Knowledge of lysosomal transport systems provides an important perspective for understanding the various functions of lysosomes. These transport systems mediate the release of lysosomal degradation products which can be reutilized for biosynthesis. Defects in lysosomal transport, as accentuated in the genetic disease, cystinosis, lead to lysosomal accumulation of the metabolite for which transport is impaired. Transport systems may also play an important role for the delivery of required cofactors or metabolites into lysosomes. Lysosomes contain enzymatic activities capable of completely degrading nucleic acids to their constituent nucleosides, but the manner by which these degradation products are released from lysosomes is virtually unknown. The major goal of this proposal is to define the characteristics of nucleoside and nucleobase transport in lysosomes of eukaryotic cells. Lysosomes, purified on Percoll density gradients, will be used to study the uptake and exodus of radiolabeled nucleosides and nucleobases. These studies will determine: (1) the existence of saturable nucleoside and nucleobase transport systems in lysosomes, (2) Km and Vmax of substrates (3) the substrate specificity of a given transport system (4) effects of pH, osmolarity, temperature and various chemical modifying reagents on transport. Whether lysosomal and plasma membrane nucleoside transport proteins share the same genetic elements will be explored by examining lysosomal nucleoside transport in mutant cells defective in their plasma membrane nucleoside transport. None of the lysosomal transport proteins have been labeled or isolated. Thus, the molecular aspects of lysosomal transport are poorly understood. The specificity and ability of nitrobenzylthioinosine to covalently link with the plasma membrane nucleoside transport protein upon uv irradiation, will be utilized to specifically label lysosomal nucleoside transport proteins and determine: (1) the molecular weight of the labeled transport protein by SDS-PAGE, (2) the number of lysosomal nucleoside transport proteins per cell and how these may change during nutritional deprivation or cellular transformation, (3) turnover rate of this nucleoside transport protein and (4) effects of glycosylation inhibitors on targeting of the nucleoside transport protein to the lysosome. Lysosomal amino acid studies have aided our understanding of the basis of cysteamine treatment of cystinosis. Similarly, knowledge of the characteristics of lysosomal nucleoside transport could lead to the design of new chemotherapeutic agents specifically delivered to lysosomes when coupled to ligands recognized by receptor- mediated endocytosis.
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