Determination of the Degradation Pathway of Organeller Membrane Proteins in the Context of Reticulocyte Maturation

Erwin Schrödinger Fellowship J 1998 Degradation Pathway of Organellar Membrane Proteins Slawomir SWKA 09.10.2000 One of the important questions in the context of organelle degradation is the fate of membrane proteins during/after the action of 15-lipoxygenase on organellar membranes. We made the observation that some microsomal membrane proteins including Sec61a, Sec61ß, Sec63, KDEL receptor, and TRAM aggregate during treatment with 15-lipoxygenase even in the absence of cytosol. At the same time, the treatment of microsomal membranes with 15-lipoxygenase renders Sec6lct more susceptible to degradation by chymotrypsin. In this proposal we address the question whether aggregation by 15-lipoxygenase constitutes a signal for degradation. If preincubation with 15- lipoxygenase increases the degradation rate in reticulocyte lysates, we will examine the effect of 15-lipoxygenase on the turnover of different membrane proteins in vivo using a lipoxygenase-producing stable cell line. Furthermore we want to determine the proteolytic pathway used to degrade organellar membrane proteins. We will perform degradation assays using a series of proteasomal inhibitors to determine whether degradation of Sec6lct is proteasome-dependent. If the degradation is proteasome- independent, we will use fractions of reticulocyte lysates to define and isolate the involved protelytic system. By cultivating rabbit reticulocytes we will address the question whether 15-lipoxygenase is required for organelle degradation in vivo. We will also examine whether microinjection or overexpression of 15-lipoxygenase in murine erythrolukemia cells, a model system used for red cell differentiation, can induce organelle degradation. Using a lipoxygenase-producing HeLa Tet-Off cell line we will determine whether 15-lipoxygenase alone is sufficient for organelle degradation in cells where organelle degradation does not normally occur.