The overall goals of this research are to understand the B cell machinery that directs immunoglobulin (Ig) to its destination, either as a plasma membrane protein or as a secretory product. Previous results indicate that two stages in the transport of newly synthesized membrane and secreted Ig are sensitive to the protonophore, CCCP. The first stage seems to be entry of Ig into the Golgi complex and the second stage corresponds to transport between the trans-Golgi and the cell surface. The first CCCP-sensitive stage is indistinguishable biochemically from the stage of Ig processing in which several non-secreting myeloma mutants are arrested. The two current goals of this project are: 1) to clarify the mechanism of action of CCCP on intracellular Ig transport. Effects of CCCP on ion fluxes through the plasma membrane, on the pH of organelles involved in protein traffic, and on cellular ATP levels will be measured and compared with its effects on the turnover of non- secreted Ig. 2) to determine the intracellular sites where non- secreted Ig is arrested in several mutant cell lines. Available biochemical data suggest that Ig in all these mutants fails to reach the mid-Golgi compartment, but biochemical approaches cannot provide further resolution. Immunocytochemistry at the EM level, using immuno-gold staining on ultrathin frozen sections, will be used to localize the sites of accumulation of Ig in the mutants and in CCCP blocked cells. The results of this research should increase our understanding of the regulation of the intracellular pathway of newly synthesized antibody molecules. These experiments will also provide new information concerning the mechanism of action of the chemical agent, CCCP, which has been widely used as a tool in studies of cellular metabolism and secretion.
