©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Myristoylation of Calcineurin B Is Not Required for Function or Interaction with Immunophilin-Immunosuppressant Complexes in the Yeast Saccharomyces cerevisiae(*)

(Received for publication, April 7, 1995; and in revised form, August 9, 1995)

Dahai Zhu (1) (3) Maria E. Cardenas (1) Joseph Heitman (1) (3) (2)(§)

From the  (1)Departments of Genetics and (2)Pharmacology and the (3)Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Calcineurin is a heterodimeric Ca/calmodulin-dependent protein phosphatase that regulates signal transduction and is the target of immunophilin-immunosuppressive drug complexes in T-lymphocytes and in yeast. Calcineurin is composed of a catalytic A subunit and a regulatory B subunit that is myristoylated at its amino terminus. We employed genetic and biochemical approaches to investigate the functional roles of myristoylation of calcineurin B (CNB1) in Saccharomyces cerevisiae. A calcineurin B mutant in which glycine 2 was substituted by alanine (CNB1-G2A) did not incorporate [^3H]myristate when expressed in yeast. Both wild-type calcineurin B and the CNB1-G2A mutant protein are partially associated with membranes and cytoskeletal structures; hence, myristoylation is not required for these associations. In several independent genetic assays of calcineurin functions (recovery from alpha-factor arrest, survival during cation stress, and viability of a calcineurin-dependent strain), the nonmyristoylated CNB1-G2A mutant protein exhibited full biological activity. In vitro, both wild-type and CNB1-G2A mutant proteins formed complexes with both cyclophilin A-cyclosporin A (CsA) and FKBP12-FK506 that contained calcineurin A. Interestingly, expression of the nonmyristoylated CNB1-G2A mutant protein rendered yeast cells partially resistant to the immunosuppressant CsA, but not to FK506. This study demonstrates that calcineurin B myristoylation is not required for function, but may participate in inhibition by the cyclophilin A-CsA complex.


INTRODUCTION

Calcineurin is a Ca/calmodulin-regulated serine/threonine-specific protein phosphatase. The fully active enzyme is a heterotrimer composed of a 60-kDa catalytic A subunit, a 19-kDa regulatory B subunit, and calmodulin(1) . The regulatory B subunit is a calcium-binding protein with four EF hands and marked similarity to calmodulin. Recent NMR studies confirm that calcineurin B and calmodulin share similar secondary structures(2) . However, these two calcium-binding proteins cannot replace each other, and both are required for full activation of calcineurin(1) . In addition, Ca binding to calcineurin B and calmodulin has different effects on calcineurin activity(3) .

Calcineurin plays important roles in mediating different biological responses including regulation of the vacuolar Ca-ATPase PMC1 in yeast(4) , the renal Na,K-ATPase in mammals (5) , a potassium channel in plants(6) , and the N-methyl D-aspartate channel in vertebrates(7) . Several independent studies have demonstrated that calcineurin is required for adaptation of the yeast Saccharomyces cerevisiae to high salt stress(8, 9, 10) . Calcineurin has also been shown to participate in neuronal signaling pathways for long-term depression(11) , neurotoxicity(12) , and neurite outgrowth(13) .

In complex with the immunophilins cyclophilin A and FKBP12, the immunosuppressants cyclosporin A (CsA) (^1)and FK506 bind to and inhibit calcineurin ((14) ; reviewed in (15, 16, 17) ). The cytoplasmic subunit of a transcription factor required for T-cell activation (NF-ATc) is a substrate for calcineurin in vitro(18) . Dephosphorylation of NF-ATc by calcineurin may promote nuclear translocation and transcription of T-cell activation genes(19) . Physical interactions between calcineurin and immunophilin-drug complexes have been previously examined by chemical cross-linking and photoaffinity labeling(20, 21, 22) . Recently, a combined molecular modeling and mutagenic approach has implicated a latch region of calcineurin B in both immunophilin-drug complex binding and calcineurin phosphatase activity(23) .

Both immunophilins and calcineurin are highly conserved. The yeast S. cerevisiae expresses homologs of mammalian cyclophilin A (24) , FKBP12(25) , calcineurin A (CMP1 and CMP2 isoforms), and calcineurin B (CNB1)(26, 27, 28, 29) . Genetic studies demonstrated that calcineurin is required for recovery from alpha-factor mating pheromone in yeast(26, 29, 30) . Recent studies have provided direct evidence that yeast cyclophilin A and FKBP12 are required for CsA and FK506 cytotoxicity, respectively, and that calcineurin is essential in CsA- and FK506-sensitive strains(9, 31) . These results demonstrate that, as previously established in T-cells, the yeast target of the cyclophilin A-CsA and FKBP12-FK506 complexes is also calcineurin. In addition, Cardenas et al.(32) have recently demonstrated that immunophilins and calcineurin functionally interact in the absence of exogenous immunosuppressants.

In both fungi and vertebrates, the calcineurin B regulatory subunit is modified by amino-terminal myristoylation(26, 33) . A variety of observations suggest that myristoylation functions in protein-protein interactions, membrane association, and protein folding(34) . Several N-myristoylproteins are kinases or phosphatases that play crucial roles in signal transduction. For instance, myristoylation of the yeast GTP-binding protein alpha-subunit is essential for membrane association (35) and inhibition of G(36) . Myristoylation is required for transactivation by the FBR v-Fos protein, a retrovirally transduced c-fos proto-oncogene homolog(37) . Myristoylation of pp60 is required for membrane association and oncogenic transformation(38) . Finally, only myristoylated forms of the abl tyrosine kinase recruit phosphatidylinositol 3-kinase to the plasma membrane(39) .

In this report, we present genetic and biochemical evidence that myristoylation of calcineurin B is not required for biological activity or for association with the calcineurin A catalytic subunit, with membranes, with a Triton-insoluble cytoskeletal fraction, or with the immunosuppressive FKBP12-FK506 and cyclophilin A-CsA complexes. Thus, we conclude that myristoylation is not required for several calcineurin functions in yeast.


MATERIALS AND METHODS

Yeast Strains, Culture Medium, and Transformation

Yeast strains for this study are listed in Table 1or have been described (25, 32) . Yeast media were prepared as described(9, 40, 41) . The FK506- and CsA-sensitive calcineurin-dependent cev1 mutant strain 204 was crossed to isogenic strains JK9-3da (CNB1, wild-type), TB85 (Deltacnb1/pRS316), TB85 (Deltacnb1/CNB1), and TB85 (Deltacnb1/CNB1-G2A). As described below, wild-type CNB1 and the CNB1-G2A mutant were expressed from pRS316-derived plasmids pYDZ3 (CNB1) and pYDZ10 (CNB1-G2A). Matings were on YPD medium (1% yeast extract, 2% Bacto-peptone, 2% glucose, 2% agar) and Leu Ura Trp diploids were selected on YNB plus His (0.67% yeast nitrogen base without amino acids, 2% glucose, 2% agar, 20 mg/liter histidine), sporulated, and dissected to determine if the CNB1-G2A mutant protein supports calcineurin-dependent growth of strain 204. Yeast cells were transformed with lithium acetate (42) .



Isolation of the Yeast Calcineurin B Gene (CNB1) by PCR

The yeast CNB1 gene with 5`-regulatory sequences was PCR-cloned with primers based on published sequence(10, 27, 29) . Primers 194 (5`-CCAAAGCTTCTTATTGTTTGTTACATATAC-3`) and 196 (5`-CGCGGGATCCACGCTATGAAAAAGGAAAACAAATGA-3`) contain a HindIII site and a BamHI site, respectively. PCR was 35 times 30 s at 94 °C, 30 s at 45 °C, and 90 s at 72 °C. The 1064-base pair CNB1 PCR fragment was purified, digested with BamHI/HindIII, cloned in CEN URA3 plasmid pRS316(43) , confirmed by sequencing, and named pYDZ3.

Site-directed Mutagenesis of Calcineurin B

The CNB1 Gly-2 codon was mutated to Ala (GGT to GCT) by PCR overlap site-directed mutagenesis (44) with external primers 194/196 (see above) and internal primers 197 (5`-TTTGGAAGGAGCAGCAGCCATTTTAAGAATAAA-3`) and 198 (5`-TTTATTCTTAAAATGGCTGCTGCTCCTTCCAAA-3`). The two first round site-directed mutagenesis PCRs used primers 194/197 and 196/198. PCR products were gel-purified, mixed, and used as templates for second round PCR with external primers 194/196. This PCR product was purified, BamHI/HindIII-digested, cloned in the BamHI/HindIII sites of plasmid pRS316, confirmed by sequencing the entire CNB1 gene, and named pYDZ10.

Expression, Purification, and Production of Antisera against His(6)-tagged CNB1

The yeast CNB1 cDNA was PCR-cloned with primers 181 (5`-CGCGGGATCCGATGGGTGCTGCTCCTTCCAAAATTG-3`) and 182 (5`-CCCTGAATTCTTACACATCGTATTGCAATGTC-3`), which contain a BamHI site and an EcoRI site, respectively, as described above. The 757-base pair PCR product was purified, BamHI/EcoRI-digested, ligated into BamHI/EcoRI sites of His6 expression plasmid pTrcHisB (Invitrogen), confirmed by sequencing, and introduced into Invitrogen Escherichia coli strain Top10. Bacterial cells expressing His6-CNB1 were grown overnight at 37 °C in 20 ml of FB medium with 100 µg/ml ampicillin. Cells were centrifuged and resuspended in 1 liter of FB medium (25 g/liter tryptone, 7.5 g/liter yeast extract, 1 g/liter glucose, 6 g/liter NaCl, 50 ml/liter 1 M Tris-HCl, pH 7.6) with 100 µg/ml ampicillin. At A = 0.6, 2 mM isopropyl-1-thio-beta-D-galactopyranoside was added and incubated for 5 h. Cells were centrifuged, resuspended in 10 ml of lysis buffer (40 mM Hepes, pH 7.4, 200 mM KCl, 10% glycerol, 0.5 mM phenylmethylsulfonyl fluoride), lysed by sonicating 10 times 1 min with cooling, and centrifuged for 30 min at 40,000 rpm in a Beckman Ti-70 rotor. Clarified cell lysate was mixed with 4 ml of Ni-nitriloacetic acid-agarose resin (QIAGEN Inc.) pre-equilibrated in lysis buffer; stirred for 30 min at 4 °C; transferred to an Econo-Column (Bio-Rad); and washed with 250 ml of lysis buffer, 100 ml of 100 mM imidazole/lysis buffer, and 20 ml of 20 mM imidazole/lysis buffer. His6-CNB1 was eluted in 4 ml of 200 mM imidazole/lysis buffer. His6-CNB1 was purified by electroelution after SDS-PAGE and used to generate a rabbit antiserum by standard procedures(45) .

In Vivo Labeling with [^3H]Myristate and Immunoprecipitation Analysis

In vivo [^3H]myristate labeling of yeast proteins was as described(29) . Cell extracts for immunoblotting were prepared as described below. Yeast CNB1 was immunoprecipitated by incubating equal amounts of cell extracts with anti-yeast CNB1 antisera for 1 h at 4 °C with shaking. Protein A-Sepharose was added and incubated for 1 h at 4 °C. Immunoprecipitates were washed four times with 1 ml of wash buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Nonidet P-40, 1 mM EDTA, pH 8.0, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin, 100 units of aprotinin) and one time with phosphate-buffered saline. Proteins were eluted with SDS sample buffer for 4 min at 95 °C and fractionated by 15% SDS-PAGE. The gel was treated with fluorographic enhancer (Amplify, Amersham Corp.) for 1 h, dried, and analyzed by autoradiography.

Preparation and Fractionation of Yeast Protein Extracts

Yeast cultures were grown in 10 ml of YPD medium for 12 h at 30 °C. Cells were diluted in 50 ml of YPD medium to A = 0.2 and incubated until A = 1.0. Cells were collected, washed with H(2)O, and resuspended in 500 µl of ice-cold buffer (20 mM Hepes, pH 7, 2 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride). An equal volume of glass beads was added, and cells were broken by bead beating in 5 times 45 s with cooling. Supernatant was collected by microcentrifugation for 30 min at 4 °C. Yeast total proteins were fractionated by SDS-PAGE, transferred to nitrocellulose, probed with anti-CNB1 antisera for 1 h at room temperature, incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody, and detected by ECL (Amersham Corp.).

For subcellular localization, yeast cultures were grown as described above. Cells were broken with a bead beater, and unbroken cells were pelleted at 5000 rpm for 5 min at 4 °C. Clarified cell lysate was centrifuged at 100,000 times g for 30 min at 4 °C, and supernatant was collected. The pellet was resuspended in 500 µl of lysis buffer and is referred to as the particulate fraction. Particulate fractions (100 µl) were further extracted with 1% Triton X-100 or 1% sodium cholate for 1 h at 0 °C. The extracted mixture was centrifuged at 100,000 times g for 30 min at 4 °C, and the clear supernatant is referred to as the soluble fraction. The pellet was resuspended in 100 µl of lysis buffer and is referred to as the resistant pellet fraction. Yeast cell fractions were subjected to Western blot analysis as described above.

Analysis of Calcium-dependent Mobility of Wild-type and Mutant Calcineurin B Proteins

The calcium-dependent gel mobility shift of wild-type and G2A mutant calcineurin B proteins was tested by addition of Ca (5 mM) or EGTA (5 mM) to the total protein solution in SDS sample buffer prior to SDS-PAGE and Western blot analysis as described previously(14) .

Assay of Pheromone Response

Yeast cells of isogenic MATa strains were grown overnight in YPD medium at 30 °C. Cultures were diluted 10-fold in YPD medium, and 10^5 cells/plate were added to 4 ml of YPD top agar medium and poured on YPD plates. Discs saturated with 10 µg of alpha-factor (synthesized and desalted by the Howard Hughes Medical Institute peptide facility) were placed on the surface of the medium. Plates were photographed after 96 h of incubation at 30 °C.

Binding of Calcineurin to Immunophilin-Affi-Gel 10 in Yeast Cell Extracts

His6-FKBP12 and His6-cyclophilin A purification, FKBP12- and cyclophilin A-affigel 10 affinity matrix preparation, cell extracts and binding experiments with and without FK506 and CsA, and Western blotting with antisera against calcineurin subunits CNB1 and CMP1 were as described(32) .


RESULTS

Generation of a Nonmyristoylated Calcineurin B Mutant

The regulatory B subunit of calcineurin (CNB1) is myristoylated at the amino-terminal glycine residue in both yeast (29) and mammals(33) . The physiological functions of this lipid modification, if any, are not yet known. The yeast CNB1 gene with its 5`-regulatory elements was cloned by PCR from yeast genomic DNA using the known sequence of the gene (see ``Materials and Methods''). The authenticity of the PCR-generated CNB1 gene was confirmed by DNA sequence analysis and functional complementation of a cnb1 deletion strain (described below).

To test for functional roles of amino-terminal myristoylation of calcineurin B, a G2A mutation was generated by site-directed mutagenesis of the cloned CNB1 gene. Plasmids expressing the CNB1-G2A mutant protein were introduced into a host strain deleted for the calcineurin B gene, and expression and myristoylation were analyzed by Western blotting with rabbit anti-CNB1 antisera and in vivo labeling with [^3H]myristic acid performed in parallel with the same samples. As shown in Fig. 1A, both wild-type CNB1 (lanes 1 and 3) and the CNB1-G2A mutant protein (lane 4) were expressed at equivalent levels, whereas no CNB1 protein was detectable in the Deltacnb1 mutant host strain transformed with the control vector alone (lane 2). To demonstrate that the yeast CNB1-G2A mutant protein was not myristoylated, equal amounts of protein extracts from cells metabolically labeled with [^3H]myristic acid were immunoprecipitated with antisera against yeast CNB1, and the immunoprecipitates were analyzed by SDS-PAGE and autoradiography. Whereas the wild-type CNB1 protein was labeled with [^3H]myristic acid (Fig. 1B, lanes 1 and 3), the CNB1-G2A mutant protein was not (lane 4). Thus, the G2A mutation abolishes myristoylation of the yeast calcineurin B protein.


Figure 1: Wild-type yeast calcineurin B is myristoylated; the CNB1-G2A mutant protein is not. Isogenic yeast strains with the indicated genotypes were metabolically labeled with [^3H]myristate. A, proteins were extracted and subjected to 15% SDS-PAGE followed by Western blotting with rabbit anti-yeast CNB1 antiserum. B, calcineurin B was immunoprecipitated from equal amounts of protein extracts prepared from cells metabolically labeled with [^3H]myristic acid. Immunoprecipitates were fractionated by 15% SDS-PAGE and analyzed by autoradiography with fluorography (see ``Materials and Methods''). Exposure was for 30 days at -70 °C with an enhancing screen. Isogenic yeast strains were JK9-3da (wild-type (WT)), TB85 (Deltacnb1), TB85/pYDZ3 (Deltacnb1/CNB1), and TB85/pYDZ10 (Deltacnb1/CNB1-G2A) (see Table 1and ``Materials and Methods'').



Calcineurin B Does Not Require Myristoylation to Associate with Membranes or the Cytoskeleton

Myristoylation of some proteins, such as Ras-related proteins, is required for association with cellular membranes and transformation(46, 47) . Furthermore, it is known that the mammalian calcineurin B regulatory subunit is myristoylated and fractionates with both the cytosol and membranes(1) . To test if calcineurin B also associates with cellular membranes in yeast and whether myristoylation participates in any such association, wild-type CNB1 and the CNB1-G2A mutant protein were expressed in yeast cells from centromeric plasmids and examined by subcellular fractionation and Western blot analysis as described above. Both wild-type CNB1 and the nonmyristoylated CNB1-G2A mutant protein were recovered in both yeast cytosol and particulate fractions (Fig. 2A); however, by densitometric quantification, the ratio of nonmyristoylated CNB1-G2A mutant protein associated with the particulate versus the soluble fraction was found to be 2.5-fold decreased compared with wild-type CNB1, suggesting that the myristoyl group does contribute to membrane association of CNB1. The associated calcineurin A catalytic subunit CMP1 also fractionated with both the particulate and soluble fractions (Fig. 2B). As expected, the yeast plasma membrane H-ATPase PMA1 (48) was found exclusively in the particulate fraction (Fig. 2C), whereas yeast cyclophilin A was found to be almost entirely cytosolic (Fig. 2D).


Figure 2: Myristoylated CNB1 and nonmyristoylated CNB1-G2A are recovered in both cytosolic and membrane fractions. Total cell-free lysates (T) or cytosolic (supernatant (S)) and particulate (P) fractions (see ``Materials and Methods'') were analyzed by SDS-PAGE and Western blotting with antisera against calcineurin B CNB1 (A), calcineurin A CMP1 (B), PMA1 (C), and yeast cyclophilin A CPR1 (D) as described for Fig. 1A. Strains were JK9-3da (wild-type (WT)) and TB85/pYDZ10 (Deltacnb1/CNB1-G2A).



The particulate fraction includes both membranes and components of the cytoskeletal matrix. Densitometric quantification of the Western blot shown in Fig. 2A revealed that 65% of CNB1 was associated with the particulate fraction. To test whether CNB1 in the particulate fraction is associated with the cytoskeletal matrix, the particulate fractions from strains expressing wild-type CNB1 or nonmyristoylated CNB1-G2A were further treated with either 1% Triton X-100 or 1% sodium cholate, and the soluble and resistant fractions were recovered by centrifugation and analyzed by Western blotting. As shown in Fig. 3and quantified by densitometry, 40% of CNB1 and CNB1-G2A was extracted by a mock extraction with buffer alone (Fig. 3, A and B, lanes 1 and 2). In contrast, 80% of wild-type CNB1 and the CNB1-G2A mutant protein was extracted by Triton X-100, and similar amounts of both wild-type CNB1 and the CNB1-G2A mutant protein were detected in the Triton-soluble and Triton-resistant fractions (Fig. 3, A and B, lanes 3 and 4). Based on previous reports, Triton-insoluble proteins are associated with cytoskeletal structures in yeast and other organisms(49, 50, 51, 52, 53) . As a control for cytoskeletal association, control Western blotting was performed, revealing that a similar proportion of the known yeast cytoskeletal protein actin was resistant to Triton extraction (Fig. 3, C and D, lanes 3 and 4). The cytoskeleton association of yeast CNB1 was further tested by treatment of the particulate fractions with 1% sodium cholate, which removes proteins associated with the cytoskeleton. The majority (80-90%) of both wild-type CNB1 and the CNB1-G2A mutant protein was extracted by sodium cholate (Fig. 3, A and B, lanes 5 and 6). The small fraction not extractable by sodium cholate may represent insoluble protein. Taken together, these findings demonstrate that, in yeast, CNB1 associates with both membranes and cytoskeletal structures and that myristoylation is not required for either association.


Figure 3: Membrane and cytoskeletal association of wild-type CNB1 and the CNB1-G2A mutant protein. Equal amounts of the particulate fraction (P) were extracted with buffer alone or with buffer containing 1% Triton X-100 or 1% sodium cholate (see ``Materials and Methods''). Samples analyzed were buffer-soluble (S; lane 1), buffer-resistant (P; lane 2), Triton-soluble (S; lane 3), Triton-resistant (P; lane 4), sodium cholate-soluble (S; lane 5), and sodium cholate-resistant (P; lane 6). Fractions were analyzed by Western blotting with antisera against the calcineurin B subunit CNB1 (A and B) or yeast actin (C and D) as described for Fig. 1A. Proteins were from isogenic wild-type CNB1 strain JK9-3da (wild-type (WT)) (A and C) and CNB1-G2A mutant strain TB85/pYDZ10 (Deltacnb1/CNB1-G2A) (B and D).



Myristoylation Is Not Required for CaBinding by Calcineurin B

Calcineurin B shares significant identity with calmodulin, including four EF hands that chelate Ca ions. To test if myristoylation of calcineurin B is involved in Ca binding, we analyzed the Ca-dependent mobility of wild-type CNB1 and the CNB1-G2A mutant protein by SDS-PAGE and Western blot analysis (Fig. 4). Addition of Ca prior to electrophoresis had no effect on the mobility of either wild-type or G2A mutant CNB1, whereas prior addition of EGTA led to a marked reduction in the electrophoretic mobility of both wild-type and mutant CNB1 proteins. These findings indicate that both proteins are Ca-bound in total yeast cell extracts. In addition, we noted a slight reduction in the electrophoretic mobility of the CNB1-G2A mutant protein compared with wild-type CNB1 (Fig. 4); similar alterations in apparent molecular weight have been previously described for other nonmyristoylated mutant proteins(36, 54, 55) .


Figure 4: Ca-dependent mobility shift of wild-type CNB1 and the CNB1-G2A mutant protein. EGTA (5 mM) or Ca (5 mM) was added to protein extracts in SDS sample loading buffer, and proteins were separated by 15% SDS-PAGE and analyzed by Western blotting with anti-CNB1 antisera as described for Fig. 1A. Protein extracts were from isogenic wild-type CNB1 strain JK9-3da (wild-type (WT)) and CNB1-G2A mutant strain TB85/pYDZ10 (Deltacnb1/CNB1-G2A).



Myristoylation Is Not Required for Calcineurin Function in Recovery from alpha-Factor Arrest, Cation Resistance, or Viability

Loss of calcineurin function renders yeast cells hypersensitive to cation stress by either LiCl or NaCl(8, 9) . Biological function of the CNB1-G2A mutant protein was investigated by complementation of the LiCl-hypersensitive phenotype of a Deltacnb1 mutant strain. As shown in Fig. 5, yeast cells expressing the CNB1-G2A mutant protein from a centromeric plasmid grew as well as cells expressing wild-type CNB1 (Fig. 5, A and B), indicating that the CNB1-G2A mutant complements a Deltacnb1 mutation and renders yeast cells resistant to LiCl toxicity. In addition, expression of the CNB1-G2A mutant protein rendered either a cnb1 cmp1 CMP2 (strain EL1, Table 1) or a cnb1 cmp2 CMP1 (strain EL2, Table 1) mutant strain LiCl-resistant, indicating that the CNB1-G2A mutant protein functions with either the calcineurin A catalytic subunit CMP1 or CMP2 (data not shown).


Figure 5: The nonmyristoylated CNB1-G2A mutant protein is functional and renders yeast cells partially resistant to CsA, but not to FK506. Isogenic yeast strains lacking FKBP12 (Deltafpr1, JHY3-3D), cyclophilin A (Deltacpr1, MH250-2C), and calcineurin B (Deltacnb1, TB85) and the calcineurin B-deficient strain TB85 (Deltacnb1) expressing either wild-type CNB1 (Deltacnb1/CNB1) or the CNB1-G2A mutant (Deltacnb1/CNB1-G2A) were grown for 96 h at 30 °C on the following: A, YPD medium; B, YPD medium containing 200 mM LiCl; C, YPD medium containing 200 mM LiCl plus 100 µg/ml CsA; and D, YPD medium containing 200 mM LiCl plus 1 µg/ml FK506.



Calcineurin is also required for yeast cells to recover from alpha-factor pheromone-induced cell cycle arrest(26, 29, 30, 32) . The ability of the CNB1-G2A mutant protein to function and promote adaptation to pheromone was tested by halo assay. Cells lacking calcineurin B (Deltacnb1) were unable to recover from pheromone arrest (Fig. 6). In contrast, expression of either the wild-type or the G2A mutant calcineurin B protein enabled a calcineurin B mutant host strain to recover and resume growth following growth arrest by alpha-factor (Fig. 6). Thus, by a second measure, the nonmyristoylated CNB1-G2A mutant protein can functionally replace wild-type yeast calcineurin B.


Figure 6: Yeast cells expressing nonmyristoylated CNB1-G2A recover from alpha-factor-induced G(1) arrest. Cells of isogenic MATa yeast strains were plated in YPD top agar medium, and discs soaked with 10 µg of synthetic alpha-factor were placed on the surface. Arrest of cell growth by alpha-factor results in a zone of growth inhibition (halo) surrounding the disc. With prolonged incubation, wild-type MATa cells overcome this initial arrest, resume growth, and form colonies within the halo. In contrast, mutant strains that cannot recover from cell cycle arrest induced by alpha-factor yield halos that, even with prolonged incubation, remain clear. Portions of halos were photographed after 96 h of incubation at 30 °C. Isogenic strains were as follows: wild-type (WT), JK9-3da; Deltacnb1, TB85; Deltacnb1/CNB1, TB85 expressing wild-type CNB1; and Deltacnb1/CNB1-G2A, TB85 expressing the nonmyristoylated CNB1-G2A mutant protein.



Biological function of the nonmyristoylated calcineurin B mutant protein was also assessed in a yeast strain whose viability is calcineurin-dependent. In an independent study, we isolated a recessive mutation, cev1, which exhibits CsA-FK506 sensitivity and synthetic lethality with a calcineurin cnb1 mutation. (^2)Thus, the cev1 mutation renders calcineurin essential for viability, as in other CsA-FK506-sensitive strains(9, 31, 56, 57) . We tested if the CNB1-G2A calcineurin B mutant subunit supports calcineurin-dependent growth in a cev1 cnb1 double mutant strain. The cev1 mutant strain was mated to isogenic Deltacnb1::LEU2 strains of opposite mating type containing the control plasmid pRS316 or plasmids expressing wild-type CNB1 (plasmid pYDZ3) or the CNB1-G2A mutant protein (plasmid pYDZ10). The resulting diploids were sporulated, and meiotic segregants were analyzed by tetrad analysis. As previously observed, the cev1 and cnb1 mutations exhibited synthetic lethality. In contrast, with the cev1/CEV1 cnb1/CNB1 (CNB1 plasmid) and the cev1/CEV1 Deltacnb1/CNB1 (CNB1-G2A plasmid) diploids, dissection yielded 11 tetrads with 4 viable:0 inviable, six with 3 viable:1 inviable, and three with 2 viable:2 inviable segregants and 12 with 4 viable:0 inviable, seven with 3 viable:1 inviable, and one with 2 viable:2 inviable segregants, respectively. More important, many segregants were CsA^S FK506^S (cev1), Leu (Deltacnb1::LEU2), and Ura 5-FOA^S (CNB1 or CNB1-G2A plasmid present and essential). Thus, either wild-type CNB1 or the CNB1-G2A mutant protein restores viability to an inviable cev1 cnb1 mutant strain. In summary, as determined by three independent tests (cation resistance, recovery from alpha-factor, and viability in a calcineurin-dependent strain), myristoylation of calcineurin B appears not to be required for function.

Myristoylation Is Not Required for Calcineurin Association with the Cyclophilin A-CsA or FKBP12-FK506 Complex

We investigated whether myristoylation of yeast CNB1 is required for formation of immunophilin-drug-calcineurin complexes. Cell extracts from a strain lacking the chromosomal calcineurin B gene (cnb1Delta::LEU2) and from this mutant expressing wild-type CNB1 or the CNB1-G2A mutant protein from a low copy number plasmid were incubated with FKBP12 and cyclophilin A affinity matrices. Bound proteins were eluted from the matrices and analyzed by SDS-PAGE and Western blotting with antibodies against either yeast calcineurin B (Fig. 7B) or the yeast calcineurin A catalytic subunit CMP1 (Fig. 7A)(28, 32) . As shown in Fig. 7, wild-type CNB1 and the CNB1-G2A mutant protein bound equally well to both the FKBP12-FK506 and cyclophilin A-CsA complexes. In addition, FKBP12-FK506 and cyclophilin A-CsA binding to the calcineurin A catalytic subunit was not affected by the status of calcineurin B myristoylation. No binding occurred with cell extracts lacking calcineurin B, indicating that the regulatory subunit is required for the interaction between immunophilin and calcineurin A in the presence of drugs in vitro. Furthermore, the observation that both CMP1 and CNB1 are recovered to the same extent in the binding assays with wild-type and CNB1-G2A mutant proteins indicates that the nonmyristoylated CNB1 protein can associate with the calcineurin A CMP1 catalytic subunit as efficiently as the wild-type CNB1 protein does.


Figure 7: Myristoylated and nonmyristoylated calcineurin B proteins interact with FKBP12-FK506 and cyclophilin A-CsA complexes. Binding assays to His(6)-FKBP12-affigel 10 and His(6)-cyclophilin A-affigel 10 of cell extracts prepared from strain TB85 lacking CNB1 (Deltacnb1) and from strain TB85 expressing wild-type CNB1 (Deltacnb1/CNB1) or CNB1-G2A (Deltacnb1/CNB1-G2A) protein from a CEN plasmid were carried out in the absence or presence of 20 µM FK506 or 100 µM CsA. Following elution of the affinity matrices, proteins were analyzed by SDS-PAGE and Western blotting. The upper half of the blot (A) was probed with an affinity-purified calcineurin A antibody, and the lower half of the blot (B) was probed with calcineurin B antiserum. Arrows indicate the positions of migration of calcineurins A and B.



We next tested if myristoylation is required for calcineurin inhibition by immunophilin-drug complexes in vivo. A yeast calcineurin B-deficient strain was transformed with plasmid pYDZ3 expressing wild-type CNB1 and with plasmid pYDZ10 expressing the CNB1-G2A mutant. Sensitivity of the transformed yeast cells to CsA and FK506 was tested in the presence of LiCl. Yeast strains lacking cyclophilin A or FKBP12 were resistant to CsA plus LiCl or FK506 plus LiCl, respectively (Fig. 5, C and D). Growth of yeast cells expressing wild-type CNB1 was inhibited by either CsA or FK506 (Fig. 5, C and D). Interestingly, cells expressing the CNB1-G2A mutant protein were partially resistant to CsA plus LiCl (Fig. 5C), but not to FK506 plus LiCl (Fig. 5D). Although myristoylation of CNB1 is not required for cyclophilin A-CsA binding to calcineurin (Fig. 7), this observation suggests that it may be involved in inhibition of calcineurin by the cyclophilin A-CsA complex.


DISCUSSION

Physiological functions of myristoylation have been studied for viral and cellular proteins including Ras- and Src-related proteins (58) and G protein alpha-subunits(59) . Myristoylation can participate in protein-membrane associations and, in some cases, is required for biological function(59) . However, not all myristoylated proteins associate with membranes(60) , and for some proteins, such as the catalytic subunit of protein kinase A, myristoylation is not required for membrane association or biological function(58, 61) .

The calcineurin B regulatory subunit from mammals and yeast is covalently myristoylated at its amino-terminal glycine residue(29, 33) . In addition to the hydrophobic myristate, calcineurin is the target for two hydrophobic inhibitors, CsA and FK506, and acidic phospholipids bind to calcineurin B and activate calcineurin(62, 63) . Interactions between these hydrophobic moieties could influence calcineurin folding, assembly, activity, or association with proteins, substrates, or membranes. To test this, we introduced a G2A mutation to prevent myristoylation of yeast calcineurin B in vivo and used genetic and biochemical approaches to examine whether myristoylation of the calcineurin B regulatory subunit is required for cellular localization, biological functions, or association with and inhibition by the cyclophilin A-CsA or FKBP12-FK506 immunophilin-drug complex.

We find that both wild-type calcineurin B and the G2A mutant protein are localized to both cytosol and particulate fractions (Fig. 2). A portion of calcineurin B in the particulate fraction is detergent-extractable, indicating that calcineurin B is membrane-associated and that myristoylation is not required for this association (Fig. 3). A smaller fraction of the CNB1-G2A mutant protein was pelletable (Fig. 2), suggesting that the myristoyl group at least partially contributes to membrane association of yeast calcineurin B. Recent studies of Src-related proteins suggest how nonmyristoylated mutants of myristoylated proteins continue to associate with membranes. Three alternating amino-terminal lysine residues function, in conjunction with myristate, to bind proteins to membranes by electrostatic interactions with negatively charged membrane phospholipids(58, 64, 65, 66, 67, 68) . A similar amino-terminal basic amino acid motif in yeast calcineurin B (NH(2)-GAAPSKIDRDEIERLRKRFMKLDRD-COOH) may participate in membrane association.

Recoverin, a myristoylated member of the EF-hand superfamily that is a Ca sensor in the visual system, associates with membranes by a Ca-myristoyl switch in which Ca binding promotes extrusion of the myristoyl group and exposure of hydrophobic groups in recoverin(69, 70) . A similar GTP-myristoyl switch mediates GTP-dependent association of the ADP-ribosylation factor with membranes(71, 72) . A Ca-myristoyl switch could also operate in the calcineurin B regulatory subunit; purified calcineurin B contains exposed hydrophobic patches that predispose to aggregation(2) .

Because of the association of yeast CNB1 with the particulate fraction, we examined the possible association of yeast CNB1 with cytoskeletal matrices by detergent treatment of particulate fractions. This technique has been used to analyze cytoskeletal association of p60(49, 51, 73) , v-Fgr(74) , p120(75) , Nef of human immunodeficiency virus type 1(52, 76) , and the yeast SEC1 protein(53) . By Triton extraction of the yeast particulate fraction, a fraction of the yeast calcineurin B subunit is cytoskeleton-associated. The nonmyristoylated CNB1-G2A calcineurin B mutant protein was also associated with the cytoskeleton (Fig. 3), indicating that myristoylation is not required for this association. In mammals, calcineurin has also been found to be associated with the cytoskeleton in adrenal cells (77, 78) and neurons(79) .

Although we found that myristoylation of the calcineurin B regulatory subunit was not essential for association with cellular membranes or the cytoskeleton, myristoylation could have been required for calcineurin subunit interactions or activity. However, our genetic studies reveal that the CNB1-G2A mutant is fully functional in several independent biological assays, and we conclude that myristoylation of calcineurin B is not required for calcineurin function in yeast. Our findings are in accord with previous biochemical studies that revealed myristoylation of calcineurin B was not required for association of calcineurin A and B subunits (80, 81) or for association of heterologous calcineurin subunits and reconstitution of phosphatase activity (82) and with the recent demonstration that reconstituted nonmyristoylated calcineurin is enzymatically active in vitro(83) .

Calcineurin transduces signals required for T-cell activation and yeast pheromone response and regulates the renal Na,K-ATPase(5) , a potassium channel in plants(6) , and cation fluxes in yeast(4, 8, 9) . Myristoylation of the calcineurin B regulatory subunit could be required for only a subset of functions or under special conditions not explored in our assays. Alternatively, as in other examples where myristoylation is not detectably required for function, such as protein kinase A, the nonmyristoylated mutant could confer only subtle defects. In the case of protein kinase A, myristoylation is not required for activity, but does contribute to stability(84, 85) .

Numerous studies have established that the calcineurin B subunit is required for and plays a central role in FKBP12-FK506 and cyclophilin A-CsA binding and inhibition of calcineurin(20, 21, 23, 32, 86, 87, 88) . However, it had not been tested whether myristoylation of the calcineurin B subunit participated in binding or inhibition of calcineurin by the cyclophilin A-CsA or FKBP12-FK506 complex. We found that both wild-type calcineurin B and the nonmyristoylated G2A mutant protein bound to FKBP12-FK506 and cyclophilin A-CsA affinity matrices (Fig. 7B). In addition, the calcineurin A catalytic subunit CMP1 associated with either immunophilin-drug complex irrespective of whether calcineurin B was myristoylated or not (Fig. 7A). Thus, myristoylation is not required for association of calcineurin B with calcineurin A or with either immunophilin-drug complex.

We next tested whether myristoylation is required for inhibition of calcineurin by CsA or FK506. Expression of the nonmyristoylated G2A calcineurin B mutant protein rendered yeast cells partially resistant to CsA, but not to FK506 (Fig. 5). In vitro, the CNB1-G2A mutant protein bound the cyclophilin A-CsA complex to the same extent as wild-type CNB1 (Fig. 7). One possibility is that our in vitro assay is not sensitive enough to detect a subtle decrease in binding affinity or does not faithfully mimic in vivo binding conditions. Alternatively, the CNB1-G2A mutation may confer partial CsA resistance by inhibiting some step subsequent to binding required for full inhibition of calcineurin. The cyclophilin A-CsA complex is a noncompetitive calcineurin inhibitor(89) , and binding of cyclophilin A-CsA stimulates calcineurin activity toward the substrate p-nitrophenyl phosphate(14, 90) . Thus, binding of cyclophilin A-CsA likely induces conformational changes that inhibit or activate calcineurin. The calcineurin B myristoyl group may participate in these conformational changes.


FOOTNOTES

*
This work was supported in part by Council for Tobacco Research Grant 4050 (to M. E. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Depts. of Genetics and Pharmacology, Duke University Medical Center, P. O. Box 3546, CARL 322, Research Dr., Durham, NC 27710. Tel.: 919-684-2824; Fax: 919-684-5458.

(^1)
The abbreviations used are: CsA, cyclosporin A; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.

(^2)
C. Hemenway and J. Heitman, unpublished results.


ACKNOWLEDGEMENTS

We thank S. Muir and T. Breuder for technical assistance, R. Ye and T. Bretscher for antisera against CMP1 and actin, C. Hemenway for the cev1 mutant, J. Nevins for support, T. Starzl for assistance, T. Means and P. Casey for advice and comments, and S. Bowling for assistance with the manuscript. FK506 was from Fujisawa.


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