Kazuaki Ohashi

Further extension of mammalian GATA‐6

Mammalian GATA‐6, which has conserved tandem zinc fingers (CVNC‐X17‐CNAC)‐X29‐(CXNC‐X17‐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.

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.

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.