Correspondence to: Peter N. Devreotes, Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205. Tel:(410) 955-4699 Fax:(410) 955-5759 E-mail:pnd{at}welch.jhu.edu.
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Abstract |
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We have identified a novel gene, Tortoise (TorA), that is required for the efficient chemotaxis of Dictyostelium discoideum cells. Cells lacking TorA sense chemoattractant gradients as indicated by the presence of periodic waves of cell shape changes and the localized translocation of cytosolic PH domains to the membrane. However, they are unable to migrate directionally up spatial gradients of cAMP. Cells lacking Mek1 display a similar phenotype. Overexpression of Mek1 in torA- partially restores chemotaxis, whereas overexpression of TorA in mek1- does not rescue the chemotactic phenotype. Regardless of the genetic background, TorA overexpressing cells stop growing when separated from a substrate. Surprisingly, TorAgreen fluorescent protein (GFP) is clustered near one end of mitochondria. Deletion analysis of the TorA protein reveals distinct regions for chemotactic function, mitochondrial localization, and the formation of clusters. TorA is associated with a round structure within the mitochondrion that shows enhanced staining with the mitochondrial dye Mitotracker. Cells overexpressing TorA contain many more of these structures than do wild-type cells. These TorA-containing structures resist extraction with Triton X-100, which dissolves the mitochondria. The characterization of TorA demonstrates an unexpected link between mitochondrial function, the chemotactic response, and the capacity to grow in suspension.
Key Words: TorA, Mek1, chemotaxis, mitochondria, Dictyostelium
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Introduction |
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Chemotaxis is a fundamental property of living cells that enables them to move appropriately during inflammation, angiogenesis, metastasis, and embryonic and neuronal development. The well-characterized chemotactic responses of Dictyostelium discoideum amoebae provide a powerful system for genetic analysis of this fascinating process. The chemotactic behavior and underlying biochemical responses of these amoebae are remarkably similar to those of leukocytes (for review see
A series of genetic and cell biological analyses has identified many of the components involved in directional sensing and movement. For example, all of the responses to chemoattractants are absent in cells lacking surface receptors or G protein subunits (
To further elucidate mechanisms of directional movement, we have isolated a series of chemotaxis mutants. Our screen relied on scoring the phenotypes of plaques on bacterial lawns, derived from clonally seeded cells. Wild-type cells aggregate and differentiate with characteristic morphology, and cells with defects in the chemoattractant-mediated events that control these developmental processes can be readily visualized. We selected clones that resembled those of cells lacking the G protein ß subunit (
A screen for small plaque mutants resulted in isolation of Tortoise (TorA), which is required for efficient chemotaxis and the demonstration that TorA and Mek1 are in a related pathway. These mutants are able to sense chemoattractant gradients but cannot move towards the higher concentration efficiently. Remarkably, TorA is clustered in a novel round structure near one end of many mitochondria. Our results point to an unanticipated role for mitochondria in the chemotactic response.
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Materials and Methods |
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cAMP, 2'deoxy-cAMP, DAPI, and G418 were from Sigma-Aldrich. Mitotracker red CMXRos was from Molecular Probes. Blasticidin S was from Calbiochem. Monoclonal antigreen fluorescent protein (GFP) antibody was from Babco Covance. Multiwell chemotax chambers were from Neuroprobe.
Dictyostelium Growth, Development, Clonal Selection, and Plaque Analysis on Bacterial Lawns
Dictyostelium cells were grown in axenic medium (
Motility in Buffer
The behavior of single cells in buffer was performed as described (
Motility in a Spatial Gradient
Aggregation-competent cells were washed from filters and deposited on the bridge of a gradient chamber (
Motility in Natural cAMP Waves
For analyzing motility in natural aggregation territories, exponentially growing cells were washed free of nutrients and suspended in BSS at a density of 2.4 x 106 cells/ml according to methods described previously (
Two-dimensional Computer-assisted Analysis of Cell Motility
Digitized images of cells in buffer and in spatial gradients of cAMP were automatically outlined using the gray scale threshold option of DIAS ( (area/perimeter2). The chemotactic index was computed as the net distance moved to the source of chemoattractant divided by the total distance moved in the time period. Percent positive chemotaxis was computed as the proportion of the cell population exhibiting a positive chemotactic index.
Restriction Enzymemediated Integration (REMI) Mutagenesis and Plasmid Rescue
Restriction enzymemediated integration (REMI) mutagenesis was performed according to
Cloning of Full-Length TorA Gene and Construction of torA Null Cell Lines and TorA Expression Constructs
A partial cDNA was cloned from a gt11 cDNA library containing the 3' end of the TorA gene. A 0.9-kb fragment of this cDNA was fused at the TthIII site to a genomic fragment containing the 5' end plus 1 kb noncoding sequence upstream of the TorA gene. This generated a 2.4-kb ORF containing the full-length gene. TorA null cell lines were constructed using the 8-kb rescued plasmid, linearized by HindIII. Another knock-out construct was made by inserting the Bsr cassette as an XbaI fragment into the SpeI site of the TorA gene. Both constructs were used to disrupt the TorA gene in wild-type AX3 cells by transforming the DNA by electroporation and selecting for transformants at 5 µg/ml blasticidin. A TorA expression construct was made by adding a BglII site and ribosome binding site to the 5' end of the coding sequence using PCR and the use of a BamHI site at the 3' end of the gene, which allowed cloning of the gene under an actin15 promoter in the BglII site of B18 (
Northern Analysis
Total RNA of 107 cells was isolated (-32Plabeled dATP DNA fragments.
Preparation of Triton- and Triton/Sodium Carbonateinsoluble Fractions, Immunoblot Analysis, and Silver Staining
Triton-insoluble actin cytoskeletons were prepared from 107 cells according to
Mitochondria Staining with DAPI and Mitotracker
For DAPI staining, cells were fixed for 5 min with 2% formaldehyde in HL5, and then for 20 min with 1% formaldehyde in methanol at -20°C. Cells were washed with PBS and stained for 15 min with 5 µM DAPI and washed with PBS. For Mitotracker red staining, cells were incubated 15 min with 500 nM Mitotracker red and washed with 10 mM Na/K phosphate buffer.
cAMP, cGMP, F-actin, and Chemotaxis Measurements
For each assay, cells were developed for 5 h in DB while pulsing with 100 nM cAMP at 6-min intervals and were treated with 3 mM caffeine for 20 min. After two washes in cold DB, 5 x 106 cells were stimulated with 5 x 10-6 2' deoxy-cAMP (cAMP accumulation) and 10-6 M cAMP (cGMP accumulation). For F-actin, 2 x 106 cells were stimulated with 10-7 M cAMP. cAMP amounts were detected using a [3H]cAMP detection kit (Amersham Pharmacia Biotech). cGMP levels were measured using a radioimmuno assay, [3H]cGMP detection kit (Amersham Pharmacia Biotech). F-actin measurements were performed as described (
Fluorescence and Electron Microscopy on TorAGFP-expressing Cells
Fluorescence microscopy on live cells was performed on a ZEISS microscope (Axiovert 135 TV) as described previously (
Online Supplemental Material
Video 1 shows a time-lapse movie of aggregating control cells on agar after 5 h of development. Frames were taken every 8 s. Video 2 shows a time-lapse movie of aggregating torA- cells on agar after 5 h of development. Frames were taken every 8 s. Video 3 shows a time-lapse movie of aggregating Mek1/torA- cells on agar after 5 h of development. Frames were taken every 8 s. Video 4 shows TorAGFP-expressing cells. Frames were taken every 3 s. Video 5 shows deconvoluted images for GFP (green) and DAPI (blue) in TorAGFP-expressing cells. Time-lapse movies are available at http://www.jcb.org/cgi/content/full/152/3/621/DC1.
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Results |
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Isolation of Tortoise and Cloning of the Corresponding Gene
To isolate novel mutants with defects in chemotaxis, we used REMI to generate 30,000 transformants with random insertions in the genome. These were clonally seeded on bacterial lawns, and plaques that expanded slowly under these conditions were selected. 18 small plaque mutants were isolated, and Tortoise (torA-) was chosen for further study. The plaque size and additional phenotypes of torA-, described in detail below, closely resembled those of mek1- (Fig 1A and Fig B).
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Time-lapse videos more clearly revealed the mutant phenotype. The coordinated motions of the cells indicated that torA- generated cAMP waves with frequency and speed nearly identical to those of wild-type cells. However, the mutant aggregated slowly because the distances covered by the chemotactic movement steps were significantly less than those of wild-type cells (see Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/152/3/621/DC1). Typically, wild-type cells aggregated into mounds after the passage of 20 waves. In contrast, torA- had not completed aggregation after 30 or 40 waves (data not shown). Eventually, the incompletely aggregated cells reorganized into much smaller territories and formed tiny mounds and fruiting bodies. A similar phenotype has been described for mek1- (
Expression of Mek1 almost completely suppressed the phenotype of torA- (Fig 1A and Fig B). The sizes of all the torA- plaques on bacterial lawns were increased nearly to those of wild type. Within the plaques, there was a decrease in the number and a corresponding increase in the size of the multicellular structures formed for Mek1/torA-. In a time-lapse video, Mek1/torA- also displayed a pattern of aggregation that closely resembled wild-type cells (see Video 3, available at http://www.jcb.org/cgi/content/full/152/3/621/DC1). As shown in Fig 1 C, the levels of Mek1 were not lower in Tortoise than in wild type, suggesting that the observed phenotypic suppression is due to overexpression of Mek1 in torA-.
Next, we isolated and identified the gene mutated in torA- (Fig 2). We cloned the regions flanking the insertions and determined the sequence as described in Materials and Methods. To verify whether the observed phenotypes were caused by the REMI insertions, we linearized the recovered plasmids, transformed them into wild-type Ax3 cells, and recreated the genotype through homologous recombination. Fig 1 A shows that disruption of TorA in fresh wild-type cells regenerates the small plaque and tiny structure phenotype on bacterial lawns. This mutant was used for all further analyses.
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The genomic sequence of TorA contained an ORF of 808 amino acids, which was disrupted at amino acid 746 and caused a 500-bp deletion. To prove that disruption of this ORF is responsible for the phenotype, we created insertions at amino acids 501 and 746 by homologous recombination in wild-type cells. The new mutants displayed the small plaque phenotype. The ORF of TorA predicts a largely -helical protein with a predicted coiled coil domain from residues 689736. It bears weak homology to other proteins with coiled coil domains such as myosin. To understand this intriguing phenotype represented by these mutants, we turned to a thorough analysis of the torA- mutant.
The Phenotypes of Tortoise and Their Suppression by Mek1 Can Be Traced to Alterations in Chemotaxis
The defects in motility and/or chemotaxis suggested from time-lapse movies were further analyzed by computer-assisted methods (Table 1 and Fig 3) (
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In Fig 3 A, perimeter tracks are presented of control, torA-, and Mek1/torA- cells responding to spatial gradients of cAMP. Although control and Mek1/torA- cells moved in a smooth directional fashion towards the source of chemoattractant, torA- cells moved in a zig-zag fashion in random directions (Fig 3 A). The rough tracks reflect high frequencies of lateral pseudopod formation. The frequency of lateral pseudopod formation, which was low in control and Mek1/torA- cells, has previously been demonstrated to be inversely proportional to the chemotactic index (
Finally, single-cell behavior was analyzed in submerged cultures during natural aggregation. Control cells moved in a cyclic fashion, reflected by peaks and troughs at constant intervals in velocity plots (Fig 3 C). These movement steps are in response to the periodic cAMP waves. Plots of the positions of three representative control cells in Fig 3 C reflect the movement steps. The points are well-separated in the front and clustered at the peak and in the back of each deduced wave. In contrast, peak velocity behavior was sporadic for three representative torA- cells in close proximity (Fig 3 C). Interestingly, the velocity between peaks that reflect the nonchemotactic motility between two cAMP waves was only slightly reduced. This suggests that torA- cells are defective in increasing their speed in response to the approaching cAMP gradient. These results demonstrate that in natural aggregation territories, torA- cells move in all directions (i.e., in a nonchemotactic fashion), exhibit a less constant period, and move with depressed peak velocities.
Also, we studied motility of the three cell types in the absence of a cAMP gradient on a glass surface. The morphology parameters (length, area, and roundness) were similar in all three cell types. Under these conditions, the velocity of torA- cells was lower and the rate of turning was higher than that of control cells (Table 1). These basic motility defects were only partially reversed in Mek1/torA- cells (Table 1).
The following results further show that torA- is specifically defective in chemotaxis. First, the appearance of coordinated cell movements caused by the propagation of cAMP waves after 34 h of development indicates that torA- differentiates properly and expresses a cAMP signaling system. Consistent with this, wild-type and torA- cells accumulate equal amounts of cAMP in response to cAMP stimulation, indicating that torA- cells have normal chemoattractant-induced activation of adenylyl cyclase (Fig 4 A). Previous studies suggest that appropriate activation of adenylyl cyclase requires wild-type levels of surface receptors, G protein subunits, adenylyl cyclase, and several cytosolic regulators. Second, since it has previously been reported that mek1- cells fail to accumulate cGMP in response to chemoattractant stimulation, we tested whether torA- cells had a defect in cGMP metabolism. Under our conditions, both the torA- and the mek1- cells displayed a wild-type cGMP response. Third, chemoattractant-induced polymerization of actin was normal in both mutants (data shown for torA- only). Since the chemotaxis defect was caused by failure to detect the gradient or a failure to respond properly, we assessed whether the cells can sense the direction of a gradient. We transformed torA- and wild-type cells with a GFP fusion of the PH domain of cytosolic regulator of adenylyl cyclase (Crac). When wild-type cells are placed in a cAMP gradient this fusion protein translocates to the side of the highest concentration (
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Based on the results described above, the slow rate of plaque expansion in torA mutants may be caused by defects in motility or chemotaxis towards bacteria. This phenotype was not caused by a phagocytosis defect, since the rates of uptake of heat-killed yeast particles were identical in torA- and wild-type cells (data not shown). To assess whether the torA- cells were able to move towards bacterially produced chemoattractants, we filmed the edges of expanding plaques. We found that wild-type cells often made forays into the surrounding bacterial lawn, whereas torA- cells did not (data not shown).
Ectopic Expression of TorA Leads to Surface-dependent Growth
In performing control experiments to confirm that the Tortoise phenotype was caused by a deletion in the isolated gene, we discovered a fascinating new phenotype caused by overexpression of the TorA gene. Cells with high levels of expression lost their ability to grow in shaken suspension and were entirely dependent on the surface of a petri dish for growth. We expressed the full-length gene under a constitutive Actin 15 promoter in torA- cells. The resulting TorA/torA- cell line formed larger plaques and fruiting bodies; however, the fruiting bodies were slightly smaller than those of wild type (Fig 5 A). On growth plates, detached TorA-overexpressing cells did not proliferate but became smaller and rounder, whereas the attached cells continued to grow. Typically, wild-type cells grow at a similar rate whether attached or in suspension. We next designed an experiment to directly show that the mutant grows normally when attached to the surface of a petri dish but cannot grow in suspension. Wild-type cells and cells overexpressing TorA were grown to confluency in petri dishes, harvested, and shaken in flasks. Under these conditions, cells overexpressing TorA stopped growing, whereas wild-type cells displayed normal growth rates. Thus, TorA overexpression leads to surface-dependent growth (Fig 5 B).
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TorA Is Clustered to One End of Mitochondria and Is Associated with the Cytoskeleton
To localize TorA in living cells, we fused GFP to its COOH terminus and expressed the construct under the Actin 15 promoter. The phenotype of the TorAGFP/torA- cell line was identical to that of TorA/torA- (Fig 5 A), including the acquisition of surface-dependent growth, indicating that the GFP fusion did not significantly affect TorA function (data not shown). The observed TorAGFP fluorescence was localized to punctate regions throughout the cell (Fig 6 A; see Video 4, available at http://www.jcb.org/cgi/content/full/152/3/621/DC1).
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Staining with Mitotracker of TorAGFP-expressing cells showed identical staining patterns, suggesting that TorAGFP localizes to mitochondria (data not shown). Costaining with DAPI of fixed cells showed that TorAGFP was localized to one end of mitochondria (Fig 6 B; see Video 5, available at http://www.jcb.org/cgi/content/full/152/3/621/DC1).
Deletion Analysis of TorA Reveals Domains Required for Function and Localization
To identify domains required for TorA function and localization, we made a series of COOH terminally truncated proteins, which were fused to GFP. To study whether these mutant proteins were functional, we tested whether expression of these constructs could suppress the torA- phenotype. As shown in Fig 7, truncation of the 63 COOH-terminal amino acids results in the inability to restore the torA- phenotype, indicating that the protein is not functional. Interestingly, this protein and shorter NH2-terminal fragments that lack the coiled coil domain localize to mitochondrial clusters. As more of the COOH-terminal sequence was deleted, staining of the clusters decreases, and staining of the entire mitochondrion increases. The NH2-terminal 156 amino acids are necessary and sufficient to target GFP to the mitochondrion. When this region is deleted, the protein targets to the cytosol and does not complement the torA- mutant. Interestingly, the NH2 terminally truncated protein does not form clusters in the cytosol, suggesting that the formation of these structures requires the association with certain mitochondrial proteins.
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Expression of TorA Leads to the Formation of Clusters That Stain with Mitotracker
The clusters appeared to be an intriguing novel mitochondrial structure that stained with the mitochondrial dye Mitotracker (Fig 8). Expression of wild-type TorA or TorAGFP resulted in the formation of several structures in the mitochondria that showed intense staining with this dye (Fig 8 B). The Mitotracker and GFP signals display identical patterns, indicating that the structures observed after Mitotracker staining indeed contain TorAGFP (Fig 8C and Fig D). Importantly, wild-type cells also contained these structures. About 2030% of wild-type cells showed punctate staining with Mitotracker, but in only a few mitochondria per cell (Fig 8 A). Thus, the number or size of these structures is enhanced by overexpression of TorA. Mitotracker staining depends on the mitochondrial membrane potential to be specifically imported in mitochondria, suggesting that the novel structure may be a region with high electron potential. Mitotracker does not accumulate at any site outside the mitochondrion and at only one site in each mitochondrion, so the staining of this structure is specific. We also detected some of these structures in torA- mitochondria (data not shown), indicating that the clusters contain other proteins besides TorA. We could not determine whether the null had fewer structures. Using electron microscopy, we visualized the clusters as a round electron-dense mass in the mitochondrion (Fig 8, EG). The Mitotracker results suggest that these structures also exist in wild-type cells, but they may be harder to find in electron microscopy.
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TorA Localizes to a Triton X-100/Sodium CarbonateInsoluble Fraction
Investigation of the subcellular localization of TorAGFP and its colocalization with mitochondria revealed that TorA displayed some unusual properties for an integral component of mitochondria. Using immunoblots (Fig 9 A), we showed that nearly all TorAGFP was localized to the particulate fraction. After extraction of the particulate fraction with Triton X-100, TorAGFP localized to the Triton X-100insoluble fraction. Under these conditions, 70% of the mitochondrial marker TopA (
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Discussion |
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In our search for novel proteins that link the chemotactic signal to the actin cytoskeleton during chemotaxis, we discovered a novel mitochondrial protein, TorA, whose function is essential for chemotaxis. This unexpected finding indicates that mitochondria may have a role in directional movement. Importantly, these mutants have no obvious defects in signal transduction or movement, indicating that the chemotaxis defect is very specific. TorA was associated with a novel round structure in the mitochondrion. Overexpression of TorA induces the number of these structures and simultaneously causes cell growth to become surface dependent.
Interestingly, the torA- phenotype strongly resembles that of the previously identified mek1- (
The torA- mutant does not display obvious defects in cAMP signaling. The cells produce wild-type amounts of cAMP in response to exogenous cAMP, and the frequency of the cAMP waves is similar to that of wild-type cells. In addition, other signaling responses that have been implicated in chemotaxis, such as cGMP accumulation and actin polymerization, are not altered in torA-. Several data indicate that torA- cells are not impaired in sensing cAMP gradients. First, they express wild-type levels of cAMP receptor. Second, the sensitivity of the actin polymerization response is not altered in torA- cells. Third, the mutant is capable of directional sensing of the cAMP gradient, since it recruits PH domains to the side of the highest concentration.
However, torA- cells are incapable of responding to a spatial gradient with directed movement. This aberrant behavior was also manifested in natural aggregation territories in submerged cultures. Control cells suppress lateral pseudopod formation when chemotaxing up a spatial gradient (
The unexpected localization of TorA to a single punctate region of the mitochondrion raises several interesting questions about the role of TorA in chemotaxis and surface-dependent growth. This is the first description of a protein that localizes near the end of a mitochondrion. This suggests that mitochondria are not symmetric. Interestingly, TorA colocalizes with a structure in the mitochondrion that shows enhanced staining with the mitochondrial dye Mitotracker. These staining patterns are also observed in cells that do not overexpress TorA, but the number of mitochondria that display this structure is much lower. Similarly, many mitochondria from TorAGFP-overexpressing cells displayed an electron dense spot in electron micrographs. These data suggest that TorA overexpression enhances the formation or enlargement of these structures. In addition, TorA is completely resistant to both Triton X-100 and sodium carbonate extraction, which completely dissolves the mitochondrion. Only very few proteins have these properties (Fig 9 C) and it remains to be investigated whether they are part of the structure that contains TorA.
Deletion analysis shows that the ability to form clusters resides in the NH2-terminal half of the TorA protein. A shorter construct, containing only the NH2-terminal 156 residues fused to GFP, still targets the protein to mitochondria but forms only very few clusters resulting in diffuse mitochondrial staining. The sequence contains many positively charged residues, which may represent a mitochondrial import signal. Expression of a mutant protein that lacks 63 COOH-terminal residues is able to form clusters but does not rescue the torA- phenotype, indicating that cluster formation is not sufficient for TorA function.
Overexpression of TorA causes the inability to grow in suspension, and nonadherent cells die. This growth defect correlates with increased staining of the submitochondrial structures with Mitotracker and may be the result of impaired mitochondrial function due to a general increase in number or size of these structures. The growth defect is not a cytokinesis defect as has been described for a variety of Dictyostelium mutants (
We can only speculate on the mechanism by which disruption of TorA affects chemotaxis. One possibility is that TorA is necessary for normal mitochondrial function. However, the specificity of the chemotaxis defect suggests mitochondria of torA- are still producing wild-type levels of ATP. Evidently, we cannot rule out that mitochondria in torA- cells are impaired in other functions that may affect chemotaxis. Chemotaxis may demand a sudden increase in mitochondrial function or a repositioning of mitochondria, which requires TorA. TorA disruption or overexpression may affect the shape of mitochondria. Living Ax3-, torA--, and TorA-overexpressing cells display similar numbers and shapes of mitochondria. However, in fixed cells, mitochondria from torA- appeared larger and rounder when stained with DAPI or Mitotracker (van Es, S., unpublished observations). This suggests that there may be a subtle defect in mitochondrial structure that is not readily visualized in living cells.
Mek1 and TorA mutants display similar impairments in chemotaxis and the capacity of Mek1 to suppress the chemotactic defects in TorA is intriguing. Mek1 mutants may have a similar mitochondrial defect as TorA mutants. Alternatively, TorA may somehow affect the Mek1 activity or localization. We are currently studying the localization of Mek1 in wild-type cells and TorA mutants to investigate this possibility.
The unexpected finding that a protein with specific roles in chemotaxis and substrate sensing is localized to mitochondria is fascinating and suggests that processes like chemotaxis also require mitochondrial proteins in other species. Thus far, no strong homologues of TorA have been found in other species, but it remains possible that functional homologues in other species exist that share a similar structure but have little obvious sequence homology. For example, recent data in Drosophila and mammalian cells suggest that functional homologues exist between species that do not share any sequence homology (
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Footnotes |
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The online version of this article contains supplemental material.
1 Abbreviations used in this paper: cGMP, guanosine 3', 5' cyclic monophosphate; Crac, cytosolic regulator of adenylyl cyclase; DIAS, dynamic image analyzing system; GFP, green fluorescent protein; REMI, restriction enzymemediated integration; TorA, Tortoise.
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Acknowledgements |
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The authors wish to thank Dr. Rick Firtel (University of California at San Diego, La Jolla, CA) for the Mek1 expression plasmid, Dr. Carole Parent (National Institutes of Health, Bethesda, MD) for the PH(Crac)-GFP plasmid, and Dr. Margaret Clarke (Oklahoma Medical Research Foundation, Oklahoma City, OK) for the TopA-GFP expression plasmid.
Part of this work was performed by D. Wessels and D.R. Soll who used the WM Keck Dynamic Image Analysis Facility at the University of Iowa, which is funded by the WM Keck Foundation. This work was supported by the Netherlands Organization for Scientific Research (NWO) grant to S. van Es, and National Institutes of Health grants GM28007 to P.N. Devreotes and HD18577 to D.R. Soll.
Submitted: 7 September 2000
Revised: 8 December 2000
Accepted: 8 December 2000
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References |
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