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

1.人ABC转运蛋白TAPL在培养的细胞中稳定表达，并测定其细胞内定位。TAPL定位于溶酶体膜上。此外，通过用不同荧光蛋白标记的TAPL的表达确定，TAPL形成同源二聚体。氨基端4个跨膜片段足以使TAPL定位于溶酶体。对TAPL的ATP结合的重要残基进行突变，消除了酵母对ATP的高敏感性. TAPL具有羧基端剪接异构体（在人和大鼠中分别为3和4），这将产生TAPL的功能多样性.我们还证明了C.在肠细胞的颗粒膜上表达优雅。这些同源基因的缺失进一步表明它们对于这种肠细胞内颗粒的形成和/或维持是重要的。FXYD家族成员Mat-8与Nat，K+-ATP酶共定位于结直肠癌细胞质膜上，提示Mat-8可能是膜转运纳米机器的调控组分。将突变引入跨膜区的保守残基后，Mat-8表达于细胞内膜而非质膜.在本项目中，我们构建了膜蛋白TAPL和Mat-8的各种表达系统以及荧光蛋白标记的表达系统。采用这些系统在荧光显微镜下观察转运蛋白及其调节蛋白。突变体的进一步构建及其表达将为我们提供关于膜转运蛋白复合物的缔合和解离的重要信息。总之，这些发现将是有价值的，并成为未来的膜运输纳米机器的功能和结构的研究的基础知识。

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.