A new microsporidium Percutemincola moriokae gen. nov., sp. nov. from Oscheius tipulae: A novel model of microsporidia-nematode associations.

Here, we describe a new microsporidium Percutemincola moriokae gen. nov., sp. nov., which was discovered in the intestinal and hypodermal cells of a wild strain of the nematode Oscheius tipulae that inhabits in the soil of Morioka, Iwate Prefecture, Japan. The spores of Pe. moriokae had an average size of 1.0 × 3.8 µm and 1.3 × 3.2 µm in the intestine and hypodermis, respectively, and electron microscopy revealed that they exhibited distinguishing features with morphological diversity in the hypodermis. Isolated spores were able to infect a reference strain of O. tipulae (CEW1) through horizontal transmission but not the nematode Caenorhabditis elegans. Upon infection, the spores were first observed in the hypodermis and then in the intestine the following day, suggesting a unique infectious route among nematode-infective microsporidia. Molecular phylogenetic analysis grouped this new species with the recently identified nematode-infective parasites Enteropsectra and Pancytospora forming a monophyletic sister clade to Orthosomella in clade IV, which also includes human pathogens such as Enterocytozoon and Vittaforma. We believe that this newly discovered species and its host could have application as a new model in microsporidia-nematode association studies.

Molecular dissection of Caenorhabditis elegans ATP-binding cassette transporter protein HAF-4 to investigate its subcellular localization and dimerization.

Caenorhabditis elegans HAF-4 and HAF-9 are half-type ATP-binding cassette (ABC) transporter proteins, which are highly homologous to the human peptide transporter protein, transporter associated with antigen processing-like (TAPL, ABCB9). TAPL forms homodimers and localizes to lysosomes, whereas HAF-4 and HAF-9 form heterodimers and localize to intestine-specific non-acidified organelles. Both TAPL and HAF-4/HAF-9 are predicted to have four amino-terminal transmembrane helices [transmembrane domain 0 (TMD0)] additional to the six transmembrane helices that form the canonical core domain of ABC transporters with a cytosolic ABC region. TAPL requires its amino-terminal domain for localization to lysosomes; however, molecular mechanisms underlying HAF-4 and HAF-9 localization to their target organelles had not been elucidated. Here, we demonstrate that the mechanisms underlying HAF-4 localization differ from those underlying TAPL localization. Using transgenic C. elegans expressing mutant HAF-4 proteins labeled with green fluorescent protein, we reveal that the TMD0 of HAF-4 was not sufficient for proper localization of the protein. The mutant HAF-4, which lacked TMD0, localized to intracellular organelles similarly to the wild-type protein and functioned normally in the biogenesis of its localizing organelles, indicating that the TMD0 of HAF-4 is dispensable for both its localization and function.

Characterization of HAF-4- and HAF-9-localizing organelles as distinct organelles in Caenorhabditis elegans intestinal cells.

BACKGROUND: The intestinal cells of Caenorhabditis elegans are filled with heterogeneous granular organelles that are associated with specific organ functions. The best studied of these organelles are lipid droplets and acidified gut granules associated with GLO-1, a homolog of the small GTPase Rab38. In this study, we characterized a subset of the intestinal granules in which HAF-4 and HAF-9 localize on the membrane. HAF-4 and HAF-9 are ATP-binding cassette (ABC) transporter proteins that are homologous to the mammalian lysosomal peptide transporter TAPL (transporter associated with antigen processing-like, ABCB9). RESULTS: Using transgenic worms expressing fluorescent protein-tagged marker proteins, we demonstrated that the HAF-4- and HAF-9-localizing organelles are not lipid droplets and do not participate in yolk protein transport. They were also ruled out as GLO-1-positive acidified gut granules. Furthermore, we clarified that the late endosomal protein RAB-7 localizes to the HAF-4- and HAF-9-localizing organelles and is required for their biogenesis. CONCLUSIONS: Our results indicate that the HAF-4- and HAF-9-localizing organelles are distinct intestinal organelles associated with the endocytic pathway.

Amino-terminal extension of 146 residues of L-type GATA-6 is required for transcriptional activation but not for self-association.

Transcription factor GATA-6 plays essential roles in developmental processes and tissue specific functions through regulation of gene expression. GATA-6 mRNA utilizes two Met-codons in frame as translational initiation codons. Deletion of the nucleotide sequence encoding the PEST sequence (Glu(31)-Cys(46)) between the two initiation codons unusually reduced the protein molecular size on SDS-polyacrylamide gel-electrophoresis, and re-introduction of this sequence reversed this change. The long-type (L-type) GATA-6 containing this PEST sequence self-associated similarly to the short-type (S-type) GATA-6, as determined on co-immunoprecipitation of Myc-tagged GATA-6 with HA-tagged GATA-6. The L-type and S-type GATA-6 also interacted mutually. The L-type GATA-6 without the PEST sequence also self-associated and interacted with the S-type GATA-6. The transcriptional activation potential of L-type GATA-6 is higher than that of S-type GATA-6. When the PEST sequence (Glu(31)-Cys(46)) was inserted into the L-type GATA-6 without Arg(13)-Gly(101), the resultant recombinant protein showed significantly higher transcriptional activity, while the construct with an unrelated sequence exhibited lower activity. These results suggest that the Glu(31)-Cys(46) segment plays an important role in the transcriptional activation, although it does not participate in the self-association.

Co-operative function and mutual stabilization of the half ATP-binding cassette transporters HAF-4 and HAF-9 in Caenorhabditis elegans.

Caenorhabditis elegans HAF-4 and HAF-9 are half ABC (ATP-binding-cassette) transporters that are highly homologous to the human lysosomal peptide transporter TAPL [TAP (transporter associated with antigen processing)-like; ABCB9]. We reported previously that both HAF-4 and HAF-9 localize to the membrane of a subset of intestinal organelles, and are required for the formation of these organelles and other physiological aspects. In the present paper, we report the genetic and physical interactions between HAF-4 and HAF-9. Overexpression of HAF-4 and HAF-9 did not rescue the intestinal organelle defect of the haf-9 and haf-4 deletion mutants respectively, indicating that they cannot substitute for each other. Double haf-4 and haf-9 mutants do not exhibit more severe phenotypes than the single mutants, suggesting their co-operative function. Immunoprecipitation experiments demonstrated their physical interaction. The results of the present study suggest that HAF-4 and HAF-9 form a heterodimer. Furthermore, Western blot analysis of the deletion mutants and RNAi (RNA interference) knockdown experiments in GFP (green fluorescent protein)-tagged HAF-4 or HAF-9 transgenic worms suggest that HAF-4-HAF-9 heterodimer formation is required for their stabilization. The findings provide a clue as to how ABC transporters adopt a stable functional form.

Transporter associated with antigen processing-like (ABCB9) stably expressed in Chinese hamster ovary-K1 cells is sorted to the microdomains of lysosomal membranes.

The carboxyl terminus of a human ATP-binding cassette (ABC) transporter, transporter associated with antigen processing (TAP)-like (TAPL), was tagged with green fluorescence protein (GFP), and the resulting fusion protein (TAPL-GFP) was stably expressed in Chinese hamster ovary (CHO)-K1 cells. The GFP signal was co-localized with that of LysoTracker but not that of MitoTracker, as visualized under a microscope. TAPL-GFP was co-sedimented with lysosomal marker cathepsin D on Percoll density gradient centrifugation. These results indicated that TAPL is a lysosomal ABC transporter but not a mitochondrial one. It was not solubilized completely with a non-ionic detergent under ice-cold conditions, and was co-sedimented with flotillin-1 on sucrose density gradient centrifugation. A similar result was obtained with high pH-treatment. Furthermore, treatment with methyl-ß-cyclodextrin resulted in an altered distribution of TAPL-GFP. These results suggest that TAPL may be localized to the microdomains (lipid rafts) of lysosomal membranes enriched in cholesterol.

Tissue and developmental expression of SRAM, an unconventional Rel-family protein.

Previously we have reported the purification and cDNA cloning of a novel Rel/Ankyrin-family protein named SRAM from the flesh fly, Sarcophaga peregrina. Rel proteins generally translocate into the nucleus upon immune stimuli by dissociating from an inhibitory ankyrin domain, while SRAM is unique in terms of its constitutive nuclear localization with its internal ankyrin domain accompanied, at least in a Sarcophaga cell line and fat body cells. Although SRAM had been originally identified as a sole factor that binds to the κB motif of the inducible Sarcophaga lectin gene promoter, its transcriptional activity remained controversial. Moreover, homologues of SRAM have not been found in any other established model organisms including Drosophila. Here we report that the developmental expression of SRAM was up-regulated at the early stages of embryogenesis and metamorphosis. Furthermore, SRAM expression was prominent in the digestive tracts of the third instar larvae. We argue the hypothesis that SRAM has evolved as a quite unconventional Rel-family protein in Sarcophaga.

Convergent synthesis of fluorescence-labeled probes of Annonaceous acetogenins and visualization of their cell distribution.

The convergent synthesis of fluorescence-labeled solamin, an antitumor Annonaceous acetogenin, was accomplished by two asymmetric alkynylations of 2,5-diformyl tetrahydrofuran with an alkyne tagged with fluorescent groups and another alkyne with an α,ß-unsaturated γ-lactone. Assay for the growth inhibitory activity against human cancer cell lines revealed that the probe with the fluorescent groups at the end of the hydrocarbon chain may have the same mode of action as natural acetogenins. The merged fluorescence of dansyl-labeled solamin and MitoTracker Red suggests that Annonaceous acetogenins localize in the mitochondria.

Normal formation of a subset of intestinal granules in Caenorhabditis elegans requires ATP-binding cassette transporters HAF-4 and HAF-9, which are highly homologous to human lysosomal peptide transporter TAP-like.

TAP-like (TAPL; ABCB9) is a half-type ATP-binding cassette (ABC) transporter that localizes in lysosome and putatively conveys peptides from cytosol to lysosome. However, the physiological role of this transporter remains to be elucidated. Comparison of genome databases reveals that TAPL is conserved in various species from a simple model organism, Caenorhabditis elegans, to mammals. C. elegans possesses homologous TAPL genes: haf-4 and haf-9. In this study, we examined the tissue-specific expression of these two genes and analyzed the phenotypes of the loss-of-function mutants for haf-4 and haf-9 to elucidate the in vivo function of these genes. Both HAF-4 and HAF-9 tagged with green fluorescent protein (GFP) were mainly localized on the membrane of nonacidic but lysosome-associated membrane protein homologue (LMP-1)-positive intestinal granules from larval to adult stage. The mutants for haf-4 and haf-9 exhibited granular defects in late larval and young adult intestinal cells, associated with decreased brood size, prolonged defecation cycle, and slow growth. The intestinal granular phenotype was rescued by the overexpression of the GFP-tagged wild-type protein, but not by the ATP-unbound form of HAF-4. These results demonstrate that two ABC transporters, HAF-4 and HAF-9, are related to intestinal granular formation and some other physiological aspects.

Differential activation of the lectin and antimicrobial peptide genes in Sarcophaga peregrina (the flesh fly).

Sarcophaga lectin is an immune defense protein which is transcriptionally induced upon immune challenge in the flesh fly, Sarcophaga peregrina. So far, we have revealed that the Sarcophaga lectin gene has multiple NF-kappaB -binding motifs in its promoter. Here we showed that the nuclear extracts from Sarcophaga-derived culture cells, NIH-Sape-4, and larval fat bodies have binding activity to the multiple kappaB motifs in the lectin gene promoter, some of which were responsive to immune stimuli. We also compared the expression profiles of the lectin gene with those of the antibacterial peptide genes from the point of view of inducers, expression tissues and local induction in digestive tracts. In each case, the lectin gene was activated in different manners from other inducible defense genes. These results indicate the complex regulation of the lectin gene, possibly by NF-kappaB -related transcription factors.

Functional dissection of transmembrane domains of human TAP-like (ABCB9).

An ABC transporter, TAP-Like (TAPL), was dissected into its amino-terminal transmembrane domain and the following core domain. When these domains were transiently expressed as tagged proteins with a His6- or Myc-epitope tag, the amino-terminal ones (Met(1)-Lys(182)) could not associate with each other, or with the full-length transporter (Met(1)-Ala(766)). However, both the core domain (Arg(141)-Ala(766)) and full-length protein mutually interacted. The amino-terminal domain (Met(1)-Arg(141)) as well as the full-length transporter fused with fluorescent protein GFP was sorted to lysosomal membranes upon their stable expression, as visualized by means of fluorescent microscopy, while the core domain (Arg(141)-Ala(766)) was broadly distributed in the intra-cellular membranes. These results suggest that the sorting signal for lysosomes is present within the amino-terminal transmembrane domain (Met(1)-Arg(141)) of the TAPL molecule.

Biochemical characterization of transporter associated with antigen processing (TAP)-like (ABCB9) expressed in insect cells.

The ATP-binding cassette (ABC) transporter, transporter associated with antigen processing (TAP)-like (TAPL) tagged with a histidine cluster was overexpressed, amounting to as much as 1-2% of total membrane proteins in Drosophila cell line S2. TAPL was effectively solubilized from membranes by Triton X-100, NP-40 and n-dodecyl-beta-D-maltoside. Solubilized TAPL bound ATP-agarose and adenosine 5'-diphosphate (ADP)-agarose but not adenosine 5'-monophosphate (AMP)-agarose. The binding was competed for by excess free ATP, ADP, guanosine 5'-triphosphate (GTP) and dATP but not by AMP. Pyrimidine nucleotides such as uridine 5'-triphosphate (UTP) and cytidine 5'-triphosphate (CTP) were less effective competitors, suggesting that purine nucleotide triphosphates are substrates for TAPL. The ATP-binding of TAPL required Mg(2+), and was observed at neutral pH. Chemical cross-linking experiments suggested that TAPL forms a homodimer in the membrane and under the solubilized conditions.

Cyclic AMP-dependent proteolysis of GATA-6 expressed on the intracellular membrane.

Cyclic AMP-dependent proteolysis of GATA-6 was characterized by fusing GATA-6 with the carboxyl-terminal membrane domain of SREBP-2. When the fusion protein was stably expressed in CHO-K1 cells, it was recovered in the ER membrane. This protein was processed in a similar manner to SREBP-2 upon cholesterol starvation, and the GATA-6 moiety moved into the nucleus. The GATA-6 moiety on the membrane became undetectable in the presence of dbcAMP or cholera toxin. However, H-89, K-252a, MG115 and lactacystin inhibited this decrease, suggesting that the cytoplasmic GATA-6 moiety of the fusion protein was degraded by proteasomes though A-kinase upon elevation of the cellular cAMP concentration.

Interaction of Mat-8 (FXYD-3) with Na+/K+-ATPase in colorectal cancer cells.

Mat-8 was fused with a Myc-tag or green fluorescent protein at its carboxyl terminus, and then expressed in Chinese hamster ovary K1 cells. Determination of the cellular localization of the tagged proteins suggested that they were localized on the intracellular membrane, being not only detected around the nuclear envelope but also partly overlapping with markers for endosomes and Golgi bodies. However, Mat-8 with the Myc-tag was detected on the plasma membrane as well as the intracellular membrane, when it was expressed in colorectal cancer cells. The membrane fraction of the cancer cells was solubilized and immuno-precipitated with an antibody for the Myc-tag. Western-blotting analysis demonstrated that the Na+/K+-ATPase alpha subunit was present in the precipitate. Furthermore, the immuno-precipitate obtained with an antibody for the Na+/K+-ATPase alpha subunit reacted with that for the Myc-tag. These results suggested that Mat-8 could be associated with Na+/K+-ATPase similar to other FXYD family members. The Gly41-->Arg mutation in the transmembrane region of Mat-8 inhibited its association with the Na+/K+-ATPase alpha subunit and localization on the plasma membrane, whereas the Cys44-->Ala or Cys49-->Ala substitution did not. Thus the conserved Gly41 residue in the transmembrane domain could be indispensable for localization of Mat-8 on the cell surface.

GATA-4 gene organization and analysis of its promoter.

The mouse GATA-4 gene is separated by six introns, and this gene organization is conserved in rodents and man. The transcriptional start site of the GATA-4 gene is essentially the same in rat heart, stomach and testis, and in cultured cells expressing GATA-4 such as TM3, TM4, I-10 and P19.CL6 cells. The 5'-upstream of the GATA-4 gene is also conserved in rodents and man. We examined its promoter activity by means of luciferase reporter gene assay using testis-derived TM3 and TM4 cells. The GC-boxes and E-box located in the several tens of base pairs upstream of the transcriptional start sites of the GATA-4 gene were found to be critical for its promoter activity in these cells, consistent with the mode of transcription characteristics of the TATA-less promoter. P19.CL6 cells differentiate into beating cardiomyocytes upon induction by DMSO, accompanied by stimulation of the transcription of heart-specific genes including GATA-4. Interestingly, they exhibit increased luciferase reporter gene activity upon induction by DMSO. Both proximal tandem GC-boxes and the E-box are also contributed to the reporter gene activity in P19.CL6 cells.

Examination of drug resistance activity of human TAP-like (ABCB9) expressed in yeast.

A half-type ABC transporter, human TAP-like (hTAPL) tagged with histidine cluster, was expressed in budding yeast protease-deficient strain BJ5457, and the effect of expression for resistance to peptide compounds including antibiotics and proteinase inhibitor was examined. Among these compounds, the yeast expressing hTAPL exhibits high sensitivity to valinomycin, a monovalent cation ionophore. A mutation in Walker A motif, which lost ATP-binding activity of hTAPL, eliminated the enhanced sensitivity to valinomycin. These findings suggest that the transport activity of hTAPL is important for conferring high valinomycin-sensitive phenotype to yeast.

Participation of a galactose-specific C-type lectin in Drosophila immunity.

A galactose-specific C-type lectin has been purified from a pupal extract of Drosophila melanogaster. This lectin gene, named DL1 (Drosophila lectin 1), is part of a gene cluster with the other two galactose-specific C-type lectin genes, named DL2 (Drosophila lectin 2) and DL3 (Drosophila lectin 3). These three genes are expressed differentially in fruit fly, but show similar haemagglutinating activities. The present study characterized the biochemical and biological properties of the DL1 protein. The recombinant DL1 protein bound to Escherichia coli and Erwinia chrysanthemi, but not to other Gram-negative or any other kinds of microbial strains that have been investigated. In addition, DL1 agglutinated E. coli and markedly intensified the association of a Drosophila haemocytes-derived cell line with E. coli. For in vivo genetic analysis of the lectin genes, we also established a null-mutant Drosophila. The induction of inducible antibacterial peptide genes was not impaired in the DL1 mutant, suggesting that the galactose-specific C-type lectin does not participate in the induction of antibacterial peptides, but possibly participates in the immune response via the haemocyte-mediated mechanism.

Further extension of mammalian GATA-6.

Mammalian GATA-6, which has conserved tandem zinc fingers (CVNC-X(17)-CNAC)-X(29)-(CXNC-X(17)-CNAC), is essential for the development and specific gene regulation of the heart, gastrointestinal tract and other tissues. GATA-6 recognizes the (A/T/C)GAT(A/T)(A) sequence, and interacts with other transcriptional regulators through its zinc-finger region. The mRNA of GATA-6 uses two Met codons in frame as translational initiation codons, and produces L- and S-type GATA-6 through leaky ribosome scanning. GATA-6 is subjected to cAMP-dependent proteolysis by a proteasome in a heterologous expression system. These protein-based characteristics of GATA-6 will be helpful for the identification of target genes, together with determination of the in vivo binding sites for GATA-6 and understanding of the complex network of gene regulation mediated by GATA-6.