Investigating the role of Reep5 in formation, maintenance, and function of the endoplasmic reticulum

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***The Endoplasmic Reticulum (ER) is a dynamic multi-functional organelle found in eukaryotes. It is a specialized organ responsible for many functions, including Ca2+ cycling, protein translation and trafficking. An important group of proteins that mediate formation and maintenance of the ER are proteins with reticulon homology domains (RHD). One group of RHD proteins are the receptor expression-enhancing proteins (REEPs). The precise role of REEPs in ER formation, maintenance, and responses to ER stress is not well understood. Our ongoing proteomic studies have identified REEP5 to be an evolutionary-conserved, cardiac cell-enriched, membrane protein that is the dominant REEP in the heart.******Hypothesis and Aim: We hypothesize that REEP5 controls the ER homeostatic environment and ER integrity, affecting cardiac function through its regulation of Ca2+ signaling, and is centrally involved in cardiac ER stress responses. My primary goal is to investigate the mechanism of how REEP5 contributes to normal ER formation, maintenance and function. We propose utilizing cell biological, biochemical, mass spectrometry, and advanced cellular imaging approaches to address the role of REEP5.******Examine the effect of Reep5 knockdown on HEK and myocyte ER. We will perform immunofluorescence and super resolution microscopy to examine REEP5-localization in cells. Site-directed mutation of REEP5 will be performed and consequences of mutations will be assessed on ER morphology and localization. To study ER stress responses, we will assess WT and knockdown of REEP5 using lentiviral vectors with/without ER stress induction induced by H2O2 and tunicamycin. Reactive oxygen species (ROS) reflecting ER stress, will be measured, as will cell viability and apoptosis.***Identify REEP5 interacting proteins. Using standard*immunoprecipitation studies and mass spectrometry we will identify REEP5*interacting proteins involved in ER formation and maintenance. We*generated a REEP5-myc-flag-his expressing stable HEK cell line, and will*purify REEP5 and interactors. Analyses will be performed in the*presence and absence of ER stresses. Immobilized proteins will be*visualized by Coomassie staining and identified by mass spectrometry.***Examine the effect of REEP5 deletion in the zebrafish. To investigate the role of REEP5 in heart development, we have generated a morpholino zebrafish for REEP5. REEP5 is expressed in the zebrafish myocardium during embryonic development, and the zebrafish REEP5 protein has high homology (~80%) to the human REEP5. We have successfully silenced REEP5 expression in zebrafish. The initial characterization of the morphants show perturbed embryonic development with abnormal heart formation and dysfunction. Fish and tissues will be assessed by immunoblotting, immunofluorescence, histology and bright field imaging.**************