Abstract
Inositol phosphate-containing molecules play an important role in a broad range of cellular processes. Inositol 5-phosphatases participate in the regulation of these signaling molecules. We have identified four inositol 5-phosphatases in Dictyostelium discoideum, Dd5P1-4, showing a high diversity in domain composition. Dd5P1 possesses only a inositol 5-phosphatase catalytic domain. An unique domain composition is present in Dd5P2 containing a RCC1-like domain. RCC1 has a seven-bladed propeller structure and interacts with G-proteins. Dd5P3 and Dd5P4 have a domain composition similar to human Synaptojanin with a SacI domain and OCRL with a RhoGAP domain, respectively. We have expressed the catalytic domains and show that these inositol 5-phosphatases have different substrate preferences. Single and double gene inactivation suggest a functional redundancy for Dd5P1, Dd5P2, and Dd5P3. Inactivation of the gene coding for Dd5P4 leads to defects in growth and development. These defects are restored by the expression of the complete protein but not by the 5-phosphatase catalytic domain.
Highlights
EXPERIMENTAL PROCEDURESIdentification and Sequence Analysis—The first putative inositol 5-phosphatase sequence was obtained using degenerated primers complementary to the conserved motifs I and II present in inositol 5-phosphatases (see “Results”), and the PCR product was used to screen a cDNA library kindly provided by Dr R
Inositol phosphate-containing molecules play an important role in a broad range of cellular processes
We have identified four inositol 5-phosphatases in Dictyostelium discoideum, Dd5P1– 4, showing a high diversity in domain composition
Summary
Identification and Sequence Analysis—The first putative inositol 5-phosphatase sequence was obtained using degenerated primers complementary to the conserved motifs I and II present in inositol 5-phosphatases (see “Results”), and the PCR product was used to screen a cDNA library kindly provided by Dr R. A comparison of the sequence obtained from cDNA clones with the genomic data base sequences revealed the presence of one intron in each inositol 5-phosphatase gene (Dd5P1: nucleotides 397– 499; Dd5P2: nucleotides 53– 187; Dd5P3: nucleotides 241–384; and Dd5P4: nucleotides 1357–1622) (GenBankTM accession numbers AY184992, AY184993, AY184994, and AY184995, respectively). Western Blot Analysis—The purified inositol 5-phosphatase catalytic domains were analyzed by 10% SDS-PAGE followed by immunostaining with Penta-His Antibody (Qiagen) and Anti-mouse IgG peroxidase conjugate (Sigma). Expression of Proteins in D. discoideum—To express proteins in D. discoideum, the desired DNA fragment was cloned into an extrachromosomal vector containing a neomycin cassette (pMB74 or pAH2). To express the full-length Dd5P4 in Dd5P4Ϫ cells, the obtained full-length cDNA clone was inserted into the BglII site of pMB74. After 30 min, the reaction was stopped and lipids were extracted
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