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Address correspondence to Karl Ekwall, University College Sodertorn, Dept. of Natural Sciences, Alfred Nobels Alle 3, S-141 52 Huddinge, Sweden. Tel.: 46-8-608-4713. Fax: 46-8-608-4510. E-mail: karl.ekwall{at}cbt.ki.se
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Abstract |
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Key Words: Schizosaccharomyces pombe; P-type ATPase; endoplasmic reticulum; calcium; microtubule
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Introduction |
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Within the protein family of P-type ATPases, the Ca2+-ATPases are of special interest, since calcium plays a key role in signal transduction in eukaryotic cells (Clapham, 1995). The transient elevation in cytosolic-free calcium stimulates several Ca2+-binding proteins and their targets that act to elicit downstream signaling pathways. Ubiquitous effectors of calcium signaling are Ca2+/calmodulin (CaM)*-dependent protein kinases and Ca2+/CaM-dependent phosphatase, calcineurin, which act by modulating the phosphorylation state of diverse proteins including transcription factors (Stull, 2001). There is a growing body of evidence that the cellular response to a rise in calcium depends on the amplitude, frequency, duration, and location of the Ca2+ signal (Sanders et al., 1999). The calcium signal is terminated when cytosolic-free Ca2+ concentration is reduced to basal levels by Ca2+-ATPases and Ca2+/H+ exchangers that transport calcium from the cell or sequester it in organelles. In the last case, refilling of intracellular compartments safeguards the Ca2+ release during subsequent signaling events and provides a lumenal space with specific Ca2+ concentration required for diverse biochemical reactions taking place in those compartments (Corbett and Michalak, 2000).
Two main classes of Ca2+-ATPases have been described: sarco/ER Ca2+-ATPases (SERCA) and plasma membrane Ca2+-ATPases, which differ from one another in their subcellular distribution, biochemical characteristics, and mode of regulation (Guerini and Carafoli, 1999; Carafoli and Brini, 2000). In addition, the secretory pathway Ca2+-ATPases initially characterized in budding yeast (Rudolph et al., 1989) has emerged as a separate class (Gunteski-Hamblin et al., 1992; Sorin et al., 1997).
The fission yeast Schizosaccharomyces pombe is an excellent model system for eukaryotic cell biology. Several components of Ca2+-mediated signaling of animal cells have been identified and characterized in fission yeast. A temperature-sensitive Ca2+-binding site CaM mutant exhibits broken spindles and defects in chromosome segregation (Moser et al., 1997). CaM is localized to the spindle pole bodies and sites of polarized cell growth in S. pombe (Moser et al., 1997). In cells undergoing cytokinesis, CaM was found on both sides of septum. Similar CaM redistribution at the cell equator was observed in dividing animal cells where CaM activation by elevation of free Ca2+ was proposed to trigger the formation of the cleavage furrow (Li et al., 1999). Thus, there are several indications that Ca2+ and CaM have an important role in regulating aspects of the cytokinesis both in animal cells and fission yeast, but more direct evidence for Ca2+ affecting this process is lacking.
Recently, the gene ppb1+ encoding for the catalytic subunit of calcineurin was isolated from S. pombe (Yoshida et al., 1994; Plochocka-Zulinska et al., 1995). Mating, microtubule distribution, chromosome segregation, spindle pole body, and nuclear positioning were impaired in calcineurin-deficient cells (Yoshida et al., 1994). The lack of ppb1+ resulted in branched cells with multiple septa that fail to cleave. These observations further indicate that Ca2+ homeostasis can have profound effects on cytoskeleton functions in fission yeast.
In spite of these findings, little is known about how calcium signals are generated and controlled in S. pombe, and so far little is known about the molecular identity of transporters, which deplete cytosolic calcium. The cta3+ gene product was identified previously as a Ca2+-ATPase (Ghislain et al., 1990) and is related to the ENA1 Na+-ATPase of Saccharomyces cerevisiae. Cta3p is required for Na+ tolerance in S. pombe (Nishikawa et al., 1999). The Ca2+/H+ exchangers were shown to be responsible for Ca2+ transport in membranes of the secretory pathway organelles, but Ca2+-ATPase activity has so far not been detected in membrane preparations (Okorokov et al., 2001). Although the genes encoding for several putative calcium ATPases were identified by the S. pombe genome-sequencing project, no genetic analysis has been performed on the corresponding null mutants. Thus, the involvement of each individual pump in calcium homeostasis and the role of the pumps in signal transduction and diverse cellular functions have not been established. Toward this end, we have here determined the subcellular localization of the putative calcium ATPase SPAC2E11.07C and analyzed the physiological consequences of its gene deletion. To follow the preexisting nomenclature of P-type ATPases in fission yeast, SPAC2E11.07C was named cta4+ (calcium/cation transporting ATPase). The cta4+ gene encodes a novel member of the P4 family of P-type ATPases that is localized in the ER. cta4+ is not an essential gene, but cta4 mutants display several morphological defects, an imbalance in cation homeostasis and are temperature sensitive and cold sensitive lethal. Microtubules are generally destabilized in cells lacking Cta4p. The microtubule length is decreased, and the number of microtubules per cell is increased concomitant with an increase in the number of microtubule catastrophe events in the midzone of the cell. Fluorescence resonance energy transfer (FRET) experiments in living cells using the fluorescent yellow cameleon indicator for Ca2+ indicated that a deletion of cta4+ causes an elevation of cellular calcium levels. Our results reveal a link between control of cell shape, microtubule dynamics, cytokinesis, and cation homeostasis, and appoint Ca2+ as a key regulatory ion. Hence, our data points to a new level of control over these important processes.
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Results |
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To verify the ER localization of Cta4 ATPase, the antibody against another ER organelle marker, chaperone BiP (Pidoux and Armstrong, 1993), was used. IF microscopy demonstrated colocalization of Cta4-GFP and BiP (Fig. 1 B). Distribution of BiP in membrane fractions was more restricted than that of Sec61-myc and was similar to that of Cta4. Both BiP and Cta4 proteins were concentrated in fractions 2022 (Fig. 1 C). Therefore, it could be concluded from these results that Cta4 ATPase is localized to the ER compartment.
cta4+ regulates cation homeostasis
To further investigate the function of Cta4p in fission yeast, the cta4+ gene was disrupted with the ura4+ marker gene, and the resulting cta4 phenotype was compared with that of wild type. Tetrad analysis revealed that cta4+ was not an essential gene; however, cta4
cells exhibited poor growth at 25°C compared with that at 30°C and died at 36°C, indicating that some aspect of cell cycle progression was impaired in these conditions (Fig. 2 A). Growth of Cta4-GFP cells was not impaired at 25 and 36°C, pointing out that this allele is fully functional.
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The Ca2+/CaM-dependent protein phosphatase 2B, calcineurin, was shown to be responsible for a maintenance of cation homeostasis in S. cerevisiae by regulating the expression of Ca2+ and Na+ ATPases (Nakamura et al., 1993; Cunningham and Fink, 1996; Mendoza et al., 1996). In S. pombe, the calcineurin A subunit-like protein encoded by the ppb1+ gene is the target of cyclosporin A (CsA) (Yoshida et al., 1994). cta4 cells were found to be susceptible to 10 µg/ml CsA (Fig. 3), whereas wild-type strain remained insensitive to threefold higher drug concentration. This result indicates that cta4+ acts in the same or parallel pathways as calcineurin, since ppb1+ seems necessary for the viability of the cta4
mutant.
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Discussion |
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Several lines of evidence suggest that cta4+ is involved in Ca2+ homeostasis. First, cta4 was unable to grow when calcium was supplied to medium and when it was chelated by EGTA (unpublished data). Second, calcium measurements in cta4
cells using fluorescent indicator yellow cameleon showed that nuclear calcium levels were increased in cta4-null cells in comparison with wild-type cells. It is likely that nuclear Ca2+ levels are indicative of the cytoplasmic Ca2+ levels, since Ca2+ ions can diffuse through the nuclear pores (Lipp et al., 1997). Thus, loss of cta4+ function reduces the ability to sequester Ca2+ to internal stores, presumably the ER (see below). The consequence of this is an increase in intracellular Ca2+.
The cta4 mutant cells were sensitive to inhibition of calcineurin, Ca2+/CaM-dependent protein phosphatase type 2B, by CsA. The ppb1+ gene of S. pombe encodes a catalytic subunit of calcineurin. In this respect, it is noteworthy that pleiotropic phenotypes of cta4 were reminiscent of those reported previously for ppb1
mutants regarding growth characteristics, septation, cell shape defects, and microtubule integrity (Yoshida et al., 1994). Moreover, S. pombe mutants lacking cta4+ were, like S. cerevisiae calcineurin mutants, sensitive to Ca2+ and Mn2+ cation stress. Thus, it is possible that calcineurin function is defective in cta4-null cells. It remains to be determined if the cta4+ and ppb1+ genes interact and if these two gene products share an essential overlapping function. Finally, cta4+ overexpression was toxic to wild-type cells, and this could be overcome by addition of Ca2+ (unpublished data). Therefore, both elevated and lowered Ca2+ cytosolic levels may be deleterious to S. pombe. Since cta4+ deletion is not lethal to S. pombe cells, it could be concluded that other Ca2+ transporters deplete cytosolic Ca2+ in this genetic background, although to a lesser extent than cta4+ normally does. Therefore, the precise regulation of Ca2+ homeostasis fails and, consequently, Ca2+ signals.
From our results it could be presumed that Cta4p might transport Ca2+. However, direct biochemical evidence is needed to establish the exact substrate specificity of Cta4p. Our attempts to measure ATP-dependent Ca2+ transport in isolated membranes of S. pombe 972 have shown that all Ca2+ uptake was abolished by protonophore FCCP, indicating that Ca2+ transport was due to Ca2+/H+ exchange (Okorokov et al., 2001). The similar result was obtained with S. pombe strain Hu237 used for determination of subcellular localization of Cta4p (unpublished data). Further experiments will be necessary to find the conditions favoring a detection of biochemical activity of Cta4 ATPase. The biochemical characterization of Cta4p will contribute to our comprehension of the physiological function of P4 ATPases.
Cta4p localizes to the ER in S. pombe. In animal cells, ER is equipped with SERCA-type Ca2+-ATPase and is a main Ca2+ store compartment (Mendolesi and Pozzan, 1998; Carafoli and Brini, 2000). The ATPases belonging to SERCA-type were also found in plant, protozoa, and insect (Liang et al., 1997; Lockyer et al., 1998; Talla et al., 1998). Interestingly, the gene encoding for SERCA-type Ca2+ pump has not been identified in yeast, although Ca2+-ATPase activity could be detected in the S. cerevisiae membranes derived from the ER (Okorokov and Lehle, 1998; unpublished data). These observations raise a possibility that Cta4p could represent a primary ancient pump serving the ER. It is likely that the evolution of the ER as an organelle was driven by a wide range of functions supporting the development of complex signaling networks within the eukaryotic cell. This may have been the driving force leading to the appearance of additional specialized ATPases, such as SERCA, which may sequester calcium ions into the ER.
The pleiotropic defects exhibited by cta4 could be interpreted by either a direct or an indirect involvement of cta4+ in microtubule integrity, cell shape, and cytokinesis through regulated changes in Ca2+ concentrations. Since Ca2+ is a well-known secondary messenger, any transient elevations in the intracellular Ca2+ concentration would result in Ca2+ binding to multiple classes of Ca2+-binding proteins, each of which can, in its turn, regulate multiple downstream signaling pathways. On the other hand, Ca2+ is emerging as the regulatory ion for many ER/Golgi functions. Oscillations in free Ca2+ concentrations in the ER of animal cells were shown to control diverse processes, including protein synthesis, chaperone function, and glycoprotein processing (Corbett and Michalak, 2000). The budding yeast secretory pathway requires Ca2+ for proper glycosylation, sorting, and ER-associated protein degradation (Antebi and Fink, 1992; Durr et al., 1998; Okorokov and Lehle, 1998). Further studies are required to identify components of the Ca2+ signaling machinery, which depend on Cta4p. However, some speculations about possible downstream targets can be made already.
We showed that loss of cta4+ enables yeast cells to complete cytokinesis. Previously, this process was shown to be dependent, in part, on the cps1+ gene encoding ß-(1,3)-D-glucan synthase (Ishiguro et al., 1997; Liu et al., 2000). Expression of a homologue of cps1+ in budding yeast, FKS2, is induced by PKC together with calcineurin in a Ca2+-dependent manner (Zhao et al., 1998). cps1+ also appears to be dependent on calcineurin, since the cps1ts mutant is hypersensitive to CsA (Ishiguro et al., 1997). In addition, cps1ts mutants are, like cta4, multiseptated and branched. Considering our supposition that calcineurin function might be compromised in cta4
, then defects in cytokinesis could be explained through changes in Cps1 activity, and this raises the possibility that cta4+ and cps1+ act in the same pathway.
There is a strong link between cell shape/polarity and microtubules in fission yeast (Sawin and Nurse, 1998). Therefore, our data provide evidence that microtubule integrity relies on cta4+ function. From this study, we cannot distinguish direct from indirect effects of Ca2+ on microtubules. Thus, additional studies will be necessary to gain a deeper comprehension of the involvement of cta4+ in this process. One of the possibilities is that cta4+ would regulate microtubule integrity, controlling the stability and/or deposition of microtubule-associated proteins. A recent study in animal cells has shown that Ca2+-binding proteins, such as S100A1 and S100B, might have a role in the in vivo regulation of the state of assembly of microtubules in a Ca2+-regulated manner (Sorci et al., 2000). Also, in S. pombe, microtubule-associated factors, such as the CLIP170-like protein Tip1, are involved in microtubule dynamics (Brunner and Nurse, 2000). The phenotypes of tip1 null and cta4
are related, since the mutants cells are not rod-shaped like wild-type S. pombe cells. Furthermore, both mutants show shorter microtubules and an increase in the frequency of microtubule catastrophe events throughout the cell rather than exclusively in the cell tips, which are the regions of polarized growth (Fig. 7) (Brunner and Nurse, 2000). In both cases, the changes in cell shape are associated with a defective guidance mechanism for microtubules. In this respect, since Tip1p is a microtubule-binding protein it would be interesting to investigate if Tip1p is directly or indirectly dependent on Ca2+ and/or Cta4p.
We provide evidence that the Ca2+ concentration is crucial for establishing the correct cell polarity by regulating microtubule dynamics. This finding is not without precedence, since studies in plant root tips demonstrate that root hair polarity is dependent on a Ca2+ gradient that increases in Ca2+ concentration toward the tip of the root hair (Bibikova et al., 1997; Wymer et al., 1997; Gadella et al., 1999). Furthermore, the polarity marked by this gradient is dependent on microtubules (Bibikova et al., 1999). Thus, the Ca2+-dependent mechanisms operating with respect to cell polarity may likely be of general significance in eukaryotes.
Further measurements of cytosolic Ca2+ are needed to clarify whether a Ca2+ gradient exists within the yeast cell and if there is a correlation between localized high Ca2+ and the sites of polarized growth. At the moment, it is tempting to speculate that increased Ca2+ at the ends of the cells would be a guiding signal for microtubule growth and a factor that induces the occurrence of microtubule catastrophe events.
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Materials and methods |
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Live analysis of S. pombe cells
We performed GFP and CFP/YFP time-lapse analysis using the ratio imaging module of Openlab software version 2.25 and a ZEISS Axioskop II imaging microscope equipped with a Hamamatsu C474295 CCD camera. The yeast cells were embedded in 10 µl 1% soft agar in PMG medium under a 22 x 22-mm no. 1 coverslip (Propper) and subjected to time-lapse video capture using a 10% neutral density filter to reduce photobleaching of the GFP.
Membrane fractionation
Yeast cells were grown to late log phase. After incubation in 1.2 M sorbitol and 30 mM mercaptoethanol, pH 8.5, for 10 min at 25°C, they were washed with 1.2 M sorbitol and 50 mM NaH2PO4 adjusted with citric acid to pH 5.8. Spheroplasts were then isolated by incubation of the cells with lytic enzymes from Tritrichoderma at 30°C in the same buffer. Spheroplasts lysis and isolation of membranes followed published procedures (Okorokov and Lehle, 1998). The resuspended total membranes were loaded onto a step gradient formed of 56, 52, 48, 45, 42, 39, 36, 33, 30, and 25% sucrose (wt/wt). After centrifugation at 140,000 g for 2 h 45 min, the membrane fractions were collected from the bottom and stored at -70°C.
Immunoblotting
Yeast membranes from the sucrose gradient fractions (10 µl) were spotted on nitrocellulose membrane and probed with antibodies. Anti-GFP and anti-myc antibodies were purchased from Molecular Probes and Sigma-Aldrich, respectively. Anti-BiP antibodies were provided by Prof. J. Armstrong (University of Sussex, Brighton, UK). The blots were developed with peroxidase-conjugated secondary antibody.
Recombinant DNA
All procedures with recombinant DNA were performed according to standard techniques (Maniatis et al., 1982). The YC2 construct was PCR amplified from the original DNA clone (Miyawaki et al., 1997) using oligonucleotides that add a PKKKRKV (SV40) nuclear localization signal fused to the YC2 NH2 terminus and cloned into pREP3x multicopy plasmid digested with SalI. The expression of cta4+ was induced in PMG medium lacking thiamine. The cta4+ gene was tagged at its endogenous site with GFP using the method from Bahler et al. (1998).
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Footnotes |
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Acknowledgments |
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This work was supported by the Swedish Medical Research Council VR-M grant no. 12562 to K. Ekwall, Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro grant E-26/171.374/99 to L. Okorokova, a Natural Science Council VR-N open postdoc grant to H. Appelgran, and a Wennergren stipend to M. Tabish.
Submitted: 5 November 2001
Revised: 28 February 2002
Accepted: 3 April 2002
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