Targeting and plasticity of mitochondrial proteins revealed by proximity-specific ribosome profiling

The wheres and whys of protein translation Localized protein synthesis is important for a broad range of biological activities, from specifying the animal body plan to coordinating entry into the secretory pathway. Few tools are available that can investigate translation at specific subcellular sites. Jan et al. present a flexible ribosome profiling–based methodology to enable precise characterization of localized protein synthesis (see the Perspective by Shao and Hegde). Proximity-specific ribosome profiling provides a high-precision tool for looking at the mechanism of localized protein targeting and synthesis in living cells. The approach yielded a high-resolution systems-level view of cotranslational translocation at the endoplasmic reticulum. Williams et al. applied the technique to look at localized mRNA translation at the mitochondrial outer membrane. Science, this issue 10.1126/science.1257521, p. 701; see also p. 748 A new method reveals exactly which proteins are synthesized in the neighborhood of the endoplasmic reticulum and mitochondria. [Also see Perspective by Shao and Hegde] Nearly all mitochondrial proteins are nuclear-encoded and are targeted to their mitochondrial destination from the cytosol. Here, we used proximity-specific ribosome profiling to comprehensively measure translation at the mitochondrial surface in yeast. Most inner-membrane proteins were cotranslationally targeted to mitochondria, reminiscent of proteins entering the endoplasmic reticulum (ER). Comparison between mitochondrial and ER localization demonstrated that the vast majority of proteins were targeted to a specific organelle. A prominent exception was the fumarate reductase Osm1, known to reside in mitochondria. We identified a conserved ER isoform of Osm1, which contributes to the oxidative protein-folding capacity of the organelle. This dual localization was enabled by alternative translation initiation sites encoding distinct targeting signals. These findings highlight the exquisite in vivo specificity of organellar targeting mechanisms.