research highlights

MOLECULAR BIOLOGY A global look at local

Proximity-specific profiling cotranslationally, which was surprising reveals the exquisite specificity of trans- because signal-recognition particle (SRP)- lation at the endoplasmic reticulum and independent proteins are also known to be mitochondrial outer membrane. capable of translocating post-translationally Subcellular compartmentalization is in vitro (Jan et al., 2014). Moreover, they crucial for the proper function of an organ- found that targeting relies more on the length ism. One way cells compartmentalize pro- of the coding region after the ER-targeting tein function is by synthesizing them when signal than on dependence on SRP. and where they are needed. However, local The team also found that the translocon translation has proven difficult to study. component Sec66 is responsible for translo- According to Jonathan Weissman at the cation of a subset of SRP-independent pro- University of California at San Francisco teins. Whereas most proteins are recognized and the Howard Hughes Medical Institute, for ER targeting once the signal sequence methods for studying local translation on emerges from the ribosome, this class of pro- the proteomic scale disrupt the and are teins contains signal sequences that require applicable only to some subcellular locations. ~120 amino acids to be targeted. This longer To study local translation in intact cells, nascent chain may adopt an alternate topol- the Weissman lab’s Calvin Jan, a postdoctoral ogy necessary for translocation. According to fellow, and Christopher Williams, a graduate Weissman, these results “show us an example student, developed proximity-specific ribo- where there really is a true functional diver- some profiling, which extends the ribosome- sity of signal sequences.” profiling method previously developed in the The researchers were also able to address Weissman lab to study a subset of translat- whether mitochondrial proteins are cotrans- ing . During ribosome profiling, lationally translocated (Williams et al., mRNA fragments protected by ribosomes 2014). Though many of the proteins tested undergoing translation are analyzed by deep are capable of being imported into mito- , providing a snapshot of transla- chondria post-translationally, Weissman’s tion in a cell. team revealed that ~30% of mitochondrial In proximity-specific ribosome profiling, reference genes are cotranslationally targeted. the biotin ligase BirA is expressed as a fusion The researchers wondered whether pro-

Nature America, Inc. All rights reserved. America, Inc. © 201 5 Nature to a protein that localizes to a specific com- teins could be targeted to both the ER and partment. This BirA fusion is coexpressed mitochondria. Although the vast majority with ribosomes that contain a biotin accep- are targeted to only a single organelle, they tor . Ribosomes that come in close observed one prominent exception, Osm1, npg contact with the biotin ligase are thus bio- which is targeted to both. By analyzing their tinylated. This subset of ribosomes is then data, they found that Osm1 has two transla- subjected to streptavidin pulldown and ribo- tional start sites, which produce two forms some profiling. of the protein with distinct targeting signals. Although the method is an extension of an These results demonstrate how precisely existing approach, Weissman notes that its cells can regulate the compartmentalization development was not without its challenges, of proteins. particularly for controlling the timing of bio- As a whole, this work highlights the power tinylation. This required finding conditions of proximity-specific ribosome profiling in in which the cells were growing happily but uncovering the secrets of local translation on biotin levels were too low to label ribosomes, the global scale. Future use of this technique appropriately pulsing in biotin, and effec- should greatly enhance our understanding of tively quenching biotinylation after cell lysis. how cells such as neurons compartmentalize The team studied local translation in the reactions to ensure proper protein function. endoplasmic reticulum (ER) and the mito- Rita Strack chondrial outer membrane. The method RESEARCH PAPERS allowed them to shed light on many ques- Jan, C.H. et al. Principles of ER cotranslational tions regarding cotranslational translocation translocation revealed by proximity-specific ribosome profiling. Science 346, 1257521 (2014). of membrane proteins. Williams, C.C. et al. Targeting and plasticity of First, the researchers found that most mitochondrial proteins revealed by proximity-specific secretory proteins are targeted to the ER ribosome profiling. Science 346, 748–751 (2014).

nature methods | VOL.12 NO.1 | JANUARY 2015 | 17