The mammalian ribosome receptor,p180, mediates RER membrane biogenesis and establishment of a secretory phenotype

A remarkable event occurs during a brief interval late in the process of terminal differentiation of secretory cells from a precursor cell. During this short time period, a cytoplasm virtually devoid of ER membranes becomes populated with rough endoplasmic reticulum (RER), and the precursor cell acquires a phenotype characterized by the secretion of specific macromolecules into the extracellular milieu. To date, little is known about the mechanism by which the RER first appears and then extensively proliferates during the terminal differentiation of secretory cells. Published reports from our lab have demonstrated that the expression in yeast of a ribosome receptor, p180, is sufficient for RER membrane biogenesis and increased secretory capacity.; To prove that p180 mediates RER membrane biogenesis and the establishment of a secretory phenotype in mammalian cells, we have utilized a model system using the THP-1 monocytic line. THP-1 cells recapitulate the process of terminal differentiation into macrophage-like secretory cells. To assess the involvement of p180 in this process, we employed RNA interference technology. Electron micrographs of p180-deficient cells revealed the appearance of ER membranes with a vesiculated morphology. The membranes were devoid of bound ribosomes as well. ELISA assays demonstrated that decreased p180 levels led to lower levels of ApoE secretion.; To demonstrate that p180 is sufficient to promote RER membrane biogenesis and acquisition of a secretory-cell phenotype we utilized a mammalian non-secretory cell system, Human Embryonic Kidney 293 (HEK293) cells. Electron micrographs of HEK293 cells expressing p180 showed a significant increase in proliferation of RER membranes as well as Golgi complexes. Biochemical studies demonstrated that a stretch of seven amino acids enables p180 to hydrolyze ATP. We show here that electron micrographs of HEK293 cells expressing ATPase-defective mutant forms of p180 showed proliferation of smooth membranes.; Based on results obtained from these knockdown and over-expression experiments we conclude that p180 is both necessary and sufficient to induce a secretory phenotype in mammalian cells. This dissertation will provide a mechanistic perspective, describing the initial steps that lead to the biogenesis of RER membranes, and how precursor cells terminally differentiate into secretory cells.