Proximity-specific ribosome profiling reveals the logic of localized mitochondrial translation

Localized translation broadly enables spatiotemporal control of gene expression. Here we present LOCL-TL (LOV-domain-Controlled Ligase for Translation Localization), an optogenetic approach for monitoring translation with codon resolution at any defined subcellular location under physiological conditions. Application of LOCL-TL to mitochondrially-localized translation revealed that ~20% of human nuclear-encoded mitochondrial genes are translated on the outer mitochondrial membrane (OMM). Mitochondrially-translated messages form two classes distinguished by encoded protein length, recruitment mechanism, and cellular function. An evolutionarily ancient mechanism allows nascent chains to drive cotranslational recruitment of long proteins via an unanticipated bipartite targeting signal. Conversely, mRNAs of short proteins, especially eukaryotic origin electron transport chain (ETC) components, are specifically recruited by the OMM protein A-Kinase Anchoring Protein 1 (AKAP1), in a translation-independent manner that depends on mRNA splicing. AKAP1 loss lowers ETC levels. LOCL-TL thus reveals a hierarchical strategy that enables preferential translation of a subset of proteins on the OMM. Ribosome profiling data were generated from cell lines engineered with LOCL-TL components. Two replicates were used for each group: 1) ER-LOV-BirA and 2) Mito-LOV-BirA. RNA-seq data were collected from cell lines with LOCL-TL components and LOV-BirA targeted to the mitochondria in replicates. RNA-seq data were also collected from cell lines with perturbations on AKAP1, LARP4, or negative control gRNA.