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AIP1 homolog in Dictyostelium discoideum, is required for multicellular development under low Ca2+ conditions
Gene 337 (2004) 131 – 139 www.elsevier.com/locate/gene
DdAlix, an Alix/AIP1 homolog in Dictyostelium discoideum, is required for multicellular development under low Ca2+ conditions Susumu Ohkouchi, Medhat S. El-Halawany, Fumika Aruga, Hideki Shibata, Kiyotaka Hitomi, Masatoshi Maki * Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8601, Japan Received 16 February 2004; accepted 22 April 2004 Received by T. Sekiya
Abstract Apoptosis-linked gene 2 (ALG-2) interacting protein X (Alix), also called AIP1, is a widely conserved protein in eukaryotes. Alix and its homologs are involved in various phenomena such as apoptosis, regulation of cell adhesion, protein sorting, adaptation to stress conditions, and budding of human immunodeficiency virus (HIV). To investigate the role of Alix in development, we identified an Alix homolog in the cellular slime mold Dictyostelium discoideum and disrupted the gene by homologous recombination. The growth of DdAlix deletion mutant (alx ) cells was significantly impaired in the presence of 5 mM Li+. On an agar plate, alx cells underwent normal development and formed fruiting bodies indistinguishable from those formed by wild-type cells. However, alx cells could not form fruiting bodies in the presence of 5 mM Li+. Similar results were obtained when cells were developed in the presence of 3,4,5-trimethoxybenzoic acid 8(diethylamino)octyl ester (TMB-8), which is an antagonist of intracellular Ca2 + store. Furthermore, when the extracellular free Ca2 + was reduced to 10 nM, the ability of alx cells, but not that of wild-type cells, to form fruiting bodies was impaired. The results indicate that DdAlix is essential for development under low Ca2 + conditions and suggest that DdAlix is involved in Ca2 + signaling during development. D 2004 Elsevier B.V. All rights reserved. Keywords: Dictyostelium; Ca2+; Li+; Homologous recombination
1. Introduction Mouse ALG-2 interacting protein X (Alix, also named AIP1) was identified as a binding partner for the penta EFhand Ca2 +-binding protein apoptosis-linked gene 2 (ALG-2) (Missotten et al., 1999; Vito et al., 1999). Alix is a widely conserved protein in eukaryotes. Alix homologs have a Bro1-rhophilin conserved domain (BRD; registered as BRO1 domain in the NCBI conserved domain database), a Abbreviations: ALG-2, apoptosis-linked gene 2; Alix, ALG-2 interacting protein X; BRD, Bro1-rhophilin conserved domain; CBB, Coomassie brilliant blue; CC, coiled-coil; EGTA, ethylene glycol bis(h-aminoethylether)-N,N,NV,NV-tetraacetic acid; HIV, human immunodeficiency virus; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; PRR, proline-rich region; SDS, sodium dodecyl sulfate; TMB-8, 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester. * Corresponding author. Tel.: +81-52-789-4088; fax: +81-52-7895542. E-mail address: [email protected] (M. Maki). 0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2004.04.020
potential recognition site for Src-type tyrosine kinases in higher eukaryotes, predicted coiled-coils (CC), and a proline-rich region (PRR). Recently, we showed that ALG-2 recognizes PxY repeats in the Alix PRR in a Ca2 +-dependent manner (Shibata et al., 2004). Alix has been shown to interact with focal adhesion kinase (FAK) and proline rich tyrosine kinase 2 (PYK-2) (Schmidt et al., 2003). Although the binding site has not been determined, interaction between Alix and PYK-2 increases in the presence of Ca2 +. The relationship between Alix and Ca2 + signaling is unclear. Alix is known to have various functions. Alix is thought to be involved in apoptosis, since ALG-2 was identified as a gene required for apoptosis (Vito et al., 1996). Overexpression of a truncated form of mouse Alix containing CC and PRR makes astrocytes resistant to apoptosis (Vito et al., 1999). On the other hand, overexpression of human Alix (also named Hp95) in HeLa cells results in promotion of detachment-induced apoptosis (anoikis), and inhibition of tumorigenicity (Wu et al., 2002). Accumulating evidence
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suggests that Alix plays a role in protein sorting. Alix interacts with CHMP4 proteins, which are human homologs of yeast Snf7/Vps32 (Katoh et al., 2003, 2004), and interaction between Alix and Gag late domain p6 is required for the budding of human immunodeficiency virus (HIV) (Marsh and Thai, 2003). Alix also interacts with TSG101, one of the components of endosomal sorting complex required for transport (ESCRT) complex (Marsh and Thai, 2003). Bro1 (also known as Vps31 or Npi3) is one of the Alix homologs in Saccharomyces cerevisiae and is involved in protein sorting. S. cerevisiae has another Alix homolog, named Rim20. A mutant in which Rim20 is deleted is sensitive to Na+, Li+, and alkaline pH. Similarly, PalA, an Alix homolog of Aspergillus nidulans, is involved in regulation of alkaline pH-induced gene expression (Arst and Pen˜alva, 2003). Although there are many reports on the functions of Alix in mammalian and fungal cells, there is no report on the role of Alix in development of multicellular organisms. The functions of Alix homologs in the nematode or fruit fly are not known. To elucidate the role of Alix in development, we investigated the function of the Alix homolog DdAlix in the cellular slime mold Dictyostelium discoideum, which is a useful haploid model organism for studying many basic issues in cell biology. Dictyostelium cells proliferate as unicellular amoeba cells in a rich nutrient. Upon starvation, cells aggregate using cAMP as a chemoattractant. These cells form a migrating slug that becomes a fruiting body consisting of a mass of spore cells supported by a thin column of stalk cells. Cytoplasmic Ca2 + and H+ concentrations determine the cell fate (Kubohara and Okamoto, 1994). A slow sustained increase in cytosolic Ca2 + level mediates stalk gene induction by a low-molecular-weight signaling molecule named differentiation-inducing factor DIF-1 (Schaap et al., 1996). In this study, we disrupted the DdAlix gene by homologous recombination and analyzed phenotypes of the strain. DdAlix was found to be not essential for multicellular development under normal laboratory conditions but essential in the presence of Li+, an intracellular Ca2 + store antagonist TMB-8, and under the condition of low extracellular Ca2 +. Our results suggest that DdAlix is involved in Ca2 + signaling during development.
2. Materials and methods 2.1. Cloning of DdAlix cDNA A homology search was performed using the tBLASTn program (http://www.ncbi.nlm.nih.gov/BLAST/) with a mouse Alix amino acid sequence as a query. Primers for DdAlix cDNA cloning were designed on the basis of the retrieved DNA sequence (GenBank accession no. AF360741). Total RNA was isolated from vegetative cells using a QuickPrepk Micro mRNA purification kit (Amer-
sham) according to the instructions of the manufacturer. Reverse transcription was performed using a 5V-full RACE core kit (Takara, Japan) according to the instructions of the manufacturer. Reaction mixtures were incubated at 30 jC for 10 min and at 42 jC for 30 min and then heated to 95 jC for 5 min. DdAlix cDNA was cloned by polymerase chain reaction (PCR) using pfuTurbo DNA polymerase (stratagene), a pair of primers (5V-GCGGATCCATGTTATCAATCGAGAGAAAAAG-3V, the attached BamHI site and two additional bases indicated by an underline, and 5VCGGCTCGAG CTTAATAATGTTTATTATTTGAATTA-3V, the XhoI site and three additional bases indicated by an underline), and the product of reverse transcription as a template. After digestion with BamHI/XhoI, the PCR product was ligated into pCMV-Tag2C (Stratagene) and the nucleotide sequence was determined with an automated fluorescent sequencer, ABI PRISM310 (PE Applied Biosystems), using a Big Dyek terminator cycle sequencing ready reaction kit (PE Applied Biosystems) and either universal or custom synthesized primers. The resultant plasmid was designated as pCMV-Tag2C-DdAlix. 2.2. Plasmid constructions A DdAlix cDNA fragment was excised with BamHI/ XhoI from pCMV-Tag2C-DdAlix and ligated into the BamHI/XhoI site of pTX-FLAG (Levi et al., 2000). The resultant plasmid was designated as pTX-FLAGDdAlix and used for complementation analyses. A DdAlix disruption vector was constructed as follows: genomic DNA from vegetative Dictyostelium cells was amplified by a PCR reaction with a pair of primers (5VCCCATGGACAGATTCATATAGAC-3V and 5V-GGATCCCTTGTTCTAACCCTTTTGCGTG-3V, the attached BamHI site shown by an underline), followed by ligation into pCR2.1-TOPO (Invitrogen). An EcoRI/BamHI fragment was ligated into the EcoRI/BamHI site of pUC118, and the resultant plasmid was designated as pDdAlixKO5V. Similarly, the 3V-flanking region was amplified with a pair of primers (5V-GGATCCGTAAACTTAGCCAAAACAGCACC-3V, the attached BamHI site shown by an underline and 5V-CTCTATTGGCCATCAAAGCATCC-3V) and ligated into pCR2.1-TOPO. A BamHI/PstI fragment was ligated into the BamHI/PstI site of pDdAlixKO5V, and a BamHI fragment of the blasticidin-resistant cassette from modified pBsR503 (Puta and Zeng, 1998) was inserted into the BamHI site. The resultant plasmid, designated as pDdAlixKO, was used as a DdAlix gene disruption vector. 2.3. Anti-DdAlix antibody and Western blotting Anti-DdAlix antisera were obtained from a rabbit immunized with a DdAlix peptide (TQDYAKYTSHLQHST KSDC, corresponding to amino acids 481– 498, the carboxy-terminal cysteine residue being added for cross-linking
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reaction; indicated in Fig. 1A) coupled with keyhole limpet hemocyanin using m-maleimidobenzoyl-N-hydroxysuccinimide ester. The antisera were affinity-purified using Activated Thiol Sepharosek 4B (Amersham) coupled with antigen peptide according to the manufacturer’s instructions. For Western blotting to confirm gene disruption, cells were
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collected, washed twice with PBS, and lysed in SDS-sample buffer followed by immediate boiling. After samples has been subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore), DdAlix protein was detected with an anti-DdAlix polyclonal antibody by the color development method using diaminobenzidine (DAB) as described previously (Kitaura et al., 1999). 2.4. Cell culture D. discoideum wild-type strain AX-2 and DdAlix deletion mutant (alx ) cells were cultured in HL5 medium at 22 jC. The alx cells expressing exogenous FLAG-tagged DdAlix (alx /F-DdAlix) were maintained in HL5 medium in the presence of 10 Ag/ml G418. The number of cells was calculated using a hematocytometer. 2.5. Transformation of Dictyostelium
Fig. 1. Structure and expression of DdAlix. (A) Schematic representation of DdAlix and human Alix. Bro1-rhophilin conserved domain (BRD: registered as BRO1 domain in the NCBI conserved domain database; accession number 17278; pfam03097), predicted coiled-coil region (CC) and proline-rich region (PRR) are indicated by gray, hatched and closed boxes, respectively. ‘‘Y’’ indicates the tyrosine residue in a highly conserved potential recognition site for Src type tyrosine kinases in higher eukaryotes. Degrees of similarities in BRD, PRR, middle region between the BRD domain and PRR, and full length between DdAlix and human Alix are shown as amino acid identities (%). Positions of introns in the genomic DNA of DdAlix and human Alix are indicated by arrows. The peptide used for preparation of the antibody is indicated by a bar. The breakpoint caused by homologous recombination is indicated by an open arrowhead (see Fig. 2). (B) Expression of DdAlix protein during development. Wild-type cells were developed on cellulose filters. Cells were collected at 0, 4, 8, 12, 16, 20, and 24 h after starvation. Lysates were prepared as described in Section 2.6 and subjected to 7.5% SDS-PAGE and Coomassie brilliant blue (CBB) staining (upper panel). Proteins were transferred to a PVDF membrane, and DdAlix protein was detected by Western blotting using an anti-DdAlix polyclonal antibody (lower panel). The arrow indicates endogenous DdAlix protein, and the asterisk indicates nonspecifically reacted protein.
For disruption of DdAlix, pDdAlixKO was linearized by digestion with EcoRI followed by phenol/chloroform extraction and ethanol precipitation. Vegetative Dictyostelium cells in mid-log phase were collected, washed twice with ice-cold electroporation buffer (10 mM NaHPO4, 50 mM sucrose, pH 6.1), and resuspended in 400 Al of electroporation buffer. After mixing with 20 Ag of digested pDdAlixKO and being transferred into a 2-mm gap cuvette, the suspension was electroporated using a ECM600 (BTX) under the following conditions: charging voltage, 500 V; resistance, 13 V; capacitance, 100 AF. Transformants were selected in HL5 medium containing 4 Ag/ml blasticidin S hydrochloride (Kaken Pharmaceutical). Apparent colonies were selected, and disruption of the DdAlix gene was checked by PCR. After limiting dilution of the isolated colonies, disruption of the DdAlix gene was checked again by PCR using a pair of primers (5V-AACTTTAAATGGATTAAGAGAAGATG-3V and 5V-GTGAATGTGGATTAACACCAGC-3V). alx cells was transformed with pTXFLAGDdAlix as described above and selected in a medium containing 10 Ag/ml G418. Colonies were picked up and further selected for a colony expressing DdAlix protein. One of the transformants, designated alx /F-DdAlix, was selected and used for complementation analyses. 2.6. Developmental analysis For development on a cellulose filter, cells were harvested, washed twice with MES buffer (20 mM MES (2-(Nmorpholino)ethanesulfonic acid)-KOH, pH 6.5), and resuspended at a density of 2 107/ml in MES buffer containing various reagents as indicated. A black filter composed of mixed cellulose ester (47 mm, Cat. No. A045N047A, ADVANTEC, Japan) and two filter papers for support (No. 3, Whatman) were washed twice with MES buffer containing the same reagents as those in the cell suspension.
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Cell suspensions were plated at a density of 1 107/filter and incubated for 2 days at 22 jC. When experiments were performed in the presence of LiCl or 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), cellulose filters and filter papers were washed in the presence of 5 mM LiCl or 200 AM TMB-8. LiCl or TMB-8 was added to the cell suspension after washing twice with MES buffer. Development using Ca2 +-ethylene glycol bis(h-aminoethylether)-N,N,NV,NV-tetraacetic acid (EGTA) buffer was performed similarly, but cells, cellulose filters, and filter papers were washed four times with HEPES buffer (20 mM HEPES (2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid)NaOH, pH 7.8) containing 1 mM EGTA and various concentrations of total Ca2 +ions to adjust free Ca2 + to 0, 10 nM or 1 mM. In this experiment, free Ca2 + concentration was calculated using WinMaxc v2.0 software (http://www. stanford.edu/~cpatton/maxc.html). For development on agar, cells were washed twice with KK2 buffer (17 mM KHPO4, pH 6.5) and plated onto 1.5% KK2-buffered agar. Development was observed under a stereoscopic microscope (OLYMPUS SZX12). Photographs were taken 48 h after starvation. Wild-type cells were plated onto cellulose filters and collected at 0, 4, 8, 12, 16, 20 and 24 h after starvation. Cells were resuspended in lysis buffer (10 mM Tris (tris(hydroxymethyl)aminomethane) –HCl, pH 8.0, 10 AM pepstatin, 125 Ag/ml leupeptin, 5 mM pefabloc, 5 AM E-64, and 1 mM phenylmethylsulfonyl fluoride) and then added with SDS-sample buffer followed by immediate boiling for 5 min. Acid-washed glass beads (0.1 g; Sigma) were added to the samples collected at 20 and 24 h after starvation and vortexed for 30 s followed by 30 s of boiling. This procedure was repeated three times. Samples were subjected to SDSPAGE followed by Coomassie brilliant blue (CBB) staining or Western blotting using an anti-DdAlix antibody.
3. Results 3.1. Primary structure of DdAlix and its expression A homology search of the database revealed that DdAlix, an Alix homolog in Dictyostelium, shows significant homology with human Alix (29.6% identity; Fig. 1A). The DdAlix gene has four exons and three introns, encoding a protein of 794 amino acids with a calculated molecular mass of 90.5 kDa (Fig. 1A). Like other Alix homologs, DdAlix has a potential recognition sequence (Y315) for Src-type tyrosine kinases, predicted coiled-coil regions, and a relatively proline-rich region in the amino acid sequence. The protein expression level of DdAlix during development was analyzed by Western blotting using an anti-DdAlix antibody (Fig. 1B). The DdAlix protein migrated slightly faster than expected and was detected as an immunoreactive band of about 80 kDa by SDS-PAGE. The DdAlix protein was expressed throughout the process of development. In Fig 1B, there is a nonspecifically reacted protein band around 43 kDa shown
by asterisk. This protein band is not a degraded product of DdAlix because the same band was also detected in case of the cell lysate of a DdAlix deletion mutant (data not shown). 3.2. DdAlix gene disruption by homologous recombination To establish a DdAlix deletion mutant, Ax-2 cells were transformed with the DdAlix gene disruption vector pDdAlixKO (Fig. 2A). Blasticidin-resistant colonies were selected, and disruption of the DdAlix gene was screened by PCR. After limiting dilution of a candidate colony, deletion of the DdAlix gene was confirmed by PCR and Western blotting. The PCR product of wild-type cells gave a band of 2.3 kbp, which was absent in the PCR product of the mutant clone. The PCR product of the mutant clone gave a band of 3.8 kbp (Fig. 2B). The difference of 1.5 kbp between the PCR products of the wild-type cells and the clone is in accordance with the size of the bsr cassette in the DdAlix gene. Disruption of the DdAlix gene was further confirmed by Western blotting using an anti-DdAlix antibody in terms of protein expression level (Fig. 2C, lane 2). A band of about 80 kDa was not detected in the isolated clone (the clone is referred to as alx ). Deletion of DdAlix was complemented by expression of FLAG-tagged DdAlix exogenously (the cells were referred to as alx /F-DdAlix). The expression was confirmed by Western blotting using an anti-DdAlix antibody and an anti-FLAG antibody (Fig. 2C, lane 3). 3.3. Growth of alx cells The growth rate of alx cells was not significantly different from that of wild-type and alx /F-DdAlix cells (Fig. 3A). Some monovalent metal ions that are known to affect the growth of Rim20-deficient yeast cells (Xu and Mitchell, 2001) were tested, and it was found that the presence of 5 mM LiCl resulted in significant reduction in the growth rate, especially in the case of alx cells, compared with the growth rates of wild-type and alx /FDdAlix cells. The number of alx cells increased by only threefold after 8 days of cultivation in the presence of LiCl. This reduction in growth rate was significantly recovered in the case of alx /F-DdAlix cells (Fig. 3B). The same concentration of NaCl or KCl did not affect the growth rate (data not shown), indicating that the effect of LiCl was caused by Li+, not by Cl . 3.4. Effects of Li+ and TMB-8 on development of alx cells To investigate the role of DdAlix in development, cells were starved to induce multicellular development. alx cells, as well as wild-type and alx /F-DdAlix cells, formed normal fruiting bodies both on agar plates (data not shown) and on cellulose ester filters (Fig. 4A –C). We next examined the effect of Li+ on development. In the presence of 5 mM LiCl, wild-type and alx /F-DdAlix cells formed normal fruiting bodies after 48 h, although development of
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Fig. 2. Disruption of the DdAlix gene. (A) Strategy for DdAlix gene disruption. Four exons of the DdAlix gene and the blasticidin resistance cassette (bsr) are shown by open and closed boxes, respectively. The bsr cassette was inserted into the third exon of the gene. PCR primers used for detection of homologous recombination are shown by arrows. (B) Confirmation of gene disruption by PCR. Genomic DNA was extracted and PCR was performed as described in Section 2.5. M, DNA marker; lane 1, wild type; lane 2, alx . (C) Western blotting analysis of DdAlix protein. Lysates from wild-type (lane 1), alx (lane 2), and alx /F-DdAlix (lane 3) cells were subjected to 7.5% SDS-PAGE and transferred to a PVDF membrane. DdAlix protein was detected by an anti-DdAlix polyclonal antibody (upper panel) or an anti-FLAG monoclonal antibody (lower panel).
alx /F-DdAlix was slightly delayed. In contrast, alx cells could not form fruiting bodies but formed aggregates or a fruiting body-like structure at the apex of aggregates (Fig. 4D – F). Li+ has been suggested to inhibit inositol monophosphatase, which reduces inositol 1,4,5-trisphosphate level and is thought to inhibit Ca2 + release from intracellular stores (Phiel and Klein, 2001). Therefore, we tested whether TMB-8, a Ca2 + antagonist for the intracellular store that has been shown to be effective for Dictyostelium (Europe-Finner and Newell, 1984; Europe-Finner et al., 1985), has effects on development. As in the presence of LiCl, alx cells could not form fruiting bodies in the presence of 200 AM of TMB-8 (Fig. 4G – I). In this experiment, alx /F-DdAlix formed fruiting bodies but in a slightly delayed time course compared with that in the case of wild-type cells. 3.5. Effect of extracellular Ca2+ on development of alx cells To show more clearly the relationship of the effects of Ca2 + and DdAlix deficiency during development, alx cells were developed in the presence of different extracellular Ca2 + concentrations. Free Ca2 + was adjusted to 0, 10
nM or 1 mM by using Ca2 +-EGTA buffer (Fig. 5). When cells were developed in the presence of 1 mM free Ca2 +, all cells formed fruiting bodies (Fig. 5A –C). In the absence of free Ca2 +, none of the cells formed fruiting bodies. They formed only loose aggregates (Fig. 5G – I). When cells were developed in the presence of 10 nM free Ca2 +, wild-type and alx /F-DdAlix cells formed fruiting bodies (Fig. 5D,F). On the other hand, alx cells did not form normal fruiting bodies, but formed only aggregates or small aberrant fruiting body-like structures (Fig. 5E). Again, development of alx /F-DdAlix was slightly delayed.
4. Discussion Alix has been isolated as a binding protein for ALG-2 (Missotten et al., 1999; Vito et al., 1999). Recently, many studies have shown that Alix plays roles in various phenomena, including membrane traffic (Springael et al., 2002; Katoh et al., 2003, 2004; Odorizzi et al., 2003), apoptosis (Vito et al., 1999; Wu et al., 2002), and budding of HIV (Marsh and Thai, 2003). This is the first report showing a role of Alix in development using a model eukaryote, D. discoideum. We disrupted the DdAlix gene by a homologous
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Fig. 3. Growth curve of wild-type, alx and alx /F-DdAlix cells. Cells were inoculated into HL5 medium in the absence (A) or presence (B) of 5 mM LiCl at a density of 5 104/ml, and cell numbers were counted for 6 (A) or 8 (B) days, respectively. Data for wild-type, alx and alx /FDdAlix cells are shown by circles, squares and triangles, respectively. Averages of results of duplicate experiments are shown.
recombination method (Fig. 2), and we analyzed the phenotype of the strain (alx ). alx cells could hardly grow in the presence of LiCl (Fig. 3B). The growth rate of alx /FDdAlix cells were significantly recovered, indicating that the slower growth rate of alx cells is due to a deficiency of DdAlix, not due to any unexpected mutations during the homologous recombination. However, we cannot exclude the possibility that the observed phenotype of alx is due to a potential dominant-negative effect of truncated DdAlix created by the recombination. This phenotype may be caused by perturbation of calcium signaling (see below). Upon starvation, alx cells formed fruiting bodies indistinguishable from those of wild-type cells on an agar plate (data not shown) and on filters (Fig. 4A –C), suggesting that DdAlix is not essential for multicellular development under standard experimental condition. In the presence of 5 mM LiCl, however, alx cells could not form normal fruiting bodies but formed aggregates or small fruiting body-like structures at the apex of aggregates (Fig. 4D – F). This phenotype was complemented by exogenous expression of DdAlix (Fig. 4F), indicating that strong sensitivity to Li+ is caused by disruption of the DdAlix gene. This effect is specific to Li+ because 5 mM NaCl or KCl did not
cause a developmental defect in alx cells (data not shown). Li+ has been used as an effective treatment for bipolar disorder (See Phiel and Klein, 2001 for review.). Li+ also has effects on the development of various organisms: it causes dorsalization of Xenopus and zebrafish. In Dictyostelium, Li+ prevents spore differentiation and promotes stalk differentiation (Maeda, 1970). These effects of Li+ on development are caused by inhibition of GSK-3 (Klein and Melton, 1996). We observed that wild-type cells could not form fruiting bodies in the presence of 20 mM LiCl (data not shown). In the presence of 5 mM LiCl, wild-type cells formed fruiting bodies, but alx cells did not (Fig. 4D – F). This result indicates that alx cells are more sensitive to Li+ than wild-type cells. It remains unknown whether the stronger sensitivity of alx to Li+ is related to GSK-3 or not. Li+, an inhibitor of inositol monophosphatase, has been suggested to reduce inositol 1,4,5-trisphosphate level and to inhibit Ca2 + release from intracellular stores (Phiel and Klein, 2001). We observed that alx cells treated with TMB-8, a Ca2 + antagonist for intracellular stores, could not form fruiting bodies, suggesting that the effect of Li+ on alx cells is related to Ca2 + (Fig. 4H). The speculation of a close relationship between alx cells and Ca2 + signaling was supported by the results of experiments in which extracellular Ca2 + was restricted by using a Ca2 +EGTA buffer (Fig. 5). In these experiments, a higher pH condition (pH 7.8) was used to maintain the chelating capacity of EGTA, which is significantly reduced at the usually used pH value (pH 6.5) for Dictyostelium development (Fig. 4). At pH 7.8, all cells, including alx cells, formed fruiting bodies in the absence of EGTA (data not shown) and in the presence of EGTA plus extra Ca2 + (Fig. 5A –C). However, when the extracellular free Ca2 + concentration was adjusted to 10 nM, alx cells did not form fruiting bodies, while the wild-type cells and alx /FDdAlix cells did (Fig. 5D – F). These results suggest that alx cells are sensitive to depletion of Ca2 + and that DdAlix is related to Ca2 + signaling. However, alx cells could form fruiting bodies when developed on agar (data not shown) or on filters under standard developmental conditions (Fig. 4B). This is probably because agar and supporting filter papers contain a sufficient amount of Ca2 + for alx cells to develop. Although alx /F-DdAlix cells formed fruiting bodies in the presence of Li+, TMB8 or 10 nM Ca2 +, development was slightly delayed. Similarly, exogenous expression of F-DdAlix in alx cells complemented the reduction of growth rate in the presence of Li+, but alx /F-DdAlix cells did not grow at the same rate as that of wild-type cells (Fig. 3B). alx /F-DdAlix cells express a large amount of DdAlix protein compared with endogenous one (Fig. 2C). An appropriate level of expression of DdAlix may be needed for adaptation to stress conditions. Ca2 + is known to play important roles in Dictyostelium development. Intracellular Ca2 + concentration increased
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Fig. 4. Developmental phenotypes of alx cells in the presence of LiCl or TMB-8. Wild-type (A, D, and G), alx (B, E, and H), and alx /F-DdAlix (C, F, and I) cells were washed and plated onto cellulose filters. (A – C) Normal MES buffer (pH 6.5), (D – F) MES buffer containing 5 mM LiCl, (G – I) MES buffer containing 200 AM TMB-8. Photographs were taken at 48 h after plating. Bar: 1 mm.
Fig. 5. Developmental phenotype of alx cells in the presence of low extracellular free Ca2 +. Wild-type (A, D, and G), alx (B, E, and H), and alx /FDdAlix (C, F, and I) cells were washed and plated onto cellulose filters in the presence of 1 mM (A – C) or 10 nM (D – F) or in the absence (G – I) of free extracellular Ca2 +. The concentration of free extracellular Ca2 + was adjusted by Ca2 +-EGTA in HEPES buffer (pH 7.8). Photographs were taken at 48 h after plating. Bar: 1 mm.
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when cells were stimulated with cAMP, which is a chemoattractant of Dictyostelium (Milne and Coukell, 1991; Milne and Devreotes, 1993). The differentiation-inducing factor DIF-1 has been reported to induce a slow sustained increase in the cytosolic Ca2 + level and stalk gene induction (Kubohara and Okamoto, 1994; Schaap et al., 1996; Azhar et al., 1997). Although our results suggest that there is a relationship between DdAlix and Ca2 + signaling, none of the Alix homologs, including DdAlix, have calcium-binding motifs in their amino acid sequences. The fact that mammalian Alix proteins interact with ALG-2 in a Ca2 +-dependent manner implies that there is a relationship between Alix and Ca2 + signaling (Missotten et al., 1999; Vito et al., 1999; Shibata et al., 2004). We previously identified two penta EFhand calcium-binding proteins in D. discoideum, named DdPEF-1 and DdPEF-2, which are putative homologs of ALG-2 (Ohkouchi et al., 2001; Maki et al., 2002). Like Alix and ALG-2, DdAlix may interact with DdPEF-1 and/or DdPEF-2 and regulate Ca2 + signaling through development. Aubry et al. reported that DdPEF-1 can interact with mouse Alix in vitro. However, the PRR of DdAlix does not contain PxY repeats that are essential for interaction with ALG-2 (Shibata et al., 2004). Indeed, interaction between DdAlix and the Dictyostelium ALG-2 homologs was not detected in our co-immunoprecipitation experiment (data not shown). The results suggest that DdAlix does not interact with them at all or interacts only weakly. Gene disruptions of DdPEF-1 and/or DdPEF-2 do not exhibit a developmental phenotype (Aubry et al., 2002). It would be interesting to see whether experiments carried out under Ca2 +-restricted conditions as in this study reveal any developmental phenotypes of DdPEF-1 and DdPEF-2 mutants. Bro1, one of the two yeast Alix homologs, associates with the endosomal membrane depending on Snf7/Vps32 (Odorizzi et al., 2003). Deletion of Bro1 results in the so-called class E vps phenotype: e.g., formation of a class E compartment and aberrant secretion of carboxypeptidase Y (Odorizzi et al., 2003). Bro1 also participates in the trafficking of the general amino acid permease Gap1 (Springael et al., 2002). Rim20, another Alix homolog, is required for Na+, Li+, and alkaline pH adaptation. Rim20 interacts with the transcription factor Rim101 and regulates the processing of Rim101 (Xu and Mitchell, 2001). Similarly, PalA, an Alix homolog of A. nidulans, regulates the processing of the transcription factor PacC, which is a Rim101 homolog (Arst and Pen˜alva, 2003). These two Alix homologs in S. cerevisiae are thought to be functionally independent, since a Rim20 deletion mutant did not show aberrant secretion of carboxypeptidase Y and a Bro1 deletion mutant showed normal processing of Rim101 (Odorizzi et al., 2003; Xu and Mitchell, 2001). In preliminary experiments, however, we did not observe a significant difference between incorporation of FITC-dextran in wild-type cells and that in alx cells, suggesting that endocytosis is normal in alx cells (not shown). Proteomic analyses showed that Alix exists in phagosomes in mammalian cells, suggesting that Alix plays roles in phagocytic
pathways (Garin et al., 2001). However, we did not observe a significant difference in uptake of Escherichia coli by phagocytosis in alx cells (not shown). The results of the present study imply that DdAlix is rather a functional homolog of Rim20 than of Bro1. In Dictyostelium, however, there remains a possibility that abnormal sorting of Ca2 +related protein explains the phenotype of alx cells. Furthermore, we could not find a Rim101/PacC homolog in the currently available Dictyostelium sequence database. Higher eukaryotes such as nematodes, fruit flies, and mammals also do not have a Rim101 homolog. It seems that Alix signaling in Dictyostelium is more similar to that in mammals than to that in yeast and fungi. Therefore, investigation of DdAlix signaling in Dictyostelium may shed new light on the function of Alix homologs in higher eukaryotes that may be closely related to Ca2 + signaling.
Acknowledgements We thank F. Wada for his valuable discussion. We also thank M. Maeda for reading the manuscript. This work was supported by a Grant-in-Aid for Scientific Research B (to M. M.) and a Grant-in-Aid for Young Scientists B (to H. S.).
References Arst Jr., H.N., Pen˜alva, M.A., 2003. pH regulation in Aspergillus and parallels with higher eukaryotic regulatory systems. Trends Genet. 19, 224 – 231. Aubry, L., Mattei, S., Blot, B., Sadoul, R., Satre, M., Klein, G., 2002. Biochemical characterization of two analogues of the apoptosis-linked gene 2 protein in Dictyostelium discoideum and interaction with a physiological partner in mammals, murine Alix. J. Biol. Chem. 277, 21947 – 21954. Azhar, M., Kennady, P.K., Pande, G., Nanjundiah, V., 1997. Stimulation by DIF causes an increase of intracellular Ca2 + in Dictyostelium discoideum. Exp. Cell Res. 230, 403 – 406. Europe-Finner, G.N., Newell, P.C., 1984. Inhibition of cyclic GMP formation and aggregation in Dictyostelium by the intracellular Ca2 + antagonist TMB-8. FEBS Lett. 171, 315 – 319. Europe-Finner, G.N., Tillinghast Jr., H.S., McRobbie, S.J., Newell, P.C., 1985. TMB-8 inhibits respiration and cyclic GMP formation in Dictyostelium discoideum. J. Cell. Sci. 79, 151 – 160. Garin, J., Diez, R., Kieffer, S., Dermine, J.F., Duclos, S., Gagnon, E., Sadoul, R., Rondeau, C., Desjardins, M., 2001. The phagosome proteome: insight into phagosome functions. J. Cell Biol. 152, 165 – 180. Katoh, K., Shibata, H., Suzuki, H., Nara, A., Ishidoh, K., Kominami, E., Yoshimori, T., Maki, M., 2003. The ALG-2-interacting protein Alix associates with CHMP4b, a human homologue of yeast Snf7 that is involved in multivesicular body sorting. J. Biol. Chem. 278, 39104 – 39113. Katoh, K., Shibata, H., Hatta, K., Maki, M., 2004. CHMP4b is a major binding partner of the ALG-2-interacting protein Alix among the three CHMP4 isoforms M. Arch. Biochem. Biophys. 421, 159 – 165. Kitaura, Y., Watanabe, M., Satoh, H., Kawai, T., Hitomi, K., Maki, M., 1999. Peflin, a novel member of the five-EF-hand-protein family, is similar to the apoptosis-linked gene 2 (ALG-2) protein but possesses nonapeptide repeats in the N-terminal hydrophobic region. Biochem. Biophys. Res. Commun. 263, 68 – 75.
S. Ohkouchi et al. / Gene 337 (2004) 131–139 Klein, P.S., Melton, D.A., 1996. A molecular mechanism for the effect of lithium on development. Proc. Natl. Acad. Sci. U. S. A. 93, 8455 – 8459. Kubohara, Y., Okamoto, K., 1994. Cytoplasmic Ca2 + and H+ concentrations determine cell fate in Dictyostelium discoideum. FASEB J. 8, 869 – 874. Levi, S., Polyakov, M., Egelhoff, T.T., 2000. Green fluorescent protein and epitope tag fusion vectors for Dictyostelium discoideum. Plasmid 3, 231 – 238. Maeda, Y., 1970. Influence of ionic conditions on cell differentiation and morphogenesis of the cellular slime molds. Dev. Growth Differ. 12, 217 – 227. Maki, M., Kitaura, Y., Satoh, H., Ohkouchi, S., Shibata, H., 2002. Structures, functions and molecular evolution of the penta-EF-hand Ca2 +binding proteins. Biochim. Biophys. Acta 1600, 51 – 60. Marsh, M., Thai, M., 2003. HIV’s great escape. Nat. Med. 9, 1262 – 1263. Milne, J.L., Coukell, M.B., 1991. A Ca2 + transport system associated with the plasma membrane of Dictyostelium discoideum is activated by different chemoattractant receptors. J. Cell Biol. 112, 103 – 110. Milne, J.L., Devreotes, P.N., 1993. The surface cyclic AMP receptors, cAR1, cAR2, and cAR3, promote Ca2 + influx in Dictyostelium discoideum by a G alpha 2-independent mechanism. Mol. Biol. Cell 4, 283 – 292. Missotten, M., Nichols, A., Rieger, K., Sadoul, R., 1999. Alix, a novel mouse protein undergoing calcium-dependent interaction with the apoptosis-linked-gene 2 (ALG-2) protein. Cell Death Differ. 6, 124 – 129. Odorizzi, G., Katzmann, D.J., Babst, M., Audhya, A., Emr, S.D., 2003. Bro1 is an endosome-associated protein that functions in the MVB pathway in Saccharomyces cerevisiae. J. Cell. Sci. 116, 1893 – 1903. Ohkouchi, S., Nishio, K., Maeda, M., Hitomi, K., Adachi, H., Maki, M., 2001. Identification and characterization of two penta-EF-hand Ca2 +-binding proteins in Dictyostelium discoideum. J. Biochem. 130, 207 – 215.
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Phiel, C.J., Klein, P.S., 2001. Molecular targets of lithium action. Annu. Rev. Pharmacol. Toxicol. 41, 789 – 813. Puta, F., Zeng, C., 1998. Blasticidin resistance cassette in symmetrical polylinkers for insertional inactivation of genes in Dictyostelium. Folia Biol. (Praha) 44, 185 – 188. Schaap, P., Nebl, T., Fisher, P.R., 1996. A slow sustained increase in cytosolic Ca2 + levels mediates stalk gene induction by differentiation inducing factor in Dictyostelium. EMBO J. 15, 5177 – 5183. Schmidt, M.H., Chen, B., Randazzo, L.M., Bo¨gler, O., 2003. SETA/ CIN85/Ruk and its binding partner AIP1 associate with diverse cytoskeletal elements, including FAKs, and modulate cell adhesion. J. Cell. Sci. 116, 2845 – 2855. Shibata, H., Yamada, K., Mizuno, T., Yorikawa, C., Takahashi, H., Satoh, Y., Kitaura, Y., Maki, M., 2004. The penta-EF-hand protein ALG-2 interacts with a region containing PxY repeats in Alix/AIP1, which is required for the subcellular punctuate distribution of the amino-terminal truncation form of Alix/AIP1. J. Biochem. 135, 117 – 128. Springael, J.Y., Nikko, E., Andre, B., Marini, A.M., 2002. Yeast Npi3/Bro1 is involved in ubiquitin-dependent control of permease trafficking. FEBS Lett. 517, 103 – 109. Vito, P., Lacana´, E., D’Adamio, L., 1996. Interfering with apoptosis: Ca2 +binding protein ALG-2 and Alzheimer’s disease gene ALG-3. Science 271, 521 – 525. Vito, P., Pellegrini, L., Guiet, C., D’Adamio, L., 1999. Cloning of AIP1, a novel protein that associates with the apoptosis-linked gene ALG-2 in a Ca2 +-dependent reaction. J. Biol. Chem. 274, 1533 – 1540. Wu, Y., Pan, S., Luo, W., Lin, S.H., Kuang, J., 2002. Hp95 promotes anoikis and inhibits tumorigenicity of HeLa cells. Oncogene 21, 6801 – 6808. Xu, W., Mitchell, A.P., 2001. Yeast PalA/AIP1/Alix homolog Rim20p associates with a PEST-like region and is required for its proteolytic cleavage. J. Bacteriol. 183, 6917 – 6923.
DdAlix, an Alix/AIP1 homolog in Dictyostelium discoideum, is required for multicellular development under low Ca2+ conditions Susumu Ohkouchi, Medhat S. El-Halawany, Fumika Aruga, Hideki Shibata, Kiyotaka Hitomi, Masatoshi Maki * Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8601, Japan Received 16 February 2004; accepted 22 April 2004 Received by T. Sekiya
Abstract Apoptosis-linked gene 2 (ALG-2) interacting protein X (Alix), also called AIP1, is a widely conserved protein in eukaryotes. Alix and its homologs are involved in various phenomena such as apoptosis, regulation of cell adhesion, protein sorting, adaptation to stress conditions, and budding of human immunodeficiency virus (HIV). To investigate the role of Alix in development, we identified an Alix homolog in the cellular slime mold Dictyostelium discoideum and disrupted the gene by homologous recombination. The growth of DdAlix deletion mutant (alx ) cells was significantly impaired in the presence of 5 mM Li+. On an agar plate, alx cells underwent normal development and formed fruiting bodies indistinguishable from those formed by wild-type cells. However, alx cells could not form fruiting bodies in the presence of 5 mM Li+. Similar results were obtained when cells were developed in the presence of 3,4,5-trimethoxybenzoic acid 8(diethylamino)octyl ester (TMB-8), which is an antagonist of intracellular Ca2 + store. Furthermore, when the extracellular free Ca2 + was reduced to 10 nM, the ability of alx cells, but not that of wild-type cells, to form fruiting bodies was impaired. The results indicate that DdAlix is essential for development under low Ca2 + conditions and suggest that DdAlix is involved in Ca2 + signaling during development. D 2004 Elsevier B.V. All rights reserved. Keywords: Dictyostelium; Ca2+; Li+; Homologous recombination
1. Introduction Mouse ALG-2 interacting protein X (Alix, also named AIP1) was identified as a binding partner for the penta EFhand Ca2 +-binding protein apoptosis-linked gene 2 (ALG-2) (Missotten et al., 1999; Vito et al., 1999). Alix is a widely conserved protein in eukaryotes. Alix homologs have a Bro1-rhophilin conserved domain (BRD; registered as BRO1 domain in the NCBI conserved domain database), a Abbreviations: ALG-2, apoptosis-linked gene 2; Alix, ALG-2 interacting protein X; BRD, Bro1-rhophilin conserved domain; CBB, Coomassie brilliant blue; CC, coiled-coil; EGTA, ethylene glycol bis(h-aminoethylether)-N,N,NV,NV-tetraacetic acid; HIV, human immunodeficiency virus; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; PRR, proline-rich region; SDS, sodium dodecyl sulfate; TMB-8, 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester. * Corresponding author. Tel.: +81-52-789-4088; fax: +81-52-7895542. E-mail address: [email protected] (M. Maki). 0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2004.04.020
potential recognition site for Src-type tyrosine kinases in higher eukaryotes, predicted coiled-coils (CC), and a proline-rich region (PRR). Recently, we showed that ALG-2 recognizes PxY repeats in the Alix PRR in a Ca2 +-dependent manner (Shibata et al., 2004). Alix has been shown to interact with focal adhesion kinase (FAK) and proline rich tyrosine kinase 2 (PYK-2) (Schmidt et al., 2003). Although the binding site has not been determined, interaction between Alix and PYK-2 increases in the presence of Ca2 +. The relationship between Alix and Ca2 + signaling is unclear. Alix is known to have various functions. Alix is thought to be involved in apoptosis, since ALG-2 was identified as a gene required for apoptosis (Vito et al., 1996). Overexpression of a truncated form of mouse Alix containing CC and PRR makes astrocytes resistant to apoptosis (Vito et al., 1999). On the other hand, overexpression of human Alix (also named Hp95) in HeLa cells results in promotion of detachment-induced apoptosis (anoikis), and inhibition of tumorigenicity (Wu et al., 2002). Accumulating evidence
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suggests that Alix plays a role in protein sorting. Alix interacts with CHMP4 proteins, which are human homologs of yeast Snf7/Vps32 (Katoh et al., 2003, 2004), and interaction between Alix and Gag late domain p6 is required for the budding of human immunodeficiency virus (HIV) (Marsh and Thai, 2003). Alix also interacts with TSG101, one of the components of endosomal sorting complex required for transport (ESCRT) complex (Marsh and Thai, 2003). Bro1 (also known as Vps31 or Npi3) is one of the Alix homologs in Saccharomyces cerevisiae and is involved in protein sorting. S. cerevisiae has another Alix homolog, named Rim20. A mutant in which Rim20 is deleted is sensitive to Na+, Li+, and alkaline pH. Similarly, PalA, an Alix homolog of Aspergillus nidulans, is involved in regulation of alkaline pH-induced gene expression (Arst and Pen˜alva, 2003). Although there are many reports on the functions of Alix in mammalian and fungal cells, there is no report on the role of Alix in development of multicellular organisms. The functions of Alix homologs in the nematode or fruit fly are not known. To elucidate the role of Alix in development, we investigated the function of the Alix homolog DdAlix in the cellular slime mold Dictyostelium discoideum, which is a useful haploid model organism for studying many basic issues in cell biology. Dictyostelium cells proliferate as unicellular amoeba cells in a rich nutrient. Upon starvation, cells aggregate using cAMP as a chemoattractant. These cells form a migrating slug that becomes a fruiting body consisting of a mass of spore cells supported by a thin column of stalk cells. Cytoplasmic Ca2 + and H+ concentrations determine the cell fate (Kubohara and Okamoto, 1994). A slow sustained increase in cytosolic Ca2 + level mediates stalk gene induction by a low-molecular-weight signaling molecule named differentiation-inducing factor DIF-1 (Schaap et al., 1996). In this study, we disrupted the DdAlix gene by homologous recombination and analyzed phenotypes of the strain. DdAlix was found to be not essential for multicellular development under normal laboratory conditions but essential in the presence of Li+, an intracellular Ca2 + store antagonist TMB-8, and under the condition of low extracellular Ca2 +. Our results suggest that DdAlix is involved in Ca2 + signaling during development.
2. Materials and methods 2.1. Cloning of DdAlix cDNA A homology search was performed using the tBLASTn program (http://www.ncbi.nlm.nih.gov/BLAST/) with a mouse Alix amino acid sequence as a query. Primers for DdAlix cDNA cloning were designed on the basis of the retrieved DNA sequence (GenBank accession no. AF360741). Total RNA was isolated from vegetative cells using a QuickPrepk Micro mRNA purification kit (Amer-
sham) according to the instructions of the manufacturer. Reverse transcription was performed using a 5V-full RACE core kit (Takara, Japan) according to the instructions of the manufacturer. Reaction mixtures were incubated at 30 jC for 10 min and at 42 jC for 30 min and then heated to 95 jC for 5 min. DdAlix cDNA was cloned by polymerase chain reaction (PCR) using pfuTurbo DNA polymerase (stratagene), a pair of primers (5V-GCGGATCCATGTTATCAATCGAGAGAAAAAG-3V, the attached BamHI site and two additional bases indicated by an underline, and 5VCGGCTCGAG CTTAATAATGTTTATTATTTGAATTA-3V, the XhoI site and three additional bases indicated by an underline), and the product of reverse transcription as a template. After digestion with BamHI/XhoI, the PCR product was ligated into pCMV-Tag2C (Stratagene) and the nucleotide sequence was determined with an automated fluorescent sequencer, ABI PRISM310 (PE Applied Biosystems), using a Big Dyek terminator cycle sequencing ready reaction kit (PE Applied Biosystems) and either universal or custom synthesized primers. The resultant plasmid was designated as pCMV-Tag2C-DdAlix. 2.2. Plasmid constructions A DdAlix cDNA fragment was excised with BamHI/ XhoI from pCMV-Tag2C-DdAlix and ligated into the BamHI/XhoI site of pTX-FLAG (Levi et al., 2000). The resultant plasmid was designated as pTX-FLAGDdAlix and used for complementation analyses. A DdAlix disruption vector was constructed as follows: genomic DNA from vegetative Dictyostelium cells was amplified by a PCR reaction with a pair of primers (5VCCCATGGACAGATTCATATAGAC-3V and 5V-GGATCCCTTGTTCTAACCCTTTTGCGTG-3V, the attached BamHI site shown by an underline), followed by ligation into pCR2.1-TOPO (Invitrogen). An EcoRI/BamHI fragment was ligated into the EcoRI/BamHI site of pUC118, and the resultant plasmid was designated as pDdAlixKO5V. Similarly, the 3V-flanking region was amplified with a pair of primers (5V-GGATCCGTAAACTTAGCCAAAACAGCACC-3V, the attached BamHI site shown by an underline and 5V-CTCTATTGGCCATCAAAGCATCC-3V) and ligated into pCR2.1-TOPO. A BamHI/PstI fragment was ligated into the BamHI/PstI site of pDdAlixKO5V, and a BamHI fragment of the blasticidin-resistant cassette from modified pBsR503 (Puta and Zeng, 1998) was inserted into the BamHI site. The resultant plasmid, designated as pDdAlixKO, was used as a DdAlix gene disruption vector. 2.3. Anti-DdAlix antibody and Western blotting Anti-DdAlix antisera were obtained from a rabbit immunized with a DdAlix peptide (TQDYAKYTSHLQHST KSDC, corresponding to amino acids 481– 498, the carboxy-terminal cysteine residue being added for cross-linking
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reaction; indicated in Fig. 1A) coupled with keyhole limpet hemocyanin using m-maleimidobenzoyl-N-hydroxysuccinimide ester. The antisera were affinity-purified using Activated Thiol Sepharosek 4B (Amersham) coupled with antigen peptide according to the manufacturer’s instructions. For Western blotting to confirm gene disruption, cells were
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collected, washed twice with PBS, and lysed in SDS-sample buffer followed by immediate boiling. After samples has been subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore), DdAlix protein was detected with an anti-DdAlix polyclonal antibody by the color development method using diaminobenzidine (DAB) as described previously (Kitaura et al., 1999). 2.4. Cell culture D. discoideum wild-type strain AX-2 and DdAlix deletion mutant (alx ) cells were cultured in HL5 medium at 22 jC. The alx cells expressing exogenous FLAG-tagged DdAlix (alx /F-DdAlix) were maintained in HL5 medium in the presence of 10 Ag/ml G418. The number of cells was calculated using a hematocytometer. 2.5. Transformation of Dictyostelium
Fig. 1. Structure and expression of DdAlix. (A) Schematic representation of DdAlix and human Alix. Bro1-rhophilin conserved domain (BRD: registered as BRO1 domain in the NCBI conserved domain database; accession number 17278; pfam03097), predicted coiled-coil region (CC) and proline-rich region (PRR) are indicated by gray, hatched and closed boxes, respectively. ‘‘Y’’ indicates the tyrosine residue in a highly conserved potential recognition site for Src type tyrosine kinases in higher eukaryotes. Degrees of similarities in BRD, PRR, middle region between the BRD domain and PRR, and full length between DdAlix and human Alix are shown as amino acid identities (%). Positions of introns in the genomic DNA of DdAlix and human Alix are indicated by arrows. The peptide used for preparation of the antibody is indicated by a bar. The breakpoint caused by homologous recombination is indicated by an open arrowhead (see Fig. 2). (B) Expression of DdAlix protein during development. Wild-type cells were developed on cellulose filters. Cells were collected at 0, 4, 8, 12, 16, 20, and 24 h after starvation. Lysates were prepared as described in Section 2.6 and subjected to 7.5% SDS-PAGE and Coomassie brilliant blue (CBB) staining (upper panel). Proteins were transferred to a PVDF membrane, and DdAlix protein was detected by Western blotting using an anti-DdAlix polyclonal antibody (lower panel). The arrow indicates endogenous DdAlix protein, and the asterisk indicates nonspecifically reacted protein.
For disruption of DdAlix, pDdAlixKO was linearized by digestion with EcoRI followed by phenol/chloroform extraction and ethanol precipitation. Vegetative Dictyostelium cells in mid-log phase were collected, washed twice with ice-cold electroporation buffer (10 mM NaHPO4, 50 mM sucrose, pH 6.1), and resuspended in 400 Al of electroporation buffer. After mixing with 20 Ag of digested pDdAlixKO and being transferred into a 2-mm gap cuvette, the suspension was electroporated using a ECM600 (BTX) under the following conditions: charging voltage, 500 V; resistance, 13 V; capacitance, 100 AF. Transformants were selected in HL5 medium containing 4 Ag/ml blasticidin S hydrochloride (Kaken Pharmaceutical). Apparent colonies were selected, and disruption of the DdAlix gene was checked by PCR. After limiting dilution of the isolated colonies, disruption of the DdAlix gene was checked again by PCR using a pair of primers (5V-AACTTTAAATGGATTAAGAGAAGATG-3V and 5V-GTGAATGTGGATTAACACCAGC-3V). alx cells was transformed with pTXFLAGDdAlix as described above and selected in a medium containing 10 Ag/ml G418. Colonies were picked up and further selected for a colony expressing DdAlix protein. One of the transformants, designated alx /F-DdAlix, was selected and used for complementation analyses. 2.6. Developmental analysis For development on a cellulose filter, cells were harvested, washed twice with MES buffer (20 mM MES (2-(Nmorpholino)ethanesulfonic acid)-KOH, pH 6.5), and resuspended at a density of 2 107/ml in MES buffer containing various reagents as indicated. A black filter composed of mixed cellulose ester (47 mm, Cat. No. A045N047A, ADVANTEC, Japan) and two filter papers for support (No. 3, Whatman) were washed twice with MES buffer containing the same reagents as those in the cell suspension.
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Cell suspensions were plated at a density of 1 107/filter and incubated for 2 days at 22 jC. When experiments were performed in the presence of LiCl or 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), cellulose filters and filter papers were washed in the presence of 5 mM LiCl or 200 AM TMB-8. LiCl or TMB-8 was added to the cell suspension after washing twice with MES buffer. Development using Ca2 +-ethylene glycol bis(h-aminoethylether)-N,N,NV,NV-tetraacetic acid (EGTA) buffer was performed similarly, but cells, cellulose filters, and filter papers were washed four times with HEPES buffer (20 mM HEPES (2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid)NaOH, pH 7.8) containing 1 mM EGTA and various concentrations of total Ca2 +ions to adjust free Ca2 + to 0, 10 nM or 1 mM. In this experiment, free Ca2 + concentration was calculated using WinMaxc v2.0 software (http://www. stanford.edu/~cpatton/maxc.html). For development on agar, cells were washed twice with KK2 buffer (17 mM KHPO4, pH 6.5) and plated onto 1.5% KK2-buffered agar. Development was observed under a stereoscopic microscope (OLYMPUS SZX12). Photographs were taken 48 h after starvation. Wild-type cells were plated onto cellulose filters and collected at 0, 4, 8, 12, 16, 20 and 24 h after starvation. Cells were resuspended in lysis buffer (10 mM Tris (tris(hydroxymethyl)aminomethane) –HCl, pH 8.0, 10 AM pepstatin, 125 Ag/ml leupeptin, 5 mM pefabloc, 5 AM E-64, and 1 mM phenylmethylsulfonyl fluoride) and then added with SDS-sample buffer followed by immediate boiling for 5 min. Acid-washed glass beads (0.1 g; Sigma) were added to the samples collected at 20 and 24 h after starvation and vortexed for 30 s followed by 30 s of boiling. This procedure was repeated three times. Samples were subjected to SDSPAGE followed by Coomassie brilliant blue (CBB) staining or Western blotting using an anti-DdAlix antibody.
3. Results 3.1. Primary structure of DdAlix and its expression A homology search of the database revealed that DdAlix, an Alix homolog in Dictyostelium, shows significant homology with human Alix (29.6% identity; Fig. 1A). The DdAlix gene has four exons and three introns, encoding a protein of 794 amino acids with a calculated molecular mass of 90.5 kDa (Fig. 1A). Like other Alix homologs, DdAlix has a potential recognition sequence (Y315) for Src-type tyrosine kinases, predicted coiled-coil regions, and a relatively proline-rich region in the amino acid sequence. The protein expression level of DdAlix during development was analyzed by Western blotting using an anti-DdAlix antibody (Fig. 1B). The DdAlix protein migrated slightly faster than expected and was detected as an immunoreactive band of about 80 kDa by SDS-PAGE. The DdAlix protein was expressed throughout the process of development. In Fig 1B, there is a nonspecifically reacted protein band around 43 kDa shown
by asterisk. This protein band is not a degraded product of DdAlix because the same band was also detected in case of the cell lysate of a DdAlix deletion mutant (data not shown). 3.2. DdAlix gene disruption by homologous recombination To establish a DdAlix deletion mutant, Ax-2 cells were transformed with the DdAlix gene disruption vector pDdAlixKO (Fig. 2A). Blasticidin-resistant colonies were selected, and disruption of the DdAlix gene was screened by PCR. After limiting dilution of a candidate colony, deletion of the DdAlix gene was confirmed by PCR and Western blotting. The PCR product of wild-type cells gave a band of 2.3 kbp, which was absent in the PCR product of the mutant clone. The PCR product of the mutant clone gave a band of 3.8 kbp (Fig. 2B). The difference of 1.5 kbp between the PCR products of the wild-type cells and the clone is in accordance with the size of the bsr cassette in the DdAlix gene. Disruption of the DdAlix gene was further confirmed by Western blotting using an anti-DdAlix antibody in terms of protein expression level (Fig. 2C, lane 2). A band of about 80 kDa was not detected in the isolated clone (the clone is referred to as alx ). Deletion of DdAlix was complemented by expression of FLAG-tagged DdAlix exogenously (the cells were referred to as alx /F-DdAlix). The expression was confirmed by Western blotting using an anti-DdAlix antibody and an anti-FLAG antibody (Fig. 2C, lane 3). 3.3. Growth of alx cells The growth rate of alx cells was not significantly different from that of wild-type and alx /F-DdAlix cells (Fig. 3A). Some monovalent metal ions that are known to affect the growth of Rim20-deficient yeast cells (Xu and Mitchell, 2001) were tested, and it was found that the presence of 5 mM LiCl resulted in significant reduction in the growth rate, especially in the case of alx cells, compared with the growth rates of wild-type and alx /FDdAlix cells. The number of alx cells increased by only threefold after 8 days of cultivation in the presence of LiCl. This reduction in growth rate was significantly recovered in the case of alx /F-DdAlix cells (Fig. 3B). The same concentration of NaCl or KCl did not affect the growth rate (data not shown), indicating that the effect of LiCl was caused by Li+, not by Cl . 3.4. Effects of Li+ and TMB-8 on development of alx cells To investigate the role of DdAlix in development, cells were starved to induce multicellular development. alx cells, as well as wild-type and alx /F-DdAlix cells, formed normal fruiting bodies both on agar plates (data not shown) and on cellulose ester filters (Fig. 4A –C). We next examined the effect of Li+ on development. In the presence of 5 mM LiCl, wild-type and alx /F-DdAlix cells formed normal fruiting bodies after 48 h, although development of
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Fig. 2. Disruption of the DdAlix gene. (A) Strategy for DdAlix gene disruption. Four exons of the DdAlix gene and the blasticidin resistance cassette (bsr) are shown by open and closed boxes, respectively. The bsr cassette was inserted into the third exon of the gene. PCR primers used for detection of homologous recombination are shown by arrows. (B) Confirmation of gene disruption by PCR. Genomic DNA was extracted and PCR was performed as described in Section 2.5. M, DNA marker; lane 1, wild type; lane 2, alx . (C) Western blotting analysis of DdAlix protein. Lysates from wild-type (lane 1), alx (lane 2), and alx /F-DdAlix (lane 3) cells were subjected to 7.5% SDS-PAGE and transferred to a PVDF membrane. DdAlix protein was detected by an anti-DdAlix polyclonal antibody (upper panel) or an anti-FLAG monoclonal antibody (lower panel).
alx /F-DdAlix was slightly delayed. In contrast, alx cells could not form fruiting bodies but formed aggregates or a fruiting body-like structure at the apex of aggregates (Fig. 4D – F). Li+ has been suggested to inhibit inositol monophosphatase, which reduces inositol 1,4,5-trisphosphate level and is thought to inhibit Ca2 + release from intracellular stores (Phiel and Klein, 2001). Therefore, we tested whether TMB-8, a Ca2 + antagonist for the intracellular store that has been shown to be effective for Dictyostelium (Europe-Finner and Newell, 1984; Europe-Finner et al., 1985), has effects on development. As in the presence of LiCl, alx cells could not form fruiting bodies in the presence of 200 AM of TMB-8 (Fig. 4G – I). In this experiment, alx /F-DdAlix formed fruiting bodies but in a slightly delayed time course compared with that in the case of wild-type cells. 3.5. Effect of extracellular Ca2+ on development of alx cells To show more clearly the relationship of the effects of Ca2 + and DdAlix deficiency during development, alx cells were developed in the presence of different extracellular Ca2 + concentrations. Free Ca2 + was adjusted to 0, 10
nM or 1 mM by using Ca2 +-EGTA buffer (Fig. 5). When cells were developed in the presence of 1 mM free Ca2 +, all cells formed fruiting bodies (Fig. 5A –C). In the absence of free Ca2 +, none of the cells formed fruiting bodies. They formed only loose aggregates (Fig. 5G – I). When cells were developed in the presence of 10 nM free Ca2 +, wild-type and alx /F-DdAlix cells formed fruiting bodies (Fig. 5D,F). On the other hand, alx cells did not form normal fruiting bodies, but formed only aggregates or small aberrant fruiting body-like structures (Fig. 5E). Again, development of alx /F-DdAlix was slightly delayed.
4. Discussion Alix has been isolated as a binding protein for ALG-2 (Missotten et al., 1999; Vito et al., 1999). Recently, many studies have shown that Alix plays roles in various phenomena, including membrane traffic (Springael et al., 2002; Katoh et al., 2003, 2004; Odorizzi et al., 2003), apoptosis (Vito et al., 1999; Wu et al., 2002), and budding of HIV (Marsh and Thai, 2003). This is the first report showing a role of Alix in development using a model eukaryote, D. discoideum. We disrupted the DdAlix gene by a homologous
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Fig. 3. Growth curve of wild-type, alx and alx /F-DdAlix cells. Cells were inoculated into HL5 medium in the absence (A) or presence (B) of 5 mM LiCl at a density of 5 104/ml, and cell numbers were counted for 6 (A) or 8 (B) days, respectively. Data for wild-type, alx and alx /FDdAlix cells are shown by circles, squares and triangles, respectively. Averages of results of duplicate experiments are shown.
recombination method (Fig. 2), and we analyzed the phenotype of the strain (alx ). alx cells could hardly grow in the presence of LiCl (Fig. 3B). The growth rate of alx /FDdAlix cells were significantly recovered, indicating that the slower growth rate of alx cells is due to a deficiency of DdAlix, not due to any unexpected mutations during the homologous recombination. However, we cannot exclude the possibility that the observed phenotype of alx is due to a potential dominant-negative effect of truncated DdAlix created by the recombination. This phenotype may be caused by perturbation of calcium signaling (see below). Upon starvation, alx cells formed fruiting bodies indistinguishable from those of wild-type cells on an agar plate (data not shown) and on filters (Fig. 4A –C), suggesting that DdAlix is not essential for multicellular development under standard experimental condition. In the presence of 5 mM LiCl, however, alx cells could not form normal fruiting bodies but formed aggregates or small fruiting body-like structures at the apex of aggregates (Fig. 4D – F). This phenotype was complemented by exogenous expression of DdAlix (Fig. 4F), indicating that strong sensitivity to Li+ is caused by disruption of the DdAlix gene. This effect is specific to Li+ because 5 mM NaCl or KCl did not
cause a developmental defect in alx cells (data not shown). Li+ has been used as an effective treatment for bipolar disorder (See Phiel and Klein, 2001 for review.). Li+ also has effects on the development of various organisms: it causes dorsalization of Xenopus and zebrafish. In Dictyostelium, Li+ prevents spore differentiation and promotes stalk differentiation (Maeda, 1970). These effects of Li+ on development are caused by inhibition of GSK-3 (Klein and Melton, 1996). We observed that wild-type cells could not form fruiting bodies in the presence of 20 mM LiCl (data not shown). In the presence of 5 mM LiCl, wild-type cells formed fruiting bodies, but alx cells did not (Fig. 4D – F). This result indicates that alx cells are more sensitive to Li+ than wild-type cells. It remains unknown whether the stronger sensitivity of alx to Li+ is related to GSK-3 or not. Li+, an inhibitor of inositol monophosphatase, has been suggested to reduce inositol 1,4,5-trisphosphate level and to inhibit Ca2 + release from intracellular stores (Phiel and Klein, 2001). We observed that alx cells treated with TMB-8, a Ca2 + antagonist for intracellular stores, could not form fruiting bodies, suggesting that the effect of Li+ on alx cells is related to Ca2 + (Fig. 4H). The speculation of a close relationship between alx cells and Ca2 + signaling was supported by the results of experiments in which extracellular Ca2 + was restricted by using a Ca2 +EGTA buffer (Fig. 5). In these experiments, a higher pH condition (pH 7.8) was used to maintain the chelating capacity of EGTA, which is significantly reduced at the usually used pH value (pH 6.5) for Dictyostelium development (Fig. 4). At pH 7.8, all cells, including alx cells, formed fruiting bodies in the absence of EGTA (data not shown) and in the presence of EGTA plus extra Ca2 + (Fig. 5A –C). However, when the extracellular free Ca2 + concentration was adjusted to 10 nM, alx cells did not form fruiting bodies, while the wild-type cells and alx /FDdAlix cells did (Fig. 5D – F). These results suggest that alx cells are sensitive to depletion of Ca2 + and that DdAlix is related to Ca2 + signaling. However, alx cells could form fruiting bodies when developed on agar (data not shown) or on filters under standard developmental conditions (Fig. 4B). This is probably because agar and supporting filter papers contain a sufficient amount of Ca2 + for alx cells to develop. Although alx /F-DdAlix cells formed fruiting bodies in the presence of Li+, TMB8 or 10 nM Ca2 +, development was slightly delayed. Similarly, exogenous expression of F-DdAlix in alx cells complemented the reduction of growth rate in the presence of Li+, but alx /F-DdAlix cells did not grow at the same rate as that of wild-type cells (Fig. 3B). alx /F-DdAlix cells express a large amount of DdAlix protein compared with endogenous one (Fig. 2C). An appropriate level of expression of DdAlix may be needed for adaptation to stress conditions. Ca2 + is known to play important roles in Dictyostelium development. Intracellular Ca2 + concentration increased
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Fig. 4. Developmental phenotypes of alx cells in the presence of LiCl or TMB-8. Wild-type (A, D, and G), alx (B, E, and H), and alx /F-DdAlix (C, F, and I) cells were washed and plated onto cellulose filters. (A – C) Normal MES buffer (pH 6.5), (D – F) MES buffer containing 5 mM LiCl, (G – I) MES buffer containing 200 AM TMB-8. Photographs were taken at 48 h after plating. Bar: 1 mm.
Fig. 5. Developmental phenotype of alx cells in the presence of low extracellular free Ca2 +. Wild-type (A, D, and G), alx (B, E, and H), and alx /FDdAlix (C, F, and I) cells were washed and plated onto cellulose filters in the presence of 1 mM (A – C) or 10 nM (D – F) or in the absence (G – I) of free extracellular Ca2 +. The concentration of free extracellular Ca2 + was adjusted by Ca2 +-EGTA in HEPES buffer (pH 7.8). Photographs were taken at 48 h after plating. Bar: 1 mm.
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when cells were stimulated with cAMP, which is a chemoattractant of Dictyostelium (Milne and Coukell, 1991; Milne and Devreotes, 1993). The differentiation-inducing factor DIF-1 has been reported to induce a slow sustained increase in the cytosolic Ca2 + level and stalk gene induction (Kubohara and Okamoto, 1994; Schaap et al., 1996; Azhar et al., 1997). Although our results suggest that there is a relationship between DdAlix and Ca2 + signaling, none of the Alix homologs, including DdAlix, have calcium-binding motifs in their amino acid sequences. The fact that mammalian Alix proteins interact with ALG-2 in a Ca2 +-dependent manner implies that there is a relationship between Alix and Ca2 + signaling (Missotten et al., 1999; Vito et al., 1999; Shibata et al., 2004). We previously identified two penta EFhand calcium-binding proteins in D. discoideum, named DdPEF-1 and DdPEF-2, which are putative homologs of ALG-2 (Ohkouchi et al., 2001; Maki et al., 2002). Like Alix and ALG-2, DdAlix may interact with DdPEF-1 and/or DdPEF-2 and regulate Ca2 + signaling through development. Aubry et al. reported that DdPEF-1 can interact with mouse Alix in vitro. However, the PRR of DdAlix does not contain PxY repeats that are essential for interaction with ALG-2 (Shibata et al., 2004). Indeed, interaction between DdAlix and the Dictyostelium ALG-2 homologs was not detected in our co-immunoprecipitation experiment (data not shown). The results suggest that DdAlix does not interact with them at all or interacts only weakly. Gene disruptions of DdPEF-1 and/or DdPEF-2 do not exhibit a developmental phenotype (Aubry et al., 2002). It would be interesting to see whether experiments carried out under Ca2 +-restricted conditions as in this study reveal any developmental phenotypes of DdPEF-1 and DdPEF-2 mutants. Bro1, one of the two yeast Alix homologs, associates with the endosomal membrane depending on Snf7/Vps32 (Odorizzi et al., 2003). Deletion of Bro1 results in the so-called class E vps phenotype: e.g., formation of a class E compartment and aberrant secretion of carboxypeptidase Y (Odorizzi et al., 2003). Bro1 also participates in the trafficking of the general amino acid permease Gap1 (Springael et al., 2002). Rim20, another Alix homolog, is required for Na+, Li+, and alkaline pH adaptation. Rim20 interacts with the transcription factor Rim101 and regulates the processing of Rim101 (Xu and Mitchell, 2001). Similarly, PalA, an Alix homolog of A. nidulans, regulates the processing of the transcription factor PacC, which is a Rim101 homolog (Arst and Pen˜alva, 2003). These two Alix homologs in S. cerevisiae are thought to be functionally independent, since a Rim20 deletion mutant did not show aberrant secretion of carboxypeptidase Y and a Bro1 deletion mutant showed normal processing of Rim101 (Odorizzi et al., 2003; Xu and Mitchell, 2001). In preliminary experiments, however, we did not observe a significant difference between incorporation of FITC-dextran in wild-type cells and that in alx cells, suggesting that endocytosis is normal in alx cells (not shown). Proteomic analyses showed that Alix exists in phagosomes in mammalian cells, suggesting that Alix plays roles in phagocytic
pathways (Garin et al., 2001). However, we did not observe a significant difference in uptake of Escherichia coli by phagocytosis in alx cells (not shown). The results of the present study imply that DdAlix is rather a functional homolog of Rim20 than of Bro1. In Dictyostelium, however, there remains a possibility that abnormal sorting of Ca2 +related protein explains the phenotype of alx cells. Furthermore, we could not find a Rim101/PacC homolog in the currently available Dictyostelium sequence database. Higher eukaryotes such as nematodes, fruit flies, and mammals also do not have a Rim101 homolog. It seems that Alix signaling in Dictyostelium is more similar to that in mammals than to that in yeast and fungi. Therefore, investigation of DdAlix signaling in Dictyostelium may shed new light on the function of Alix homologs in higher eukaryotes that may be closely related to Ca2 + signaling.
Acknowledgements We thank F. Wada for his valuable discussion. We also thank M. Maeda for reading the manuscript. This work was supported by a Grant-in-Aid for Scientific Research B (to M. M.) and a Grant-in-Aid for Young Scientists B (to H. S.).
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