REGULATION OF LYSOSOMAL ENZYME TRAFFICKING

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The lysosome plays an important role in eukaryotic cells as the primary site of degradation of both cellular and extracellular macromolecules. Selective delivery of proteins to the lysosome involves addition of a common address marker (the mannose 6-phosphate marker) to the proteins, and membrane-associated receptors that bind the marker and mediate translocation of the proteins to the lysosomal compartment. Recent studies suggest that under certain conditions this targeting process can be altered such that enzymes normally destined for lysosomes are secreted from the cell. This regulatory mechanism may be important physiologically since extracellular lysosomal enzymes may play important roles in tumor growth and metastasis as well as in normal cell growth. The long range goal of this proposal is to determine the molecular basis for normal and regulated trafficking of lysosomal enzymes. The proposed studies will involve molecular and cellular biological studies on MEP, a lysosomal cysteine proteinase whose trafficking is regulated by growth factors and cellular transformation, and the receptors thought to mediate lysosomal trafficking: the cation dependent mannose 6-phosphate (Man-6-P) receptor and the Man-6-P/IGF-II receptor. Site-specific mutagenesis and expression of MEP cDNA will be used to identify structural determinants on the protein responsible for addition of the Man-6-P marker. Analysis of the protein and carbohydrate portions of MEP, and examination of the effects of growth factors on the synthesis and trafficking of the two receptors, will be carried out in order to further define the basis for MEP's regulated secretion. These studies will provide information leading to determination of the recognition structure responsible for selective phosphorylation of lysosomal proteins and will help elucidate the mechanisms involved in regulated trafficking of lysosomal proteins.