This proposal is focused on understanding the molecular composition, mechanism of action, and modes of communication of mammalian endoplasmic reticulum (ER)-organelle membrane contact sites (MCSs). Increasing evidence indicates that ER MCSs are crucial regulatory hubs, but they are poorly defined on both the molecular and functional levels. I will address this deficit by examining the mammalian homologs of a newly described class of conserved ER MCS proteins, termed Loc (lipid transfer at organelle contact site), identified in our lab in yeast (yLoc). We have demonstrated that in vivo yLocs directly facilitate membrane contacts between the ER and many other organelles and in vitro yLocs mediate the non-vesicular sterol transport between liposomes. My preliminary data reveal that the mammalian Loc (mLoc) family member GRAMD2 localizes to Ca2+-dependent ER-plasma membrane (PM) MCSs, which function to regulate store-operated Ca2+ entry (SOCE). SOCE is a PM Ca2+ influx pathway that is induced by depletion of Ca2+ from ER stores and is critical for cell homeostasis. Building on this observation and our yLoc data, I will focus on understanding the molecular basis and functions of mLocs at ER MCSs. Specifically, in Aim 1, I will examine the functional role of GRAMD2 at Ca2+-regulated ER-PM contacts.
In Aim 2, I will define molecular activities and the mechanistic role of GRAMD2.
In Aim 3, I will probe the functional role of other mLoc protein family members and examine their relationships at ER MCSs. Understanding the fundamental role of mLoc will inform upon MCSs regulation and composition will provide fundamental insight into the roles of ER MCSs in cell physiology, which will improve our understanding of the etiology human disease and could reveal new targets for therapeutics.
The failure of organelles to effectively communicate and respond appropriates to cellular demands and environmental conditions underlie a wide spectrum of human disease, including neurodegenerative diseases and cancer. The endoplasmic reticulum (ER) is central to myriad cellular functions. The research proposed here will address how ER forms dynamics membrane contacts with other organelles. This work will provide insight into the composition and regulation of mammalian ER contacts, which, despite being vital regulatory hubs within the cell, remain poorly understood. The proposed research will improve our understanding of organelle physiology and is relevant to the part of the NIH's mission that fosters fundamental basic cell biology discoveries that will directly lead to the identification of new therapeutic targets and therapies for the treatment of wide array of human diseases.
