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Molecular and Cellular Biology, May 2008, p. 3336-3343, Vol. 28, No. 10
0270-7306/08/$08.00+0     doi:10.1128/MCB.00567-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Cdc1p Is an Endoplasmic Reticulum-Localized Putative Lipid Phosphatase That Affects Golgi Inheritance and Actin Polarization by Activating Ca²⁺ Signaling ^,

Eugene Losev, Effrosyni Papanikou, Olivia W. Rossanese, and Benjamin S. Glick^*

Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois 60637

Received 30 March 2007/ Returned for modification 19 June 2007/ Accepted 27 February 2008

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In the budding yeast Saccharomyces cerevisiae, mutations in the essential gene CDC1 cause defects in Golgi inheritance and actin polarization. However, the biochemical function of Cdc1p is unknown. Previous work showed that cdc1 mutants accumulate intracellular Ca²⁺ and display enhanced sensitivity to the extracellular Mn²⁺ concentration, suggesting that Cdc1p might regulate divalent cation homeostasis. By contrast, our data indicate that Cdc1p is a Mn²⁺-dependent protein that can affect Ca²⁺ levels. We identified a cdc1 allele that activates Ca²⁺ signaling but does not show enhanced sensitivity to the Mn²⁺ concentration. Furthermore, our studies show that Cdc1p is an endoplasmic reticulum-localized transmembrane protein with a putative phosphoesterase domain facing the lumen. cdc1 mutant cells accumulate an unidentified phospholipid, suggesting that Cdc1p may be a lipid phosphatase. Previous work showed that deletion of the plasma membrane Ca²⁺ channel Cch1p partially suppressed the cdc1 growth phenotype, and we find that deletion of Cch1p also suppresses the Golgi inheritance and actin polarization phenotypes. The combined data fit a model in which the cdc1 mutant phenotypes result from accumulation of a phosphorylated lipid that activates Ca²⁺ signaling.

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We identified the CDC1 gene in a screen for temperature-sensitive mutants with defects in the inheritance of late Golgi cisternae (33). All of the mutants that showed normal Golgi structure but had reproducible defects in Golgi inheritance carried mutations in CDC1. These cdc1 mutants also had depolarized actin. However, actin depolarization was not solely responsible for the Golgi inheritance phenotype because treatment of wild-type cells with the actin-depolymerizing drug latrunculin A (2) caused only a mild Golgi inheritance defect. In addition, Golgi inheritance was not affected in act1-DSE cells, which contain a mutant actin that is a poor substrate for myosin-driven transport (8, 33). On the other hand, the myo2-66 mutation, which compromises the actin-binding motor domain of the type V myosin Myo2p (24), caused a pronounced defect in late Golgi inheritance. These observations led to the following model for the inheritance of late Golgi cisternae (31, 33): late Golgi elements reach the bud by actin-dependent and -independent pathways, and Myo2p tethers them in the bud. cdc1 mutations probably disrupt both the actin-dependent transport and the Myo2p-dependent tethering, resulting in a strong defect in Golgi inheritance.

Testing this model will require knowing the biochemical function of Cdc1p. The original cdc1 mutants were isolated by Hartwell and colleagues in a screen for cell division cycle defects (15). The mutant cells were initially found to arrest with small buds, 2N DNA, and undivided nuclei, but later studies showed that most cdc1 cells actually arrested without buds (14). Subsequent work revealed that CDC1 is an essential gene and implicated its function in processes such as mating, spindle-pole body duplication, intrachromosomal recombination, and divalent cation homeostasis (6, 13, 28, 30, 39). Despite all of these connections, the biochemical function of Cdc1p remained unknown.

The strongest evidence about the function of Cdc1p came from a study linking it to Ca²⁺ regulation. Whole-cell Ca²⁺ levels in cdc1 mutants are highly elevated (29). cdc1 mutations activate calcineurin, a Ca⁺²/calmodulin-dependent protein phosphatase that mediates Ca²⁺ signaling via activation of the transcription factor Crz1p (9). Moreover, deletion of the calcineurin regulatory subunit CNB1 is synthetically lethal with cdc1 mutations (29). In cdc1 mutants, deletion of the plasma membrane Ca²⁺ channel Cch1p or its regulatory subunit Mid1p restores intracellular Ca²⁺ to wild-type levels and partially rescues the growth defect of cdc1 mutants (29). These data suggested that Cdc1p somehow affects intracellular Ca²⁺ levels and that the growth defect in cdc1 mutants is due in part to Ca²⁺ influx (29).

There is also evidence linking Cdc1p to Mn²⁺. cdc1 mutants were rescued by Mn²⁺ addition and were sensitive to the divalent cation chelator EGTA (25, 28). The EGTA sensitivity could be alleviated by overexpression of Smf1p (25), which is a metal ion transporter with high affinity for Mn²⁺ (39). Furthermore, an SMF1 deletion, which makes cells EGTA sensitive (39), could be rescued by CDC1 overexpression (28). Similarly, CDC1 overexpression rescued the EGTA sensitivity of cells carrying a deletion of PMR1, which encodes a Golgi body-localized Ca²⁺ and Mn²⁺ transporter (28). PMR1 overexpression exacerbated the cdc1 growth defect (28). Lastly, work on the frost gene, a Neurospora homolog of CDC1, revealed a connection to Mn²⁺ and Ca²⁺ (37). These observations led to the hypothesis that Cdc1p regulates Mn²⁺ homeostasis (28). This model predicted that intracellular Mn²⁺ levels in cdc1 mutants should be different from levels in wild-type cells. However, when the whole-cell Mn²⁺ content of cdc1 mutants was examined, no difference relative to wild type was observed (27). Thus, the link between Cdc1p and Mn²⁺ remained elusive.

A genetic screen for cdc1 suppressors revealed that cells are able to survive without CDC1 when PER1 (COS16) is deleted (27). Per1p is an endoplasmic reticulum (ER)-localized protein (17) that contains eight putative transmembrane domains. When the whole-cell Mn²⁺ content of per1 mutants was examined, there was no difference relative to wild-type cells, again arguing against a role of Cdc1p in Mn²⁺ homeostasis (27).

An alternative hypothesis states that Cdc1p is a Mn²⁺-dependent protein that affects Ca²⁺ levels (10). This idea was supported by a sequence analysis suggesting that Cdc1p has a phosphoesterase motif and is related to phosphatases that use Mn²⁺ as a cofactor (34). Here, we sought to test this alternative hypothesis. In addition, we asked whether the link between Cdc1p and Ca²⁺ could explain the effect of cdc1 mutations on Golgi inheritance and actin polarization.

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Yeast strains and reagents. Saccharomyces cerevisiae strains were grown in rich glucose medium (yeast extract, peptone, and dextrose [YPD]) or minimal glucose medium (synthetic dextrose [SD]) (35). SD-URA is SD medium lacking uracil. The experiments shown in Fig. 1 were performed with the haploid strain BGY103, which has the genotype leu2-3,112 ura3-52 rme1 trp1 his4 GAL⁺ HMLa SEC7-EGFP bar1::URA3 (33). All other experiments were performed with derivatives of the haploid strain JK9-3d, which has the genotype leu2-3,112 ura3-52 rme1 trp1 his4 (21).

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FIG. 1. Two cdc1 alleles show different sensitivities to Mn2+ but still exhibit activated Ca2+ signaling at the restrictive temperature. (a) Wild-type (WT), cdc1-310, and cdc1-314 mutant strains were grown to stationary phase and serially diluted (1:10) five times. The strains were replica plated on YPD medium at 25°C and 37°C, at 37°C with 2 mM MnCl2, and at 25°C with 2 mM EGTA. (b) The activation of Ca2+ signaling was measured using a CDRE-lacZ reporter. Wild-type, cdc1-310, and cdc1-314 cells were grown in SD-URA medium to early log phase and shifted to 33.5°C for the indicated times. Reporter activity was measured every hour for 3 h.

The cdc1-314 allele contains a T377I mutation (ACT to ATT), and the cdc1-310 allele contains an L356F mutation (ACT to ATT). Our CDC1 wild-type allele has a serine codon (ATC) at position 441 instead of a proline codon (ACC), as indicated in the Saccharomyces Genome Database. To make a cdc1-314 derivative of a given strain, the full-length CDC1 gene with a T377I mutation was integrated at the CDC1 locus to create a gene duplication. Successful integrant colonies were identified by PCR. One copy of CDC1 was then popped out using 5-fluoroorotic acid (Toronto Research Chemicals, Toronto, Canada), and the resulting colonies were screened for temperature sensitivity.

To delete the CCH1 gene, a fragment was PCR amplified from a cch1 strain created by the Saccharomyces Genome Deletion Project (http://www-sequence.stanford.edu/group/yeast_deletion_project/deletions3.html). This fragment, which included the kanMX gene flanked by 400 bp upstream of the CCH1 coding sequence and 500 bp downstream of the CCH1 coding sequence, was integrated into the genome by homologous recombination. A similar strategy was used to delete the MPK1 gene. The PER1 and GUP1 genes were deleted by replacement with LEU2 using PCR-based homologous recombination (3).

The inhibitor FK506 (LC Laboratories, Woburn, MA) was stored at –20°C in ethanol at 10 mg/ml. The final concentration of FK506 in cell cultures was 1 µg/ml.

Microscopy. Immunofluorescence microscopy was performed as previously described (33). Rabbit polyclonal anti-Kar2p antibody (a gift from Jack Rose) was used at a 1:5,000 dilution. Mouse monoclonal anti-myc antibody (clone 9E10; Roche, Indianapolis, IN) was used at 2 µg/ml.

Lipid analysis. Phospholipid radiolabeling, extraction, and separation by two-dimensional thin-layer chromatography (TLC) were performed as previously described (36). The lipid extracts were not dried with nitrogen because the deacetylation step was not needed. After extraction, the lipids were resuspended in solvent A (95% ethanol, water, diethyl ether, pyridine, concentrated ammonium hydroxide [15:15:5:1:0.018, by volume]) and resolved on TLC plates (Silica Gel 60-precoated plates for TLC; EMD, Germany). The results were quantified using a Molecular Dynamics Storm 860 PhosphorImager (GE Healthcare, Piscataway, NJ) with ImageQuant, version 1.2, software. For the lipid marked with the arrows in Fig. 6a, the signal was measured as a fraction of the total radioactivity present in all of the labeled species that migrated above the origin.

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FIG. 6. A phospholipid species accumulates in the cdc1-314 mutant at the restrictive temperature. (a) Wild-type (WT) and mutant strains were grown overnight in YPD medium supplemented with 32Pi. Upon reaching log phase, the cells were shifted to 33.5°C for 3 h. Lipids were extracted and run in two dimensions on TLC plates. These images from representative plates show the area where the major radioactive lipid species migrated. Arrows mark an unidentified phosphorylated lipid that was consistently present at higher levels in cdc1 cells than in wild-type cells. (b) The level of the unidentified radioactive lipid was measured in each of the indicated strains as a fraction of the total radioactive lipid, and the results were normalized by defining the signal from wild-type cells as 1.0.

β-Galactosidase and Golgi inheritance assays. β-Galactosidase reporter assays were performed using the CDRE-lacZ (38) and MPK1-lacZ (18) reporter plasmids. The assays were performed using a yeast β-galactosidase assay kit (Pierce, Rockford, IL). Error bars in the figures represent standard errors of the means.

Golgi inheritance was quantified as previously described (33). In brief, we measured the percentage of buds that contained late Golgi membranes as a function of bud size. Bud size classes III, IV, and V were combined into one class, termed III-V.

EndoH analysis. Total cellular protein was extracted by adjusting the culture to 10% trichloroacetic acid, incubating at 60°C for 5 min, chilling on ice for 5 min, centrifuging for 5 min at 3,000 x g, and resuspending in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer supplemented with 50 mM Na⁺-PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid); pH 7.5]. The samples were split into endoglycosidase H (EndoH)-treated and -untreated samples. EndoH-treated samples were incubated in 10 µl of EndoH buffer (Prozyme, San Leandro, CA), boiled for 10 min, chilled on ice for 5 min, treated with 2.5 µl of EndoH (30 µU/µl) (Prozyme), incubated at 37°C overnight, boiled for 10 min, and examined using SDS-PAGE. Untreated samples were boiled for 10 min in SDS-PAGE buffer and examined using SDS-PAGE. The separated proteins were transferred to polyvinylidene difluoride membranes, and tagged Cdc1p-myc was detected with a SuperSignal West Femto kit (Pierce, Rockford, IL) using peroxidase-conjugated anti-c-myc antibody (clone 9E10; Roche, Indianapolis, IN) at 1:1,000.

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Role of Mn²⁺ in Cdc1p function. We reasoned that if Cdc1p is a Mn²⁺-dependent protein that affects Ca²⁺ levels, then the Mn²⁺ and Ca²⁺ links could potentially be uncoupled in some cdc1 mutants. The previously described cdc1 strains had elevated intracellular Ca²⁺ at the restrictive temperature (25, 28). In addition, these strains were rescued by supplementing the medium with Mn²⁺ and were sensitive to the divalent cation chelator EGTA (28). Even though EGTA is not a specific Mn²⁺ chelator, the effect on cdc1 mutants was thought to be Mn²⁺ related because EGTA sensitivity could be rescued by Mn²⁺ addition or by overexpression of the Mn²⁺ transporter Smf1p (28). Thus, Mn²⁺ rescue and EGTA sensitivity are hallmarks of the link between Mn²⁺ and CDC1. We looked for new cdc1 alleles that did not exhibit Mn²⁺ rescue and EGTA sensitivity but still activated Ca²⁺ signaling. Fourteen different cdc1 mutants (33) were tested for their ability to grow in the presence of 2 mM MnCl₂ at 37°C or in the presence of 2 mM EGTA at 25°C. These mutants showed a wide range of sensitivities to EGTA, and some of them could be rescued by Mn²⁺. One mutant, cdc1-310, showed the best rescue response to Mn²⁺ and one of the more severe sensitivity responses to EGTA (Fig. 1a). Conversely, the cdc1-314 mutant was not rescued by Mn²⁺ and exhibited only a mild growth defect in the presence of EGTA (Fig. 1a). Despite having such different responses to Mn²⁺ and EGTA, the cdc1-314 and cdc1-310 strains had similarly elevated Ca²⁺ signaling pathway activation levels at the restrictive temperature as measured by the calcineurin-dependent response element-lacZ fusion reporter, CDRE-lacZ (38) (Fig. 1b). The assays were performed at 33.5°C to avoid any heat shock-related responses. The results indicate that the connections to Mn²⁺ and Ca²⁺ can be separated and favor the hypothesis that Cdc1p is a Mn²⁺-dependent protein that affects Ca²⁺ signaling.

This conclusion is also supported by sequence alignment data suggesting that Cdc1p has a metal ion-dependent phosphoesterase domain (34). We set out to test the importance of this domain for the function of Cdc1p. The residues Asp95, Asp144, Asn183, His184, His259, and His316 are predicted to chelate the divalent cation in the active site, so we mutated these residues individually to alanine (Fig. 2a). Whereas wild-type CDC1 on a CEN plasmid rescued the cdc1-314 mutant at the restrictive temperature, plasmids carrying the mutated versions of CDC1 failed to rescue (Fig. 2b). As a control, growth was rescued by a plasmid carrying a mutation of His287, which is not predicted to chelate the divalent cation. The wild-type and mutant Cdc1p proteins had similar localizations (see below) and expression levels (see Fig. S1 in the supplemental material). These results confirm that the putative divalent cation-chelating residues are important for Cdc1p function, suggesting that Cdc1p is indeed a phosphoesterase.

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FIG. 2. The putative phosphoesterase domain of Cdc1p is functionally important and faces the lumen of the ER. (a) Sequence analysis revealed a putative phosphoesterase domain (red box) and three hydrophobic putative transmembrane domains (green boxes). The conserved amino acid residues labeled in red are predicted to chelate the divalent metal ion. (b) Cdc1p mutants with alanine substitutions of the conserved residues indicated in panel a were expressed from a CEN plasmid in the cdc1-314 strain at 25°C and 37°C. A mutant of His287, which is not predicted to chelate the divalent metal ion, was used as a control. Lack of growth at 37°C shows that the mutated residues are important for Cdc1p function. (c) Cells expressing myc-tagged Cdc1p were immunolabeled with anti-myc antibody (red) and anti-Kar2p antibody (green). Scale bar, 5 µm. (d) The five potential N-linked glycosylation sites in Cdc1p were mutated separately or in combination in a myc-tagged Cdc1p protein, which was expressed as a second copy in the wild-type (WT) strain. The cell extracts were treated with EndoH and examined using SDS-PAGE. The lack of a mobility shift after EndoH treatment in the N215Q N233Q double mutant indicates that these residues are N glycosylated and are therefore in the lumen of the secretory pathway. (e) A diagram showing the inferred topology of Cdc1p.

The known Cdc1p temperature-sensitive mutations are in the putative phosphoesterase domain (33) (see Materials and Methods). Presumably, these mutations destabilize Cdc1p, and high Mn²⁺ levels can partially overcome the destabilization for alleles such as cdc1-310 but not for alleles such as cdc1-314. To avoid complications due to Mn²⁺ sensitivity, we used the cdc1-314 allele for the remainder of this study.

Cdc1p localization and topology. What might be the substrate of Cdc1p? To address this question, we examined the localization of myc-tagged Cdc1p. Surprisingly, Cdc1p-myc colocalized with the ER marker Kar2p (32) (Fig. 2c). A hydrophobicity analysis revealed that Cdc1p has three predicted transmembrane domains, implying that Cdc1p is an integral membrane protein of the ER. The phosphoesterase domain is between the first and second hydrophobic domains (Fig. 2a). To determine whether the phosphoesterase domain faces the cytosol or the ER lumen, we took advantage of the fact that N-linked glycosylation occurs only in the ER lumen (1). If a protein is glycosylated, treating it with EndoH will cleave off the N-linked glycan (26) and reduce the molecular weight. To determine if a potential N-linked glycosylation site is, in fact, glycosylated, we can mutate that site and ask whether EndoH treatment of the resulting protein shifts the molecular weight. Cdc1p has five potential N-linked glycosylation sites. When we simultaneously mutated Asn5, Asn434, and Asn461 to Gln and then treated with EndoH, we saw the same shift in molecular weight that was seen with the wild-type protein (Fig. 2d). When we individually mutated Asn215 or Asn233, which are located in the phosphoesterase domain, we observed a smaller shift (Fig. 2d). When Asn215 and Asn233 were simultaneously mutated, the molecular weight shift disappeared completely, and the EndoH-treated and -untreated mutant proteins had the same mobility as the EndoH-treated wild-type protein (Fig. 2d). These results indicate that only the phosphoesterase domain residues Asn215 and Asn233 are glycosylated, implying that the phosphoesterase domain faces the lumen of the secretory pathway. Our topology assignment is supported by previous data showing that the C terminus of Cdc1p is lumenally oriented (20). Together, these findings demonstrate that the phosphoesterase domain of Cdc1p faces the ER lumen (Fig. 2e).

Mpk1p activation in cdc1 cells. Our data suggest that inhibiting the enzymatic function of Cdc1p activates Ca²⁺ signaling. One possibility is that inhibition of Cdc1p triggers a cell stress response. This idea fits with the actin depolarization seen in cdc1 mutants (33) because actin becomes depolarized in cells experiencing stress (23). We tested whether cdc1 mutants were experiencing cell stress by examining the activity of Mpk1p, a key component of the cell stress signal transduction pathway (18). These experiments employed an MPK1-lacZ reporter (18, 23). The results showed that Mpk1p is activated in a cdc1 mutant at the restrictive temperature, consistent with a cell stress response (Fig. 3a).

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FIG. 3. Mpk1p is activated in the cdc1-314 mutant but is not the major cause of Ca2+ signaling activation. (a) An MPK1-lacZ reporter was used to measure Mpk1p activity in cdc1-314 mutant cells. Wild-type (WT) and mutant cells were grown in SD-URA medium to log phase and then shifted to 33.5°C for the indicated times. Reporter activity was measured every hour for 3 h. (b) A CDRE-lacZ reporter was used to measure the activation of Ca2+ signaling. Wild-type, cdc1-314, mpk1, and mpk1 cdc1-314 cells were grown in SD-URA medium to early log phase and shifted to 33.5°C for the indicated times. Reporter activity was measured every hour for 3 h.

The results presented thus far suggested that loss of Cdc1p function might cause cell stress and activate Mpk1p, which would then activate Ca²⁺ signaling. This interpretation seemed reasonable because it has been proposed that under some stress conditions, Mpk1p activates the plasma membrane Ca²⁺ channel Cch1p (4). To test whether Mpk1p activates Cch1p in a cdc1 mutant, we deleted MPK1 and then monitored Ca²⁺ signaling activation using the CDRE-lacZ reporter. Surprisingly, deletion of MPK1 caused only a small decrease in cdc1-induced Ca²⁺ signaling activation (Fig. 3b), implying that in cdc1 cells, Cch1p is activated primarily by an Mpk1p-independent pathway. These results argue against the hypothesis that a generalized cell stress response can explain the Ca²⁺ signaling activation in cdc1 mutant cells.

Relationship of Ca²⁺ signaling activation to the cdc1 mutant phenotypes. Because cdc1 mutations activate both Ca²⁺ signaling and Mpk1p, we wanted to know which of these pathways mediates the additional cdc1 phenotypes of defective Golgi inheritance and depolarization of actin. For this purpose, we deleted the Ca²⁺ channel Cch1p and performed a Golgi inheritance assay. The results showed that loss of Cch1p completely suppressed both the Golgi inheritance and actin polarization phenotypes (Fig. 4a and b). By contrast, when we deleted MPK1 in a cdc1 mutant, actin was still depolarized (Fig. 4b). We were unable to test the effect of MPK1 deletion on Golgi inheritance because many of the small-budded cells in the mpk1 mutant had lysed. Nevertheless, these results clearly indicate that in cdc1 mutants, Cch1p-dependent activation of Ca²⁺ signaling leads to defective Golgi inheritance and depolarization of actin.

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FIG. 4. Deletion of the Ca2+ channel Cch1p completely suppresses the Golgi inheritance and actin polarization phenotypes in the cdc1-314 mutant. (a) The inheritance of late Golgi cisternae was measured by assessing the presence of the late Golgi marker protein Sec7p-GFP in the buds of wild-type (WT), cdc1-314, cch1 cdc1-314, and cch1 cells after a shift to 33.5°C for 3 h. (b) Actin was visualized by staining with Alexa 594-phalloidin. Wild-type, cdc1-314, cch1 cdc1-314, cch1, mpk1, and mpk1 cdc1-314 cultures were grown at 25°C, shifted to 33.5°C for 3 h, fixed, and stained. Fluorescence images are shown. Most of the wild-type, cch1 cdc1-314, cch1, and mpk1 cells contained polarized actin patches and cables, whereas most of the cdc1-314 and mpk1 cdc1-314 cells contained depolarized patches and few visible cables. Scale bar, 5 µm.

In a further test of the importance of Ca²⁺ signaling activation for the cdc1 mutant phenotypes, we introduced the per1 mutation, which strongly suppresses the growth defect of cdc1 strains (27). As expected, a per1 cdc1 double mutant showed no Ca²⁺ signaling activation at the restrictive temperature (see Fig. S2 in the supplemental material).

One effector of Cch1p-dependent Ca²⁺ influx is the Ca²⁺/calmodulin-dependent phosphatase calcineurin (9, 29). By activating the transcription factor Crz1p, calcineurin turns on many genes (9). We tested whether the growth and actin polarization phenotypes of cdc1 cells are caused by calcineurin activation. This experiment employed the potent calcineurin inhibitor FK506 (9). When cdc1 cells were incubated at 33.5°C, the addition of FK506 did not rescue the growth defect, nor did it prevent actin depolarization (Fig. 5b and c). As a control, FK506 was added to cdc1 cells, where it completely suppressed the elevation of CDRE-lacZ activity (Fig. 5a). Thus, the growth and actin polarization phenotypes seen in cdc1 mutants are not due to the activation of calcineurin but must be mediated by a separate Ca²⁺-dependent pathway.

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FIG. 5. Treatment of cdc1-314 cells with the calcineurin inhibitor FK506 does not suppress the growth and actin polarization phenotypes. (a) Activation of Ca2+ signaling was measured using the CDRE-lacZ reporter. Wild-type (WT) and cdc1-314 cells were grown in 10 ml of SD-URA medium to early log phase and shifted to 33.5°C for the indicated times. Immediately before the shift, 10 µl of dimethyl sulfoxide or 1 mg/ml of FK506 in dimethyl sulfoxide was added. The final concentration of FK506 in cultures was 1 µg/ml. Reporter activity was measured every hour for 3 h. (b) Cell growth was measured by taking the absorbance at 600 nm. Cultures were treated as described in panel a. Readings were taken every hour for 3 h. (c) Actin was visualized by staining with Alexa 594-phalloidin. Cultures were treated as described in panel a. After 3 h, the cells were fixed and stained. Fluorescence images are shown. Most of the treated and untreated wild-type cells contained polarized actin patches and cables, whereas most of the treated and untreated cdc1-314 cells contained depolarized patches. Scale bar, 5 µm.

Accumulation of a phospholipid in cdc1 cells. To understand how Cdc1p affects Ca²⁺ signaling, it is important to clarify the enzymatic function of Cdc1p. The topology assignment constrains the possible substrates for the phosphoesterase domain. Proteins are unlikely substrates because, to our knowledge, there are no protein kinases or phosphatases in the ER lumen. However, several lipid phosphatases are present in this compartment (11, 19). We therefore hypothesized that a phosphorylated lipid might be the substrate for Cdc1p. To test this idea, we radiolabeled wild-type and cdc1 cells with ³²P_i, extracted the radioactive lipids, and performed two-dimensional TLC analysis. A phospholipid species reproducibly accumulated in the cdc1 mutant at the restrictive temperature (Fig. 6a). Quantitation indicated that after 3 h at 33.5°C, this unidentified lipid was approximately twice as concentrated in cdc1 cells as in wild-type cells (Fig. 6b).

We postulate that in cdc1 mutants, abnormal accumulation of a lipid leads to hyperactivated Cch1p-dependent Ca²⁺ signaling, which in turn causes toxicity. This model predicts that a cdc1 strain carrying a cch1 suppressor mutation should still accumulate the unidentified lipid. Indeed, in a cch1 cdc1 double mutant, the unidentified lipid was about twice as concentrated as in the cch1 single mutant (Fig. 6b). Interestingly, in a per1 strain the unidentified lipid was about twice as concentrated as in wild-type cells, and this concentration was not enhanced in a per1 cdc1 double mutant (Fig. 6b). It therefore seems likely that the per1 and cch1 mutations suppress the cdc1 phenotypes by different mechanisms.

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Our findings add to the evidence that Cdc1p is a Mn²⁺-dependent protein. Residues predicted to chelate a divalent metal ion in the active site are crucial for Cdc1p function. This interpretation explains why genes that affect Mn²⁺ levels show genetic interactions with CDC1. For example, the ability of CDC1 overexpression to rescue the EGTA sensitivity of an smf1 strain can be explained if Cdc1p is the most Mn²⁺-sensitive essential protein in the ER because loss of Smf1p would inhibit Cdc1p by reducing the Mn²⁺ concentration in the ER. However, the analysis is complicated by the effect of Cdc1p on Ca²⁺ signaling. In some cases the genetic interactors of CDC1 influence both Ca²⁺ and Mn²⁺. For example, the genetic interaction with the Golgi body-localized transporter Pmr1p might reflect the ability of Pmr1p to transport both Ca²⁺ and Mn²⁺ (22). Here, we describe a cdc1 allele that does not cause enhanced sensitivity to Mn²⁺ levels but still exhibits Ca²⁺ accumulation, indicating that the Ca²⁺- and Mn²⁺-related phenotypes can be uncoupled.

We propose that Cdc1p is a Mn²⁺-dependent ER-localized phosphatase, with the active site facing the lumen. The substrate for Cdc1p remains to be identified. An ER-localized phospholipid is an attractive candidate because phosphorylated lipids such as dolichol phosphate and sphingosine phosphate are present in the ER lumen (11, 19). The enzymes that dephosphorylate these lipids—Lcb3p and Cwh8p, respectively—resemble Cdc1p in being multispanning ER membrane proteins with phosphatase domains facing the lumen (11, 19). Our preliminary experiments with the dolichol and sphingosine metabolic pathways did not yield any clear link to Cdc1p. However, TLC analysis revealed that cdc1 cells accumulate at least one unidentified lipid. The substrate for Cdc1p might be a lipid that has not yet been detected in yeast. A very speculative candidate is ceramide phosphate, which is present in animal cells but has not been described in yeast cells (7).

Ongoing work from other groups may shed light on the biochemical activity of Cdc1p. One of the strongest genetic interactors of CDC1 is PER1 (27). Deletion of PER1 is able to suppress a CDC1 deletion (27; also unpublished results). PER1 is involved in the processing of proteins with a glycosylphosphatidylinositol (GPI) anchor (Davis Ng, personal communication), and a recent paper showed that PER1 is required for GPI-phospholipase A₂ activity (12). We suggest that PER1 is needed to generate the substrate for Cdc1p (Fig. 7a). Thus, inactivating Cdc1p leads to accumulation of a toxic phosphorylated lipid, but simultaneous deletion of PER1 blocks activity earlier in the pathway and therefore prevents toxicity. Another strong cdc1 suppressor is GUP1 (Stephen Garrett, personal communication), which we identified independently using an insertional mutagenesis screen for suppression of cdc1 temperature sensitivity (data not shown). Like Per1p, Gup1p is a transmembrane ER protein (16, 17). Gup1p is reportedly an enzyme that adds C₂₆ fatty acids to the GPI anchor (5), and a gup1 mutation is comparable to a per1 mutation in its ability to suppress the cdc1 temperature sensitivity phenotype (see Fig. S3 in the supplemental material). These observations imply that the putative lipid phosphatase activity of Cdc1p is linked in some manner to GPI remodeling.

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FIG. 7. A model for Cdc1p function and the link between Cdc1p and Golgi inheritance. (a) Cdc1p is presumed to be a lipid phosphatase, and we suggest that Per1p is needed to generate the substrate for Cdc1p. According to this model, inactivating Cdc1p causes its substrate to accumulate to toxic levels and also causes accumulation of a nontoxic precursor lipid. When Per1p is inactivated, only the nontoxic precursor accumulates, so a per1 mutation suppresses the lethality of cdc1 mutations. This nontoxic precursor may correspond to the unidentified lipid that was visualized in the experiment shown in Fig. 6. (b) In cdc1 cells, accumulation of the toxic phosphorylated lipid activates both Mpk1p- and Cch1p-dependent Ca2+ signaling. The Ca2+ signaling activates calcineurin. In a calcineurin-independent pathway, strong Ca2+ signaling depolarizes actin. We speculate that strong Ca2+ signaling also inhibits Golgi tethering. These two effects of strong Ca2+ signaling synergize to cause a pronounced defect in Golgi inheritance.

The results presented here allow us to formulate a model of how cdc1 mutations affect Golgi inheritance. In cdc1 cells, accumulation of a toxic phosphorylated lipid activates Cch1p-dependent Ca²⁺ signaling. One possible activation mechanism is that the accumulated lipid leaves the ER and directly stimulates Cch1p at the plasma membrane. Alternatively, the accumulated lipid might act locally to promote Ca²⁺ release from the ER, thereby depleting ER-associated Ca²⁺ and triggering Ca²⁺ influx via Cch1p. In either case, Ca²⁺ signaling activation leads to a defect in actin polarization. We speculate that Ca²⁺ signaling activation also inhibits Golgi tethering. Together, these two effects of Ca²⁺ signaling disrupt the inheritance of late Golgi cisternae (Fig. 7b).

It is still not entirely clear why our screen for Golgi inheritance defects uniquely and repeatedly identified cdc1 mutants because activation of Ca²⁺ signaling by other means such as hyperosmotic stress (23) did not cause Golgi inheritance defects (data not shown). We propose that in cdc1 mutants, the Ca²⁺ signaling activation is exceptionally strong and therefore results in multiple downstream defects (Fig. 7b).

 
We thank Stephen Garrett, Davis Ng, Kyle Cunningham, and members of the Glick lab for helpful discussion. We are grateful to Mark Rose, David Levin, and Martha Cyert for providing reagents.

This work was supported by NIH grant GM-61156 (B.S.G.) and NIH training grant 5-20942 (E.L.).

 
* Corresponding author. Mailing address: Department of Molecular Genetics and Cell Biology, The University of Chicago, 920 East 58th Street, CLSC 801, Chicago, IL 60637. Phone: (773) 702-5315. Fax: (773) 702-3172. E-mail: bsglick{at}uchicago.edu

Published ahead of print on 10 March 2008.

Supplemental material for this article may be found at http://mcb.asm.org/.

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Molecular and Cellular Biology, May 2008, p. 3336-3343, Vol. 28, No. 10
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