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The ER Membrane Protein Complex (EMC) Governs Endolysosomal Turnover of Mitochondrial Tail-anchored Protein, BNIP3, to Restrict Mitophagy
Christopher J Shoemaker1, Jose M Delgado1, Logan Wallace Shepard1, Sarah W Lamson1, Samantha L Liu1
1 Department of Biochemistry and Cell Biology, Geisel School of Medicine, Dartmouth College, Hanover, NH, USA
Tail-anchor (TA) proteins are a diverse class of membrane proteins (~50 in yeast, >300 in humans) containing a single, C-terminal transmembrane domain (TMD). The hydrophobicity of a TA TMD is a primary determinant of its localization, with mitochondrially-targeted TMDs having a lower hydrophobicity, on average, than those targeted to the ER. However, this relationship is not absolute, and additional models are needed to explain the observed steady state localization of TA proteins. BNIP3 is a homodimeric TA protein localized predominantly to the outer mitochondrial membrane (OMM) where it facilitates mitochondrial turnover by lysosomes (hereafter, mitophagy). We find that BNIP3 and a close homolog, NIX, are constitutively delivered to lysosomes in an autophagy-independent manner. This alternative lysosomal delivery of BNIP3 accounts for nearly all of its lysosome-mediated degradation, even upon mitophagy induction. To identify how BNIP3, a tail-anchored protein in the OMM, is delivered to lysosomes, we performed an unbiased genome-wide CRISPR screen for factors influencing BNIP3 stability. By this approach, we revealed both known modifiers of BNIP3 stability as well as a pronounced reliance on endolysosomal components, including the ER membrane protein complex (EMC) and ER-to-Golgi trafficking. Through a combination of genetics, biochemical analyses, and cell based-reporters, we find that the endolysosomal system regulates BNIP3 alongside, but independent of, the ubiquitin-proteosome system (UPS). Unstable BNIP3 monomers are extracted from membranes allowing for additional attempts at insertion or, in the case of perpetually mislocalizing monomers, degradation by the proteasome. In contrast, BNIP3 dimers are resistant to membrane extraction. However, stable BNIP3 dimers in the ER are dramatically cleared through facilitated transport to lysosomes. BNIP3 dimers in the OMM are resistant to both forms of quality control, thus explaining the observed steady-state localization of BNIP3 at the OMM in the absence of a single, high-fidelity insertion mechanism. These findings extend recent models for tail-anchored protein quality control and install endosomal trafficking and lysosomal degradation in the canon of pathways that ensure tight regulation of endogenous TA protein localization. More broadly, these data reveal an unanticipated connection between mitophagy and TA protein quality control, wherein the endolysosomal system provides a critical axis for regulating mitochondrial dynamics and cellular metabolism.
This work is supported by the National Institutes of Health General Medical Sciences (R35GM142644 to CJS, F31GM143849 to JMD)