The goal is to examine the role of organellar acidification in tyrosinase (TYR) activation, stabilization and transport through the secretory pathway. Recent findings that the level of pigmentation in melanocytes negatively correlates with acidic intracellular organelles, leading to increasingly more severe inactivation of TYR, its retention in a pre- Golgi compartment and subsequent degradation. Furthermore, we have recently found that some amelanotic melanoma cells have a higher cytosolic PH, which together with a lower organellar PH are reminiscent of the intracellular PH characteristic of tumor cells refractory to chemotherapy. Therefore, it is possible that alterations in intracellular PH homeostasis may cause not only TYRE inactivation in amelanotic melanomas, but may also underlay other phenotypes, such as immunogenicity of melanosome associated proteins, drug resistance, and possibly anti-apoptotic mechanisms in melanoma. We will address the role of intracellular PH in TYR biogenesis by the following assays: First, we will determine the subcellular localization of TYR in amelanotic melanoma cells before and after TYR activation with acidification inhibitors. Second, we will measure the PH in the cytosol and in diverse intracellular organelles in melanocytes defective in P-protein, in which a role for organelle PH was implicated. The P-protein is a putative transporter, proposed to be the """"""""guardian"""""""" of melanosomal PH, whose dysfunction causes misrouting of TYR to other sites, including the plasma membrane. These studies will test the hypothesis that variable intracellular PH homeostasis is the cause for variability in the levels of pigmentation between these cell types.
Specific aim #1 : To evaluate the intracellular location of TYR in different cell types and conditions. The steady state subcellular localization of epitope-tagged location of TYR in different cell types and conditions. The steady immunogold electron microscopy and cell fractionation, to understand the mechanism by which intracellular PH affects TYR maturation.
Specific aim #2 : To visualize the intracellular distribution of chemotherapeutic drugs and acidic compartments in living melanocytes cells. This will provide a comparative overview of the intracellular PH in melanocytes and amelanotic melanomas. This studies will be complemented by those in aim #3.
Specific aim #3 : To evaluated the pH in the cytosol and selected intracellular organelles in melanocytes defective in P-protein. PH sensitive probes will be targeted to specific intracellular compartments and will be used to specifically determine the PH in the cytosol, endoplasmic reticulum (ER), Golgi and lysosomes/melanosomes. This information will be correlated with TYR activation under different conditions that simultaneously induced alkalization and activation of TYR to determine how inactivation of wild type TYR in melanomas correlate with alterations in intracellular PH.
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