This proposal is designed to characterize the function and structure of the lysosomal anion (sulfate) transporter, a newly recognized member of a family of proteins that are critical to cell function. These investigations will provide insights into basic lysosomal function and will form a framework for the study of this and other clinically significant lysosomal transport proteins. A considerable amount of biochemical, immunological and molecular information is available concerning the functionally homologous plasma membrane anion transporters. This information will now be applied toward study of the lysosomal system according to the following specific aims. 1) Define the substrated of lysosomal anion transport: Sulfate, chloride, and molybdate have already been identified as substrates for transport. Additional divalent anions such as SeO4-2 and monovalent anions such as HCO33-, F-, Br-, I-_, and SCN-, will be examined. Effects of pH, membrane potential and temperature will be determined. Affinity constants and rates of transport will be calculated. Emphasis will be placed further defining the role of protons as co- transported species. 2) Define the role of lysosomal anion transport in cell metabolism: Transport will be examined in lysosomes form other tissues, including kidney. Using cultured cell systems, the role of the lysosomes in regulating intracellular anion concentrations through recycling will be examined. Whether in vitro substrates for transport such as molybdate are also in vivo substrates will be determined. The role of the transporter in the regulation of lysosomal pH and chloride movement will also be examined. 3) Identify inhibitors of lysosomal anion transport: Inhibitors of the erythrocyte band 3 anion transporter including stilbene sulfonic acids, NAP-taurine, and arylisothiocyanates will be screened for their effect on the lysosomal system in order to provide information on structure and function. One important goal of these studies is to identify irreversible inhibitors that can subsequently be used as probes to aid in identification of the lysosomal transport protein. 4) Identification of the lysosomal anion transport protein Strategies for identification of the lysosomal transporter include: 1) Use of irreversible inhibitors of lysosomal membrane anion transport as probes, 2) Use of immunoblots to evaluate antibodies against the highly conserved carboxyl ends of the AE1 (BAND 3) and AE3 anion transporters for evidence of cross reactivity with the lysosomal protein, and 3) Screening rat liver cDNA libraries for hybridization with the 3' portion of a mouse AE1 cDNA probe under conditions of reduced stringency. Success with any of these techniques will be followed by attempts to clone the gene for the lysosomal transporter.
| Chou, H F; Passage, M; Jonas, A J (1998) Lysosomal sulphate transport is dependent upon sulphydryl groups. Biochem J 330 ( Pt 2):713-7 |
| Chou, H F; Passage, M; Jonas, A J (1997) ATP stimulates lysosomal sulphate transport at neutral pH: evidence for phosphorylation of the lysosomal sulphate carrier. Biochem J 327 ( Pt 3):781-6 |
| Koetters, P J; Chou, H F; Jonas, A J (1995) Lysosomal sulfate transport: inhibitor studies. Biochim Biophys Acta 1235:79-84 |
| Koetters, P J; Chou, H F; Jonas, A J (1995) Reconstitution of lysosomal sulfate transport in proteoliposomes. Biochim Biophys Acta 1244:311-6 |
| Chou, H F; Passage, M; Jonas, A J (1994) Regulation of lysosomal sulfate transport by thyroid hormone. J Biol Chem 269:23524-9 |
| Shull, R M; Kakkis, E D; McEntee, M F et al. (1994) Enzyme replacement in a canine model of Hurler syndrome. Proc Natl Acad Sci U S A 91:12937-41 |
