The mechanism(s) by which proteins are translocated across membranes remains a central focus of research in modern cell biology. The cell membrane is thought to act as a selective protein barrier and with few exceptions, exogenous proteins are either excluded from the cell or enter a 'default pathway' that rapidly culminates with lysosomal degradation. Entry of an exogenous protein into the cell's interior is a rare event and thus far, appears restricted to a few bacterial toxins with unique targeting sequences that manage to escape lysosomal degradation. In contrast to any previous description of exogenous protein translocation, we recently found an abundant, 15.5 kDa protein in the rat proximal tubule that is a proteolytic cleavage product of alpha2mu-microglobulin (A2), a 19 kDa protein synthesized predominantly by the liver. A2 is a member of a protein superfamily (including retinol binding protein) thought to function as transport proteins for hydrolyphobic ligands such as long chain fatty acids. Accumulation of this hepatic protein within the proximal tubule of the kidney may be the first physiologically significant example of 'retrograde protein transport' in which a naturally synthesized, exogenous protein is accumulated in the cytosol of another cell type. We suggest that following glomerular filtration, the proximal tubule cell metabolizes A2 by a novel pathway, ultimately accumulating large amount of a proteolytic cleavage product (A2-fragment) in the cytosol. To evaluate this hypothesis, we propose to: (1) describe the process of A2 uptake and translocation by the proximal epithelial cell; (2) determine whether this process is mediated by endocytosis (receptor-mediated, fluid-phase, or adsorptive endocytosis) or simple diffusion; (3) evaluate whether binding of A2 to a hydrophobic ligand is required for, or modifies A2 uptake; (4) identify the cellular compartment(s) responsible for processing A2 so that cytosolic accumulation, rather than degradation, of A2 occurs and (5) determine whether unique structural features determine how A2 is processed by the proximal tubule cell. These studies could provide new insights regarding a previously unknown pathway for moving proteins from outside to inside the cell. This information could have relevance for targeted delivery of various agents or drugs to the kidney. In addition, A2 binds fatty acids in vitro and act as a 'fatty acid binding like-protein' in vivo. As a transport protein, A2 could modulate fatty acid oxidation rates during both normal and pathophysiologic states such as rental ischemia.
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