Structure, Regulatory and Functional Mechanisms, and Physiological Roles of Peptide Transporter

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1. Human ABC transporter TAPL was stably expressed in the cultured cells, and its intracellular localization was determined. The TAPL was localized on the lysosomal membrane. Furthermore, TAPL formed a homo-dimer as determined by the expression of TAPL tagged with different fluorescent proteins. The amino terminal 4 trans-membrane segments were enough for TAPL to be localized to lysosome. Mutation introduced into a residue important for ATP binding of TAPL abolished the high sensitivity of the yeast to an antibiotic.2. TAPL had carboxyl-terminal splicing isoforms (3 and 4 in human and rat, respectively), which would produce functional diversity of TAPL.3. We also demonstrated that two TAPL homologues of C. elegance are expressed on the granule membranes in intestinal cells. The deletion of the genes for these homologues further indicated that they are important for formation and/or maintenance of such intestinal intracellular granules.4. Mat-8, a member of FXYD family, co-localized with Nat, K+-ATPase on the plasma membrane of colorectal cancer cells, suggesting that Mat-8 could be a regulatory component of a membrane transport nano-machine. When the mutation was introduced into the conserved residue in its transmembrane domain, Mat-8 was expressed rather on the intracellular membranes instead of plasma membrane.5. ln this project, we constructed various expression systems for membrane proteins TAPL and Mat-8 together with those tagged with fluorescent proteins. These systems were adopted for visualization of transporters and their regulators under fluorescent microscope. Further construction of mutants and their expression will give us important information on the association and dissociation of the membrane transporter complexes. Taken together, these findings would be valuable and become basic knowledge for future studies on function and structure of membrane transport nano-machines.