Unveiling Novel Mechanisms Governing Organelle-Dependent Quality Control of Tail-Anchored Membrane Proteins

Project Summary/Abstract: Membrane proteins are critical for organelle identity and function. Among these, tail-anchored (TA) proteins represent a special class of membrane proteins that are post-translationally inserted into their respective membrane via a single C-terminal transmembrane (TM) domain. TA proteins are prone to mislocalization, which can disrupt cellular function and initiate proteostatic collapse, a hallmark of age-related disease. While initial TA protein studies focused on yeast, mammalian systems represent a richer diversity of TA proteins and, in tandem, regulatory mechanisms. In this proposal, I will explore how mammalian TA quality control systems function, using the TA protein BNIP3 as a model. BNIP3 dually localizes to the ER membrane and outer mitochondrial membrane (OMM) and has been broadly implicated in organismal health span and aging. BNIP3 function and stability are regulated by the homodimerization of its transmembrane domain. By generating BNIP3 variants that localize exclusively to either the ER or OMM, this project aims to dissect 1) the specific signals used to identify orphan TA proteins, 2) the cellular machinery that recognizes them, and 3) how this dictates BNIP3 fate across organelles. The project will be pursued through two main objectives: In Aim 1, I will utilize a fluorescent reporter for ER-localized BNIP3 to pinpoint the critical signals within BNIP3 necessary for its recognition and proteasomal processing from the ER membrane. In addition, I will perform an unbiased genome wide CRISPR knockout screen to identify the cellular factors mediating TA quality control at the ER. In Aim 2, I will utilize a fluorescent reporter for OMM-localized BNIP3 to identify the signals within BNIP3 necessary for its turnover from the OMM, as well as investigate the factors mediating TA quality control from the OMM, parallel to the approaches used in Aim 1. This research promises to reveal the sophisticated mechanisms cells use to control the quality of TA proteins like BNIP3, potentially highlighting novel ways in which the ER and mitochondria maintain proteostasis through additional coordination. The findings from this work could subsequently open new avenues for understanding mechanisms of age-associated proteostatic collapse.