard Hughes Medical Institute.

Manuscript received August 14, 1998; Accepted for publication November 5, 1998.

	LITERATURE CITED

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