Sub-cellular in vivo cardiac proteomics of sarcomere and ribosome interactomes identifies skeletal NACA's role in mitochondrial protein sorting.

Background: Proteostasis and the regulation of protein folding and sorting play a critical role in maintaining cellular homeostasis. The failure of proteostasis contributes to heart failure and aging, but, despite its importance, the mechanisms and factors regulating proteostasis in cardiomyocytes remain poorly characterized. Methods: Subcellular proteomes of cardiomyocytes were analyzed in vivo using biotin proximity labeling in mouse hearts. We employed a novel homology-independent targeting integration strategy for genetic tagging and for substitution of the muscle-specific skNAC isoform with the ubiquitous short isoform in cardiomyocytes. Results: We identified hundreds of proteins localized to the Z- and M-lines of sarcomeres, the ribosomes, and the desmosomes, including multiple chaperones. A universal homology-independent targeted integration strategy allowed us to genetically tag endogenous genes in the mouse heart and confirm protein localization. We identified the large muscle-specific isoform of the nascent polypeptide-associated complex protein skNAC as Z-line and ribosome associated protein. Replacement of skNAC with a ubiquitous isoform induced dilated cardiomyopathy, accompanied by altered ribosome positioning, reduced mitochondrial protein levels, and increased endoplasmic reticulum proteins. Conclusions: We unraveled the cardiomyocyte subcellular proteome and show that skNAC, an isoform down-regulated in disease, is a key ribosome and Z-line associated protein specifically responsible for the sorting of cardiomyocytes mitochondrial versus ER proteins.