1 Howard Hughes Medical Institute
2 Department of Biochemistry and Cell Biology, MS-140, Rice University, 6100 S. Main Street, Houston, TX 77005-1892, USA
* Present address: Department of Biological Sciences Hunter College, Rm 927 North Building, 695 Park Avenue, New York, NY 10021, USA
Author for correspondence (e-mail: richard{at}bioc.rice.edu)
Accepted July 19, 2001
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SUMMARY |
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Key words: ABC, Multidrug, pH, Cell-type choice, Cell fate, Musical chairs
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INTRODUCTION |
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It has been suggested that the pH of intracellular vesicles, in part, regulates initial cell-type choice (Gross et al., 1988). In support of this hypothesis, we had previously recovered a mutant of Dictyostelium that has an altered endosomal pH and accordingly an altered ratio of initial cell types (Wood et al., 1996; Brazill et al., 2000). The gene disrupted in this strain, rtoA, was originally identified using shotgun antisense transformation (Spann et al., 1996) in a screen designed to identify mutants with altered cell-type differentiation. Transformants having any observable developmental abnormality were screened by assaying for changes in the percentages of cells differentiating into prestalk and prespore cells. The rtoA mutant had an abnormally high percentage of prestalk cells (Wood et al., 1996) (R. R. Ammann and R.H.G., unpublished). The rtoA gene disruption transformant had the same phenotype as the original antisense mutant, including a high prestalk/prespore ratio, and a developmental arrest at the mound stage.
In addition to pH, it has been shown that the initial differentiation of Dictyostelium cells occurs by a combination of asymmetric cell division and a simple musical chairs mechanism based on the cell cycle (Araki et al., 1997; Araki et al., 1994; Gomer and Firtel, 1987; Krefft and Weijer, 1989; McDonald and Durston, 1984; Ohmori and Maeda, 1987; Weijer et al., 1984a; Zimmerman and Weijer, 1993). The initial cell-type decision is made at the initiation of starvation. For each pair of sister cells in S or early G2 phase, one sister differentiates into a prestalk cell whereas the other sister becomes a null cell (a cell type that initially expresses neither prespore nor prestalk markers, but which can later differentiate into either cell type). For each pair of sister cells in late G2 or M, one sister differentiates into a prespore cell and the other sister becomes a null cell (Dictyostelium grown under standard laboratory conditions has an undetectable G1 phase (Weijer et al., 1984b)). This mechanism regulates only initial differentiation. The eventual fate of the cell is still plastic, and a variety of factors such as adenosine, ammonia, a chlorinated hydrocarbon called DIF and oxygen can change the final fate (Brookman et al., 1987; Gross et al., 1983; Kay et al., 1989; Kwong and Weeks, 1989; Schaap and Wang, 1986; Sternfeld, 1988; Williams et al., 1987; Xie et al., 1991).
Cell-cycle analysis of rtoA cells demonstrated that the increase in the percentage of prestalk cells was not due to changes in S, G2 or M phases. However, prestalk and prespore cells from rtoA disruptants originate from cells in any phase of the cell cycle at starvation. As in wild-type cells, the sisters of the differentiated cells are all null in the rtoA mutant. These results suggest that RtoA is not involved in the asymmetric cell-division mechanism or cell-cycle progression per se, but rather is involved in some process that varies during the cell cycle, and which can be measured at the time of starvation to select initial cell type (Wood et al., 1996).
Examination of the amount of RtoA protein per cell uncovered a marked heterogeneity in the amount of RtoA in individual vegetative cells (Brazill et al., 2000). The cells with high RtoA are all in the prespore phase of the cell cycle, whereas the sisters of these cells have low levels of RtoA. The cells in the prestalk phase of the cell cycle have intermediate levels of RtoA, suggesting that relative levels of RtoA protein may determine initial cell type choice. Sixty per cent of the RtoA amino acid sequence is ten tandem repeats of an 11 amino acid serine-rich motif, which we found to have a random coil structure. A recombinant protein containing this domain was able to catalyze the fusion of phospholipid vesicles, suggesting a role for RtoA in vesicle fusion. Light and electron microscopy confirmed that rtoA cells have a defect in the fusion of endocytic vesicles. In wild-type cells, sister cells tend to have different vesicle processing rates, whereas in rtoA cells the processing rates are generally the same for all cells. rtoA cells also have a decreased exocytosis rate and a decreased pH of endocytic/exocytic vesicles.
The products of many genes regulate the pH of cells and organelles. One class of proteins that does this is the ATP binding cassette (ABC) transport proteins. These proteins are found in a number of organisms, but are most noted for their involvement in cystic fibrosis (Riordan et al., 1989), multidrug resistance in human cancers (Gros et al., 1986; Gros et al., 1988; Riordan et al., 1985) and chloroquine resistance in the malaria-causing paramecium Plasmodium falciparum (Krohstad et al., 1987; Reed et al., 2000). Proteins belonging to this family use the energy from hydrolysis of ATP to transport molecules from one side of a membrane to another. These transported molecules can range in size and complexity from single ions to peptides (Gilson et al., 1988; Higgins, 1992). In Dictyostelium, there exist ABC transporters that are involved in the release of signals from cells (Good and Kuspa, 2000; Shaulsky et al., 1995). The P. falciparum ABC transporter, Pgh1, which is involved in chloroquine resistance, is believed to control the pH of the food vacuole, the site of chloroquine activity (Cowman et al., 1991; van Es et al., 1994a). When Pgh1 is mutated, the food vacuole is not properly acidified. Chloroquine, being a weak base, is unable to enter the food vacuole due to a decreased pH gradient. Pgh1 expressed in mammalian cells is localized to the intercellular vesicles. In addition, lysosomal acidification and increased accumulation of drug were observed, supporting the role of Pgh1 in pH regulation (van Es et al., 1994a; van Es et al., 1994b).
To identify other genes involved in the RtoA-regulated mechanism for choice of cell type, we initiated a second-site suppressor screen of the rtoA mutant. Second-site suppressor analysis is a genetic tool that can be used to identify components of genetic pathways (Shaulsky et al., 1996). Usually, genetic suppressors rescue the defect of the original mutation by modifying a downstream or interacting component in the faulty pathway. Using restriction-enzyme-mediated integration (REMI), we created random insertional mutations in Dictyostelium cells lacking RtoA. Selecting for mutations that allowed rtoA cells to develop past their original block at the mound stage, we identified mdrA1 and mdrA2, an apparent tandem duplication that encodes proteins with high homology to ABC transporter proteins.
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MATERIALS AND METHODS |
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REMI mutagenesis and suppressor screen
Isolation of second site suppressors followed Shaulsky et al. (Shaulsky et al., 1996) with the following modifications. rtoA cells were grown to 4x106 cells per ml in liquid HL5 medium as above. Cells were cooled on ice for 15 minutes, harvested by centrifugation and resuspended at 1x107 cells per ml in ice-cold electroporation buffer (10 mM Na2HPO4, 10 mM KH2PO4 and 50 mM sucrose, pH 6.1). Five ml of cells were mixed with 10 µg/ml of BamHI-linearized pUCBsrBam and 30 units/ml of DpnII restriction endonuclease (New England Biolabs, Beverly, MA) immediately before electroporation. Aliquots of 0.8 ml were placed into 0.4 cm gap cuvettes (Bio-Rad, Pinole, CA) and electroporated at 1.0 kV and 3 µF with a Bio-Rad Gene Pulser (Bio-Rad). The cells were transferred immediately into HL5. The cells were plated at 1x106 cells per 10 cm plate and incubated for 15 hours. Blasticidin S (Calbiochem, San Diego, CA) was added to each plate to a concentration of 4 µg/ml and the cells were incubated for another 6 days. Drug-resistant colonies were collected and plated at approximately 200 cells per 10 cm plate on SM/5 agar with Klebsiella aerogenes. Plates were screened for mutants that showed more advanced development than the original rtoA mutant. Individual plaques were isolated and plated on SM/5 agar with K. aerogenes.
Nucleic acid manipulation
Cloning of the DNA sequences flanking the insertion site and recapitulation of the mutant by homologous recombination were performed using the method of Kuspa and Loomis (Kuspa and Loomis, 1992). Sequencing of this clone was performed as previously described (Wood et al., 1996). Additional sequence was obtained from the Dictyostelium Genome Sequencing Project at the Jena Genome Sequencing Center (Jena, Germany) and by PCR of genomic DNA and cDNA. Northern and Southern blot analyses were performed following Wood et al. (Wood et al., 1996) using a DNA probe to a 500 bp region near the middle of mdrA2 obtained by RT-PCR (Perkin Elmer, Branchburg, NJ) from mRNA isolated with an Oligotex Direct mRNA Isolation Kit (Qiagen, San Clarita, CA) with the primers 5'-ATCGTAAAGATCCAGTTGG-3' and 5'-TCTCTAACCGTTGACACTGG-3'.
Preparation of a recombinant MdrA1 fragment and immunofluorescence
The reverse transcriptase polymerase chain reaction (RT-PCR) was used to generate a DNA fragment for fusion protein production. Poly(A+) RNA was isolated from cells starved for 5 hours using an Oligotex Direct mRNA Isolation Kit (Qiagen, San Clarita, CA). cDNA was created by performing RT-PCR for 1 cycle (90°C for 5 minutes, 23°C for 10 minutes, 42°C for 30 minutes and 95°C for 10 minutes) with MuLV reverse transcriptase following the manufacturers directions (Perkin Elmer, Branchburg, NJ). To generate cDNA for the second ATP binding region (amino acids 745-973) of MdrA1, the primer 5'-CGGAATTCAAATGAAATCGGCTGG-3' was used. Using this cDNA as a primer, PCR was carried out for three cycles (95°C for 30 seconds, 37°C for 30 seconds, 72°C for 1 minute) followed by 27 cycles (95°C for 30 seconds, 60°C for 30 seconds, 72°C for 1 minute) with Bio-X-act polymerase following the manufacturers directions (Intermountain Scientific Corp., Salt Lake City, UT). The above primer as well as 5'-GCGGATCCGGTTATGTTAAACCGGG-3' were used to amplify the DNA. The primers created an EcoRI site at the 3' end and a BamHI site at the 5' end of the PCR product. The purified product was then digested with EcoRI and BamHI and ligated into the BamHI/EcoRI digested expression vector pGEX3 (Amersham, Piscattaway, NJ) after the enzymes had been heat inactivated. Fusion protein was induced and purified using the BPER system and a glutathione-agarose column (Pierce, Rockford, IL) following the manufacturers direction. The protein was eluted in three fractions, which were then pooled. These fractions consisted of >85% fusion protein. The fusion protein was used to immunize a rabbit as described in Jain et al. (Jain et al., 1997). Serum was collected and was purified using an E-Z-Sep kit (Pharmacia). For affinity purification of the above sera, the fusion protein was attached to a beaded agarose support using an AminoLink immobilization kit (Pierce, Rockford, IL). The antibody was purified using their suggested protocol. Immunofluorescence was performed with this antibody using the method of Zhu and Clarke (Zhu and Clarke, 1992), without the agarose overlay.
Antibody production and western blots
Because we found that the above antibody gave a high background when used for western blots, the synthetic peptide NYFNNKHNKKQNDDSD from MdrA2 was conjugated to keyhole limpet hemocyanin and this was then used to immunize a rabbit at Biosynthesis Inc. (Lewisville, TX). Serum was collected two weeks after the third injection and was purified using an E-Z-Sep kit (Pharmacia). The antibody was purified as described above using the synthetic peptide attached to agarose beads. 1x105 cells were collected at different stages of development and boiled for 3 minutes in SDS PAGE sample buffer in a final volume of 100 µl. A 20 µl aliquot was loaded onto a 7.5% PAGE gel and western blots were done following Jain and Gomer (Jain and Gomer, 1994).
Light microscopy
To monitor pinocytosis, 200 µl of cells at a density of 1x105 cells/ml in HL5 were placed onto an 8-well coverglass bottom slide (Type 136439, Nunc, Naperville, IL) and allowed to settle for 10 minutes. The medium was then gently changed to 2 mg/ml rhodamine isothiocyanide dextran (RD) (Sigma) in HL5 for 10 minutes then replaced with HL5. After 80 minutes, lysosomes were stained by adding Lysosensor Green DND-189 (Molecular Probes) to 1 mM. After 10 minutes, the medium was changed to HL5 for an additional 10 minutes. Cells were then fixed for 3 minutes in HL5 containing 1% formaldehyde, after which the medium was changed to HL5. This procedure was also performed without chasing the RD for 80 minutes with HL5. Images were taken with a Nikon Diaphot inverted microscope using a x60 1.4 NA phase/fluorescence objective lens. Phase illumination was provided by a 12V/100W lamp operating at 4 V, and the epifluorescence illumination was provided by a 12V/100W lamp operating at 11.5 V. Both lamps used external power supplies to reduce heating of the microscope. A heat-blocking filter was in the phase illumination beam. A Photometrics cooled CCD camera operating at an exposure of 16x and gain of 24 db supplied a video image to a computer and direct monitor. To ensure that signals were not saturating, an oscilloscope was used to monitor the video signal. Nuclei were stained with DAPI following Wood et al. (Wood et al., 1996). Staining of calmodulin in fixed and permeabilized cells was done following Zhu and Clarke (Zhu and Clarke, 1992), omitting the agarose overlay step.
Endocytosis, exocytosis and pH assays
Endocytosis was assayed as previously described (Buczynski et al., 1997a). Fluid phase exocytosis was measured using FITC-dextran (FD) (Sigma) as previously described (Bush et al., 1996) with slight modifications. Cells grown to 2x106 cells/ml were harvested and resuspended in HL5 containing FD at 2 mg/ml. The cells were allowed to internalize FD for 3 hours and then were collected and resuspended in fresh HL5. At the indicated times, 1 ml of cells was collected by centrifugation and washed once in HL5 and twice in 5 mM glycine-NaOH (pH 8.5) containing 100 mM sucrose. The cells were then lysed in 1 ml of the same buffer containing 0.2% Triton X-100. The fluorescence of the lysate was measured in an Aminco Bowman Series 2 Luminescence Spectrometer. Excitation and emission wavelengths for FD were 495 nm and 520 nm, respectively. Endocytic pH was measured following Aubry et al. (Aubry et al., 1993) as described by Brazill et al. (Brazill et al., 2000). BCECF staining to measure cytosolic pH was done as described previously (Brazill et al., 2000).
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RESULTS |
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Sequence similarity of MdrA1 and MdrA2 to ABC transporters
Genomic and cDNA clones were sequenced and the deduced amino acid sequences were compared with various protein databases using BLAST. The insert had affected two tandemly oriented highly similar genes, mdrA1 and mdrA2, both of which are members of the ABC transporter family. Comparison of the nucleotide sequence also showed a similarity in how these genes are coded. Each is made up of two coding regions at the 5' end of the gene, with the first being 430 and
470 bp, respectively, and the second being
270 bp in both genes; a third coding region containing
3300 and 3700 bp, respectively, is situated at the 3' end. In the two genes the first intron is 92 and 140 bp and the second intron is 104 and 135 bp, respectively. The predicted sizes of MdrA1 and MdrA2 are 149 kDa and 166 kDa, respectively. The amino acid sequences of the two proteins have a 50.2% identity over their entire length, with an 84% identity in the middle 276 amino acids. The main differences between the two proteins are three repetitive regions with a length of 10-25 aa and a
50 aa arginine-rich region found in MdrA2 but not in MdrA1.
As is found in other members of the ABC transporter family, MdrA1 and MdrA2 contain two copies each of the ABC transporter sequence motif that constitutes a nucleotide-binding fold. The sequence is found in similar locations on each gene, from amino acids 85-255 and 728-916 in MdrA1 and 98-288 and 841-1029 in MdrA2. Both contain the two characteristic six transmembrane-spanning regions and Walker A and B motifs found in ABC transporter proteins (Higgins et al., 1986; Higgins et al., 1985; Walker et al., 1982). ABC transporters can have several topologies based on the placement of the nucleotide-binding domain (NBD) and the transmembrane domain (TMD) (Van Veen and Konings, 1998). MdrA1 and MdrA2 fall into the group with the structure {NBD-TMD}2, which places them with PDR5 from Saccharomyces cerevisiae (Balzi et al., 1994) and WHITE from Drosophila melanogaster (OHare et al., 1984) (SWISS-PROT accession no. P10090). Other structures include {TMD-NBD}2, two separate peptides {TMD}2 {NBD}2 and half-size transporters {TMD-NBD} and {NBD-TMD} (Van Veen and Konings, 1997). Fig. 2 aligns the NH2-terminal and COOH-terminal ABC cassettes of MdrA1 and MdrA2 with the consensus ABC transporter cassette. Given their proximity to each other and their high sequence identity, we believe that these two genes represent a tandem duplication. Tandem duplication events have been seen in a number of Dictyostelium genes including the adhesion protein gp24 and the discoidin I gene family (Loomis and Fuller, 1990; Welker, 1988). Sequence comparisons with other proteins show a high similarity to yeast proteins Pdr5 (29% and 27% identity for MdrA1 and MdrA2, respectively) and Bfr1 (29% and 26% identities). PDR5 is a S. cerevisiae gene cloned for its ability to confer resistance to cycloheximide and sulfometuron methyl when in multicopy (Leppert et al., 1990). bfr1+ is a Schizosaccharomyces pombe gene cloned by its ability to confer resistance to Brefeldin A (SWISS-PROT accession no. P41820) when in multicopy (Nagao et al., 1995a). The Drosophila proteins Scarlet (EMBL/GenBank/DDBJ accession no. P45843) and White show 29% and 30% identity to MdrA1, and 28% and 29% to MdrA2, respectively.
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DISCUSSION |
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mdrA1/mdrA2 was selected as a suppressor of the rtoA developmental defect, suggesting some aspect of MdrA1 and MdrA2 function counteracts normal RtoA function. One defect associated with the rtoA mutation is improper endosomal/lysosomal function, including poor exocytosis and an inability to condense the endosome/lysosome in response to osmotic stress (Brazill et al., 2000). Thus, it is reasonable to expect that a mutation that suppresses the rtoA phenotype may be involved in the functioning of this system. Indeed, MdrA1 protein is localized to the endosome/lysosome. In addition, mdrA1/mdrA2 cells are partially defective in pinocytosis, an endosomal function. When the mdrA1/mdrA2 mutation is placed in the rtoA background, this defect is accentuated, suggesting that these two proteins operate in the same system. However, as RtoA is localized to the cytoplasm and possibly small vesicles (Brazill et al., 2000), and MdrA1 is associated with endosomes, these proteins probably do not form a stable complex, and their interaction is strictly genetic. Taken together, these facts argue strongly for a role of MdrA1 and MdrA2 in proper functioning of the endosome/lysosome and it is the disruption of this function that allows the mdrA1/mdrA2 mutation to suppress rtoA. Of course, we have not ruled out the possibility that MdrA1 and MdrA2 may have other functions during development, and it is the loss of these functions that suppress the rtoA phenotype, especially the arrest at the mound stage. However, the experiments presented here support the role of the mdrA1/mdrA2 mutation in suppressing the rtoA endosomal defects.
pH and cell-type choice
An important aspect of the rtoA phenotype is the inability of this strain to properly proportion its cells into prestalk and prespore cells at the initiation of development (Wood et al., 1996). This defect stems from a disruption of the musical chairs mechanism, which Dictyostelium uses to regulate initial cell-type choice. Disruption of mdrA1/mdrA2 is able to suppress the gross inability of rtoA cells to develop past the mound stage, and also causes rtoA cells to initially differentiate into the correct percentages of prestalk and prespore cells (Table 1; Table 2). Although it has not been shown that the alteration of the initial percentages of prestalk and prespore cells is directly responsible for the arrest seen at the mound stage, it is none the less significant that loss of mdrA1 and mdrA2 can correct both phenotypes. However, this is not accomplished by repairing the musical chairs mechanism (Fig. 4). Strains carrying mutations in mdrA1/mdrA2 and rtoA still recruit prestalk cells from throughout the cell cycle instead of from cells in S and early G2 phases. These strains are also unable to restrict prespore cells from initially coming from only late G2 and M phases. Thus, mutating mdrA1 and mdrA2 is able to suppress the altered prestalk/prespore phenotype of rtoA in a manner that bypasses the cell-cycle-dependent cell-type-choice mechanism.
It has been theorized that pH plays a vital role in controlling differentiation in Dictyostelium (Furukawa et al., 1988). Acidification favors prestalk development, whereas alkalization promotes prespore differentiation. However, this regulation is thought to be based not on the pH of the cytoplasm, but on the pH of a specific class of vesicles (Gross et al., 1988). In prestalk cells, these vesicles would have a lower pH, whereas the same vesicles in prespore cells would have a higher pH. Mutants with defective vacuolar H+-ATPases have abnormal endosomal pHs but normal cytosolic pHs, suggesting that there is not an absolute connection between the pH of a vesicle and cytosolic pH (Aubry et al., 1993; Davies et al., 1996). Our data support the idea that cytosolic pH does not determine initial cell-type choice, as rtoA shows a high cytosolic pH and a high prestalk/prespore ratio, whereas rtoA/mdrA1/mdrA2 cells also have a high cytosolic pH but have normal prestalk/prespore ratios. rtoA cells have endosomes with a lower pH than wild-type cells (Brazill et al., 2000). In addition, rtoA strains have a higher percentage of prestalk cells than wild-type cells. Because mdrA1/mdrA2 cells have endosomes with higher pHs than wild-type, but initially differentiate into essentially normal numbers of prestalk and prespore cells (Table 1; Table 2), our data indicate that endosomal pH is not the primary determinant of the initial cell-type choice. This suggests either that vesicles that are not part of the endosome/lysosome system are responsible for a pH regulation of differentiation, or that something such as Ca2+ levels may regulate the initial cell-type choice (Azhar et al., 1998; Azhar et al., 1996; Saran et al., 1994).
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ACKNOWLEDGMENTS |
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