Article |
Address correspondence to Randolph Y. Hampton, Division of Biology, University of California San Diego, 9500 Gilman Dr. 0347, La Jolla, CA 92093-0347. Tel.: (858) 822-0511. Fax: (858) 534-0555. E-mail: rhampton{at}biomail.ucsd.edu
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Abstract |
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Key Words: ion transport; magnesium; protein folding; endoplasmic reticulum; Saccharomyces cerevisiae
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Introduction |
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Because fungi do not posses SERCA ATPases, the mechanisms by which calcium is supplied to the yeast ER had remained enigmatic. The absence of SERCA pumps led several groups to suggest that the yeast ER is supplied with calcium solely by the action of the Golgi-localized pump Pmr1p (Duerr et al., 1998; Marchi et al., 1999; Strayle et al., 1999). This model is reasonable because pmr1 mutants have a number of phenotypes related to the function of the ER (Duerr et al., 1998; Strayle et al., 1999). Like SERCA, Pmr1p belongs to the type IIa family of P-type ATPases and some phenotypes of pmr1
mutants can be complemented by expression of SERCA1a or related ATPases (Liang et al., 1997; Duerr et al., 1998; Talla et al., 1998; Degand et al., 1999). Pmr1p has been shown to transport both calcium and manganese, but how the Golgi apparatuslocalized Pmr1p supplies calcium to the ER is unclear (Mandal et al., 2000; Wei et al., 2000).
We and others have recently identified another P-type ATPase in yeast, Cod1p/Spf1p, that adds a new dimension to the regulation of ER ion supply (Suzuki and Shimma, 1999; Cronin et al., 2000). Cod1p belongs to the type V subfamily of P-type ATPases that are broadly conserved in eukaryotes but for which no ionic substrates have yet been identified (Axelsen and Palmgren, 1998). Close homologues of Cod1p are present in Drosophila melanogaster, Caenorhabditis elegans, Dictyostelium discoideum, Arabidopsis thaliana, and humans (Costanzo et al., 2000). Cod1p is the only member of the type V subfamily for which any phenotypic information is known. We identified COD1 in a genetic screen for mutants incapable of regulating the degradation of the ER membrane protein Hmg2p (Cronin et al., 2000). The involvement of Cod1p in the degradation of an ER protein as well as other ER phenotypes implies that Cod1p functions in the ER (Suzuki and Shimma, 1999; Cronin et al., 2000). Furthermore, the cod1 mutant phenotype suggests a role for Cod1p in calcium regulation because the mutant phenotype could be partially suppressed by exogenous calcium (Cronin et al., 2000). These observations led us to propose that Cod1p functions to supply Ca2+ to the ER (Cronin et al., 2000).
In this paper, we directly examined the role of Cod1p in ER function and calcium regulation in vivo and the biochemical requirements for Cod1p ATPase activity in vitro. We have now demonstrated that Cod1p localized to the ER by both immunofluorescence and subcellular fractionation, and have provided phenotypic evidence supporting a role for Cod1p in ER function and cellular calcium regulation. Further, we have distinguished phenotypically the roles of Cod1p and Pmr1p in ER function and quality control. Finally, we purified the Cod1 protein and directly demonstrated the ATPase activity of Cod1p in vitro. Taken together, our data indicated that Cod1p plays a significant role in ER function and cellular Ca2+ homeostasis that is not equivalent to, nor redundant with, the role of Pmr1p or other P-type ATPases. Moreover, the presence of Cod1p homologues in the genomes of all sequenced metazoans suggested that the function of Cod1p is likely to be more widely conserved in eukaryotes than that of more restricted, but better known, pumps such as the SERCA ATPases.
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Results |
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Deletion of Cod1p induced expression of calcium-regulated genes
Calcium concentration within the cytosol and secretory pathway regulates the expression of a number of genes, and mutations that perturb cellular calcium induce compensatory changes in gene expression (Garrett-Engele et al., 1995; Matheos et al., 1997; Locke et al., 2000). For instance, deletion of PMR1 produces a five- to sevenfold increase in the transcription of PMC1, a vacuolar calcium ATPase (Marchi et al., 1999, Locke et al., 2000). We investigated the effect of cod1 deletion on the expression of FKS2, PMC1, and ENA1, genes known to be regulated in response to calcium concentration, using previously described ß-galactosidase reporter plasmids (Fig. 2, A and B) (Matheos et al., 1997; Locke et al., 2000). The expression of each of the three calcium-activated reporters was three- to sevenfold higher in the cod1 mutant than in wild-type cells grown under normal laboratory conditions and in the absence of any external calcium stimulus. This effect of cod1
was similar to that reported earlier for pmr1
(Locke et al., 2000). Importantly, transient treatment with 200 mM CaCl2 induced ß-galactosidase activity to the same level both in wild-type and cod1
cells, indicating that deletion of COD1 did not interfere with the ability of the reporter constructs to respond to calcium stimulation (unpublished data). Likewise, treatment of cultures with the calcineurin inhibitor cyclosporin A diminished expression of the reporter genes, indicating that the increased expression in cod1
was largely calceneurin dependent (Fig. 2 B; unpublished data). A portion of the increase may be calcineurin independent, as the treatment did not reduce expression to the same level as in wild type. The increased ß-galactosidase activity in the cod1
mutants was specific to the calcium-regulated reporters. Expression of the CYC1ß-galactosidase reporter, which is not responsive to calcium (Matheos et al., 1997), did not differ significantly between wild-type and cod1
strains (Fig. 2 A).
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Cod1p is necessary for normal ER function
The localization of Cod1p to the ER and the previously reported phenotypes of the cod1 mutants suggested that Cod1p plays a major role in the function of the ER. We tested the sensitivity of cod mutants to the reducing agent DTT and the glycosylation inhibitor tunicamycin, both of which (through distinct mechanisms) alter protein folding in the ER (Fig. 4 A). cod1-1 cells had twice the sensitivity to tunicamycin as wild-type cells, but maintained normal sensitivity to DTT. Treatment of wild-type cells with either agent provokes the accumulation of unfolded proteins in the ER, inducing the unfolded protein response (UPR) (Frand and Kaiser, 1998; Pollard et al., 1998). We measured the unfolded protein response with a UPR reporter plasmid (4xUPREgreen fluorescent protein [GFP]) consisting of four unfolded protein response elements driving expression of the GFP coding region (4xUPRE; Fig. 4 B; Pollard et al., 1998). Strikingly, the GFP reporter in the cod1 mutant was constitutively expressed at 10 times the level of expression in wild-type cells in the absence of any additional stimulus. Furthermore, in cod1
, maximum expression of the UPR reporter required only half the dose of tunicamycin (2 µg/ml) needed to stimulate maximum expression in wild-type cells. As expected from the growth sensitivity experiments, the cod1
mutant displayed wild-type sensitivity to DTT in the unfolded protein response. Maximal induction of the reporter protein required 2 mM DTT in both wild-type and cod1
.
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pmr1 mutants have phenotypes indicative of a perturbed UPR, including hypersensitivity to DTT and tunicamycin, inositol auxotrophy, and increased expression of the ER chaperone Kar2p (Duerr et al., 1998). We examined the viability of a pmr1 hac1
double mutant in the same genetic background in which cod1
hac1
cells were inviable (Fig. 4 D). Surprisingly, the pmr1
hac1
double mutants were viable. The pmr1
hac1
double mutant retained phenotypes of both parent strains: hypersensitivity to EGTA (pmr1
) and inositol auxotrophy (hac1
). The cod1
mutant was prototrophic for inositol in all strains tested (unpublished data). Thus, although Cod1p and Pmr1p had synthetic effects on whole cell calcium that implied shared functions, they also clearly had distinct, nonoverlapping ER functions.
Regulated degradation of Hmg2p does not require the UPR
The constitutive activation of the UPR and the constitutive degradation of Hmg2p in the cod1 mutant raised the possibility that UPR played a role in regulating Hmg2p degradation (cod1
mutants cannot regulate the degradation of Hmg2p (Cronin et al., 2000). In wild-type cells, treatment with lovastatin (a 3-hydroxy-3-methylglutaryl coenzyme A reductase [HMGR] inhibitor) slows Hmg2p degradation and raises Hmg2p levels (Hampton and Rine, 1994). This regulation can be measured by flow cytometry using the Hmg2pGFP reporter protein (Hampton et al., 1996; Gardner and Hampton, 1999). We tested the effect of UPR activation on the regulated degradation of the Hmg2pGFP by treatment with the glycosylation inhibitor tunicamycin (Fig. 5 A). Hmg2pGFP degradation in cells treated with 4 µg/ml tunicamycin (a dose that fully induces the UPR) still responded to treatment with lovastatin in the same manner as cells grown without tunicamycin. In fact, Hmg2pGFP degradation was moderately slowed by tunicamycin treatment. Thus, the unregulated, constitutive degradation of Hmg2p present in cod1 mutants cannot be attributed to activation of the unfolded protein response or to defective glycosylation. We deleted HAC1 to determine if the unfolded protein response was required for regulation of Hmg2p degradation (Fig. 5 B). The regulated degradation of Hmg2pGFP in hac1
cells treated with lovastatin was indistinguishable from that seen in wild-type cells under the same treatment despite reports that some degradation substrates require the UPR for ER-associated degradation.
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Biochemical characterization of Cod1p
To directly study the biochemistry of Cod1p, we constructed a gene that expressed a modified version of the Cod1 protein with nine histidine residues and an HA epitope tag inserted just after the amino-terminal methionine. The plasmid complemented the cod1 mutant phenotype (Fig. 7). Cells expressing the histidine-tagged Cod1 protein from the native COD1 promoter at single copy or the much stronger TDH3 promoter from a 2µ plasmid were wild type for regulating Hmg2pGFP degradation (Fig. 7; unpublished data). We also constructed a derivative of this plasmid expressing the mutant D487N-Cod1p. In this mutant protein, the aspartyl phosphorylation site strictly conserved in all P-type ATPases was replaced by an asparagine residue. The plasmid expressing the D487N-Cod1p was unable to complement the cod1
null phenotype as expected from previous studies (Fig. 7; Suzuki, 2001).
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It has previously been demonstrated that the ATPase activity of P-type pumps can be stimulated by the presence of its ionic substrates. Thus, stimulation of ATPase activity by an ion normally indicates that the ion is transported by the ATPase. For example, the ATPase activity of the Golgi apparatuslocalized Pmr1p is stimulated with calcium and manganese, the substrates it transports (Mandal et al., 2000). Accordingly, we tested calcium to see if it would stimulate the ATPase activity of Cod1p (Fig. 9, A and B). Surprisingly, calcium failed to increase the ATPase activity of Cod1p. In fact, higher concentrations of calcium (>10 µM free calcium) inhibited the activity of Cod1p (Fig. 9, A and B). We next tested various biologically relevant cations with the aim of identifying which ion was the substrate of Cod1p. The range of ions tested included Ca2+, Sr2+, Ba2+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, and Cd2+, none of which stimulated activity above the level seen with magnesium alone (Fig. 9, A and B; unpublished data). Replacing the K+ in the assays with Li+, Na+, Rb+, or Cs+ did not affect the activity of the protein (unpublished data). To ensure that trace amounts of any possible contaminating cations were not stimulating the activity of Cod1p, we assayed the ATPase activity in the presence of increasing concentrations of the chelators TPEN, 1,10-phenanthroline, and EDTA (Fig. 9 C). TPEN and 1,10-phenanthroline have been used to remove metals such as zinc from metalloenzymes (Arslan et al., 1985; Dawson et al., 1986; Bays et al., 2001). None of the chelators diminished the activity of Cod1p; rather, the chelating agents all stimulated the activity of the enzyme, suggesting that heavy metals may inhibit the activity of the enzyme. The only ion for which Cod1p showed any dependence was Mg2+.
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Discussion |
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Our studies clearly show that Cod1p is a functionally important ER protein. Unique among yeast P-type ATPases, Cod1p localized to the ER membrane, as measured by immunofluorescence and direct biochemical analysis. The Cod1p subcellular localization is consistent with the functions and phenotypes of this ion pump. Our study and other studies had previously demonstrated phenotypes in ER-associated processes such as N-linked glycosylation and regulation of HMGR stability (Suzuki and Shimma, 1999; Cronin et al., 2000). The experiments above showed that Cod1p was critical in maintaining the proper ER folding environment and was required for viability of mutants deficient in the UPR, presumably due to its role in maintaining ER ion balance, as discussed below. Furthermore, at least in our strains, Cod1p had a more important role in this regard than Pmr1p, because the same synthetic lethality with hac1 was not observed with a pmr1
null. Taken together, our localization and phenotypic studies showed Cod1p to be a critical and conserved participant in ER function, with numerous phenotypes that arise from its action on ER physiology.
The identity of Cod1p as a P-type ATPase and its importance in the normal functioning of the ER suggests that it functions to control the balance of one or more ions in the lumen of the ER. The idea that this ion might be calcium first arose from our previous observations of cod1 suppression by manipulating exogenous calcium (Cronin et al., 2000). In this work, we further examined the role of Cod1p in calcium maintenance in a variety of ways. Increased expression of calcineurin-related genes can indicate depletion of secretory pathway calcium store, and a cod1 null showed the expected increase in expression of the calcineurin-activated genes PMC1, ENA1, and FKS2 (Cunningham and Fink, 1996; Matheos et al., 1997). In this assay, it is interesting to note that the magnitude of the effect of COD1 inactivation was similar to that reported for pmr1
nulls (Locke et al., 2000). Similarly, the specific growth hypersensitivity of cod1
strains to calcium chloride is reminiscent of the growth sensitivities of mutants lacking calcium-specific P-type ATPases. For example, deletion of the vacuolar calcium ATPase PMC1 results in calcium hypersensitivity, and deletion of the Golgi apparatus ATPase PMR1 leads to calcium and manganese hypersensitivity (Cunningham and Fink, 1994a; Lapinskas et al., 1996; Wei et al., 2000). Finally, the synergistic effect of cod1
and pmr1
on total cellular calcium, but no other ions, also pointed to a role for COD1 in maintaining cellular calcium homeostasis.
Although the calcium-related phenotypes were suggestive of direct involvement in calcium transport, our cross-complementation experiments and biochemical data suggested that Cod1p may influence the ER in a different manner. The known Ca2+_transporting ATPases group together by sequence homology (type II) and are generally capable of substituting for each other's function in vivo. This property has been used extensively to clone new Ca2+ pumps and aid in the characterization of Ca2+ specificity of pumps cloned by other means (Liang et al., 1997; Harper et al., 1998; Talla et al., 1998; Degand et al., 1999). For example, SERCA1a from rabbit, ECA1 from A. thaliana, and SMA1 from Schistosoma mansoni all restore some functions to yeast mutants lacking the genes encoding the endogenous Ca2+ pumps Pmr1p and Pmc1p (Liang et al., 1997; Talla et al., 1998; Degand et al., 1999). It should be noted that the ability of exogenous pumps to complement yeast mutants does not strictly depend on the localization of the exogenous pump. Thus, functions of the Golgi apparatuslocalized Pmr1p and the vacuolar Pmc1p can be substituted by the exogenous SERCA1 pump localized to the ER in yeast (Degand et al., 1999). In contrast to the apparent facility with which these pumps replace each other, neither SERCA nor ACA2, nor overexpressed PMR1, could suppress phenotypic defects of the cod1 null mutant. Similarly, overexpression of Cod1p did not suppress the EGTA sensitivity of pmr1
. Thus, if Cod1p contributes to the ER by directly pumping calcium, it must be doing so in a manner distinct from the previously known type II calcium pumps.
To further examine whether or not Cod1p was directly involved in calcium transport, we purified the Cod1 protein to identify the biochemical requirements for Cod1p ATPase activity in vitro. P-type ATPases exhibit enhanced activity in the presence of their substrates, such that the ATPase activity of SERCA1 is stimulated by calcium, the sodium/potassium ATPase by sodium and potassium (Koenderink et al., 2000), Pmr1p by manganese and calcium (Mandal et al., 2000), and the Menkes protein by copper (Voskoboinik et al., 2001). Surprisingly, calcium was not required for the biochemical activity of Cod1p, and at higher concentrations (>100 µM), Ca2+ inhibited activity. In fact, in the apparent absence of any substrate, Cod1p hydrolyzed ATP at a rate similar to that of other P-type ATPases. For example, Pmr1 has a Vmax of near 200 nmol Pi/min/mg in the presence of its preferred substrates Ca2+ and Mn2+ (Mandal et al., 2000), whereas Cod1p exhibits a Vmax for ATP hydrolysis of 150 nmol Pi/min/mg in reactions containing solely the eluted product, magnesium, potassium, and micromolar quantities of sodium. The metal chelators EDTA, TPEN, and 1,10-phenanthroline each activated (or de-inhibited?) Cod1p activity when used at concentrations that should have greatly reduced the free concentration of any divalent ions in the assay with the exception of magnesium. Cod1p appears to be acting quite differently from known P-type pumps.
A possible explanation is that the in vitro ATPase activity demonstrated by Cod1p is simply uncoupled from pumping activity. This might occur if the factors coupling Cod1p to a specific ion or ions were lost during the purification of the enzyme. This model suggests that the inability of the Drosophila homologue of Cod1p to complement the cod mutant at low copy was due to poor interaction of dCod1p with the required factors in yeast. A requirement for specificity-determining factors also leaves open the possibility for a direct role for Cod1p in calcium transport. Such coupling factors would be quite unexpected. The ATPase activity of some pumps bas been uncoupled from transport by cis mutations, and several mammalian P-type ATPases have ß subunits. However none of the ß subunits appear to modify the substrate specificity of the enzyme. Additionally, although we have been able to measure calcium transport by Pmr1p, Pmc1p, rabbit SERCA, and other Ca2+-specific ATPases in yeast microsomes (Sorin et al., 1997; Marchi et al., 1999; Ton et al., 2002), we have been unable to detect calcium transport by Cod1p-containing microsomes (unpublished data).
Another possibility is that magnesium is a substrate for Cod1p. P-type ATPases in general need free magnesium in order to hydrolyze ATP. The concentration of free magnesium needed for activation depends on the concentration of ATP present, such that generally the ratio of magnesium to ATP needed falls within the range of 13 mol magnesium per mol ATP (Ahlers, 1981; Plesner and Plesner, 1981; Brooker and Slayman, 1983; Skou, 1974). In the case of Cod1p, we obtained the greatest activity at a relatively high concentration of 10 mM magnesium, a concentration 200 times the concentration of ATP in the assay mix. A role for Cod1p in Mg2+ is attractive given the biological importance of magnesium. At present, magnesium supply and regulation in yeast are poorly understood and information about the intracellular distribution of magnesium is scarce (Beeler et al., 1997). ATPases in the lumen of the ER, such as Kar2, and other enzymes within the secretory pathway would presumably need magnesium in the lumen. It would seem fitting that yeast cells would have a way to transport magnesium into the lumen of the ER. Although the high level of magnesium needed for the activity of Cod1p does not demonstrate that Cod1p pumps magnesium, it suggests magnesium may be a good starting point for further biochemical study.
A third possibility is that Cod1p transports nonmetallic substrate. Recently, several groups have gathered evidence that the type IV group of P-type ATPases are involved in the translocation of aminophospholipids necessary for the maintenance of membrane lipid asymmetry (Tang et al., 1996; Chen et al., 1999; Ding et al., 2000; Gomes et al., 2000), though this idea has been challenged (Siegmund et al., 1998; Marx et al., 1999). The type IV ATPases that have been studied, including the Golgi apparatuslocalized yeast protein Drs2p, are apparently specific for phosphatidylserine. Yeast lacking DRS2 are defective in translocating a fluorescently labeled phosphatidylserine, a defect that can be complemented by expression of the A. thaliana ALA1, and the ATPase activity of bovine ATPase II is stimulated by phosphatidylserine (Tang et al., 1996; Ding et al., 2000; Gomes et al., 2000). Although the type V pumps, such as Cod1p, are most homologous to Ca2+-transporting ATPases, when the sequence comparisons are restricted to certain core domains, the type V pumps are more similar to the type IV ATPases than to other members of the P-type ATPase family (Axelsen and Palmgren, 1998, 2001). The similar features of core domain sequences between the type IV and V groups suggest that an investigation of nonmetallic substrates for Cod1p may be fruitful.
Understanding the role of Cod1p has significance for the understanding of the possible functions of the other type V ATPases. Genes predicted to encode type V ATPases are present in all of the eukaryotic genomes that have been sequenced to date, yet none of the other members of the type V family are known other than by sequence homology. A number of genes have been identified, including one in humans, which are predicted to encode proteins with a high degree of homology to Cod1p (>35% identity and 55% similarity; Costanzo et al., 2000). Our demonstration that one of these, the D. melanogaster dCod1p, rescues the cod1 mutant phenotype indicated experimentally that the function of these proteins is conserved and these proteins are orthologous to Cod1p. More distant homologues of COD1 in the type V subgroup are even lesser known and likely have other functions. YOR291w, the second yeast type V ATPase has less homology to COD1 than dCOD1 (Costanzo et al., 2000). Deletion of YOR291w produces none of the phenotypes displayed by cod1
mutants (this paper; Cronin et al., 2000). In fact, we have been unable to detect any phenotypes associated with this gene (unpublished data). Thus, the degree of sequence similarity (or disparity) to Cod1p appears to be a useful predictor of function.
In conclusion, we have greatly extended the phenotypic characterization of Cod1p, defining its role in calcium regulation and ER function, and we have provided the first biochemical exploration of Cod1p. This study provides the foundation for further biochemical studies of Cod1p that will likely illuminate the obscurity surrounding ion regulation and enhance our understanding of the widely conserved, yet little known, type V group of P-type ATPases.
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Materials and methods |
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Plasmid construction
pRH1112 (CEN, LEU2, HA::COD1) was constructed from pRH810 by using synthetic overlap extension to add sequence coding for an HA epitope tag to the 5' end of COD1, just after the start codon. pRH1312 was constructed from pRH1112 by replacing the native promoter of COD1 between SacI and SphI sites with a PCR-amplified TDH3 (glyceraldehyde 3-phosphate dehydrogenase) promoter.
Plasmid pOT2-GH06032 containing the dCOD1 cDNA was purchased from Research Genetics. pRH1388 (CEN, LEU2, HA::dCOD1) expressing HAdCod1p from the COD1 promoter was constructed as follows. Primers oRH1328 and oRH1329 were used to amplify the proximal part of the dCOD1 ORF before the AgeI site, adding sequence coding for an HA tag just after the start codon. This PCR product was cut with AatII and AgeI and ligated with the BglIIAatII fragment of pRH1112 and the AgeIBglII fragment of pOT2-GH06032. The HAdCOD1 ORF was subsequently transferred to a number of plasmids including pRH1389 (2µ LEU2 PTDH3-HA::dCOD1) pRH1431 (2µ URA3 PPMA1-HA::dCOd1) using AatII and BglII.
pRH1432 expressed 9HISHACod1p from the TDH3 promoter on a high copy plasmid. Synthetic overlap extension was used to add sequence coding for nine histidine residues to the 5' end of PTDH3-HA::COD1, just after the start codon and before the sequence coding for the HA epitope tag. Oligonucleotide primers oRH1079, oRH1082, oRH1354, and oRH1355 were used to amplify from the template plasmid pRH1331 (2µ, LEU2, PTDH3-HA::COD1). The resulting PCR product was subcloned into pRH1332 (2µ, URA3), using BsiWI and EagI to yield pRH1432. Plasmid pRH1478 expressed the D487N mutant of 9HISHACod1p from the TDH3 promoter. It was constructed by removing the BstEIIBstEII fragment coding for the D487N mutation from pCS210 and placing it into the same sites of pRH1432.
Yeast culture and strains
Yeast strains were grown in minimal media (yeast nitrogen base without amino acids; Difco) with glucose and the appropriate supplements as described previously, except that leucine supplementation was increased to 60 mg/liter. Experiments were performed in minimal media at 30°C unless otherwise noted. For protein purification, yeast strains were grown in minimal media with glucose supplemented with 1 g/liter adenine, 1 g/liter methionine, 1.5 g/liter leucine, 1.5 g/liter lysine, 1 g/liter histidine, and 20 g/liter casamino acids (Difco). Crosses and transformations were done using standard techniques.
The 3mycCod1p allele expressed in RHY1870 (Table I) was constructed using a PCR epitope-tagging method (Schneider et al., 1995). URA3 and the flanking myc epitope tags from pMPY3xMYC were amplified with primers oRH1190 and oRH1191. The primers added sequences homologous to the 5' end of COD1. Yeast were transformed with the PCR product and selected for acquisition of uracil prototrophy. Transformants were screened by the fluorescence plate assay for the Cod- phenotype. Cod-, Ura+ candidates were grown in YPD to allow "pop-out" of URA3 mediated by the direct repeats of the myc epitope tag followed by counterselection for loss of URA3 on media containing 5-fluoroorotic acid. The resulting Ura-, Cod+ candidates were then tested for expression of the 3mycCod1p by western analysis.
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RHY1708 (1MYC::HMG2) and RHY1709 (1MYC::HMG2, cod1::LEU2) were used for ß-galactosidase assays. They were constructed by treatment of RHY791 and RHY1202 (Cronin et al., 2000) with 5-fluoroorotic acid. The ß-galactosidase reporter plasmids were transformed into the strains using standard techniques.
hac1 strains were constructed as follows. RHY1647 (hac1
::URA3, COD1) and RHY1664 (hac1
::URA3, COD1) were constructed by transforming RHY534 and RHY872, respectively, with the BamHI fragment of pHAKO (Cox and Walter, 1996). RHY534 is a diploid version of JRY1159 (Hampton and Rine, 1994). RHY1884 was constructed by transforming MYY290 (Smith and Yaffe, 1991) with the BamHI fragment of pHAKO (Cox and Walter, 1996). RHY2179 (pmr1
::HIS3) was constructed by transforming MYY291 (Smith and Yaffe, 1991) with the AatIIAatII fragment from pL127-6 (Cunningham and Fink, 1994b). RHY2277 was isolated from a cross of RHY2179 and RHY1884.
Growth curves
Susceptibility of mutants to DTT, tunicamycin, MnCl2, and other divalent cations was tested in minimal media. Susceptibility to MnCl2, CaCl2, and EGTA was tested in liquid YPD buffered with succinate to pH 5.5. Low-density liquid cultures of wild-type or mutant cells were used to serially dilute the tested agent. The resulting cultures were incubated and measured for optical density at 600 nm.
Immunofluorescence microscopy
Immunofluorescence was performed essentially as described in detail previously (Rossanese et al., 1999), except that the cells were adhered to a multiwell slide instead of a coverslip. Images were captured using a DeltaVision confocal microscope and processed with the accompanying software.
ß-Galactosidase assays
ß-Galactosidase assays were performed as previously described (Guarente, 1983), except that freezethawing in liquid nitrogen was used to lyse cells.
Subcellular fractionation
Fractionation was performed as described by Roberg et al., 1997. Protein from each fraction was separated by SDS-PAGE, transferred to nitrocellulose, probed with the appropriate antibodies, and detected by chemiluminescence using the ECL system. NIH Image 1.6 was used to determine signal intensity.
Flow cytometry
Analysis of GFP fluorescence in living cells by flow microfluorimetry was performed on a Becton Dickinson FACScalibur® flow microfluorimeter using settings appropriate for GFP. Strains were typically grown into early log phase in minimal media. After addition of drugs, cultures were allowed to incubate 4 h before analysis.
ICP-OES
The total amount of calcium and other ions in whole cells and subcellular fractions was determined by ICP-OES. To determine the total ion content of whole cells, cells were grown in YPD, pH 5.5, to OD600 of 1.5 ml of the culture was collected by vacuum filtration onto 0.4-µm filters. The cells were then washed with 5 ml deionized water. The filters were transferred to microcentrifuge tubes and digested in 500 µl of 33% nitric acid overnight at 65°C. Samples were diluted with 3 ml of deionized water before analysis using a PerkinElmer Optima 3000XL spectrometer and software.
Solubilization and purification of histidine-tagged Cod1p
9HISHACod1p was prepared by detergent extraction of the protein from ER membranes followed by nickel affinity chromatography. ER membranes were prepared by sucrose gradient centrifugation. Before harvesting, cells were treated with 10 mM sodium azide. The cells were washed in 10 mM sodium azide, 5 mM Tris, pH 7.5, and then resuspended in lysis buffer (4 M sorbitol, 1 mM EDTA, 0.1 M KxHxPO4, pH 7.5) plus protease inhibitors (10 mM PMSF, 2 µg/ml chymostatin, 1 µg/ml leupeptin, 1 µg/ml pepstatin) so that the packed yeast cells made up one third of the volume. The cells lysed with four 30-s pulses with 1-min intervals in a Bead Beater® (BioSpec Products, Inc.). The crude lysates was clarified by centrifugation at 350 g in a Sorvall SS-34 rotor. The clarified lysates were loaded onto a sucrose step gradient and centrifuged for 2 h at 174,000 g in a Beckman Coulter SW28 rotor. ER membranes containing 9HISHACod1p were collected from the interface between 30% and 54% sucrose. The collected membranes were diluted with an equal volume of P&R buffer (0.5 M sucrose, 10 mM MES/KOH, pH 6, 150 mM potassium chloride) with protease inhibitors (1 mM PMSF, 2 µg/ml chymostatin, 1 µg/ml leupeptin, 1 µg/ml pepstatin). The membranes were pelleted by centrifugation for 1 h at 174,000 g. The pelleted membranes were resuspended in P&R buffer with protease inhibitors and stored at -90°C in 110-µl aliquots. Protein concentration in the membranes was measured using the BCA assay (Pierce Chemical Co.).
Detergent extracts of the membranes were prepared as follows. 10 mg of membrane was suspended in 10 ml of S-buffer (20 mM Hepes/Tris, pH 7.0, 20% glycerol, 0.5% E. coli lipid extract, 6 mM ß-mercaptoethanol, 10 mM imidazole, protease inhibitors, and 1.5% n-octyl-ß-D-glucopyranoside) and allowed to shake gently at 4°C for 2.5 h. The resulting suspension was centrifuged at 100,000 g for 1 h to pellet insoluble material.
The detergent-solubilized 9HISHACod1p was then purified by nickel affinity chromatography. 250 µl of NTA-Ni agarose beads (QIAGEN) per milliliter of solubilized membranes was washed twice with deionized water and then twice with S-buffer. The equilibrated beads were added to the detergent extract and gently shaken at 4°C for 30 min. The extract with beads was then transferred to 2 ml Micro Bio-Spin columns (Bio-Rad Laboratories). Unbound material was allowed to flow through by gravity elution. Each column was washed twice with 1 ml of S-buffer plus 200 mM NaCl, and the column was washed a third time with S-buffer. 9HISHACod1p was eluted after a 5-min incubation with 0.2 ml per column of S-buffer plus 300 mM imidazole. The eluted material was immediately frozen in a dry iceethanol bath and stored at -90°C in 60-µl aliquots.
Assaying ATPase activity
ATPase activity was measured using the radiometric assay described previously (Bais, 1975; Mandal et al., 2000) with some modifications. Assays were done in a 50-µl volume and generally contained 50 mM Hepes/Tris, pH 7.0, 100 mM KCl, 5 mM MgCl2, 1 mM EDTA. [32P]ATP (30 Ci/mmol; Amersham Pharmacia Biotech) was diluted 10-fold with 1 mM NaATP to the final concentration desired. Variations in the assay conditions are noted in the figures. The concentrations of free ions and MgATP complexes were calculated using WINMAXC v2.05 (Bers et al., 1994) with supplementary constants from Data for Biochemical Research (Dawson et al., 1986). EDTA was excluded from experiments determining the effect of transition metals on Cod1p, because the high affinity of EDTA (and other chelators) for transition metals prevented accurate determination of the free ion within the desired range. Solutions were checked for contaminating ions by ICP-OES. Vanadate was prepared by the method of Gordon (1991).
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Footnotes |
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Acknowledgments |
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Submitted: 11 March 2002
Revised: 18 April 2002
Accepted: 18 April 2002
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References |
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