Exploring Endoplasmic Reticulum-Associated Rab GTPases

点击翻译按钮获取中文摘要

The overarching research program of our laboratory is the characterization of mechanisms that lead to the formation of membrane contact sites (MCS), contacts between two organelles. One aspect of our program is the characterization of the mitochondria-associated membrane (MAM), a connection between the endoplasmic reticulum (ER) and mitochondria. Our lab has identified MAM signaling mechanisms, MAM targeting motifs and factors that determine MAM enrichment. For instance, our research has discovered the small GTPase Rab32 as a determinant of MAM enrichment and of the flux of Ca2+ between the ER and mitochondria. Rab32 is a member of the Rab proteins, important regulatory factors for protein secretion, of which about a dozen localize to the ER. Here, these Rabs perform a diversity of functions, some well characterized and resembling the "address tag" function of Rabs of the late secretory pathway, whereas others perform functions that remain poorly understood. What all these ER-associated Rabs have in common is that they determine to varying extents and by various means the localization of ER proteins to membrane domains. ***Since the protein composition of ER membrane domains and directed tubulation of ER membranes are critical for the interaction of the ER with other organelles, we hypothesize that ER-associated Rabs play a role for the formation of MCS. Support for this hypothesis comes from recent findings generated in our lab on ER-localized Rab32 relatives. These proteins not only determine the distribution of ER proteins within the whole reticulum, but also determine ER-mitochondria interaction and localize fissure points onto mitochondria to regulate mitochondrial dynamics. ***We will test our hypothesis with the following specific aims: ***1. Determine the extent and mechanism of ER association for all human ER-localized Rab proteins. Upon quantification of ER-association for endogenous and transfected Rabs, we will generate chimera from ER-localized and ER-excluded Rabs to determine mechanisms responsible for ER localization. ***2. Role of ER-associated Rabs for ER membrane contact sites. We will test whether dominant-active and dominant-negative ER-localized Rabs can mislocalize known MCS proteins. We will also assay for their ability to influence MCS function. ***3. Identify effectors for ER-associated Rabs. We will use candidate-based approaches that will test for interaction between ER-associated Rabs and ER-shaping enzymes with known involvement in MCS formation. We will also search for unknown MCS effectors by screening for interactors that preferentially bind GTP-bound Rabs. ***We expect this proposal to continue our research program on MCS. We will provide insight into the functioning of MCS and characterize a novel family of MCS regulatory proteins. We will also continue our efforts to provide first class training for high quality personnel (HQP). **