Tom20 Mediates Localization of mRNAs to Mitochondria in a Translation-Dependent Manner

mRNAs encoding mitochondrial proteins are enriched in the vicinity of mitochondria, presumably to facilitate protein transport. A possible mechanism for enrichment may involve interaction of the translocase of the mitochondrial outer membrane (TOM) complex with the precursor protein while it is translated, thereby leading to association of polysomal mRNAs with mitochondria. To test this hypothesis, we isolated mitochondrial fractions from yeast cells lacking the major import receptor, Tom20, and compared their mRNA repertoire to that of wild-type cells by DNA microarrays. Most mRNAs encoding mitochondrial proteins were less associated with mitochondria, yet the extent of decrease varied among genes. Analysis of several mRNAs revealed that optimal association of Tom20 target mRNAs requires both translating ribosomes and features within the encoded mitochondrial targeting signal. Recently, Puf3p was implicated in the association of mRNAs with mitochondria through interaction with untranslated regions. We therefore constructed a tom20Δ puf3Δ double-knockout strain, which demonstrated growth defects under conditions where fully functional mitochondria are required. Mislocalization effects for few tested mRNAs appeared stronger in the double knockout than in the tom20Δ strain. Taken together, our data reveal a large-scale mRNA association mode that involves interaction of Tom20p with the translated mitochondrial targeting sequence and may be assisted by Puf3p.mRNA localization to distinct cellular compartments is important for the efficiency and specificity of the translation process. Synthesis of proteins at their sites of action may decrease the likelihood of ectopic protein expression and facilitate assembly of large multiprotein complexes. Two general modes for mRNA localization are known. The first, which is common for endoplasmic reticulum (ER)-associated mRNAs, necessitates translation of a short region of the protein (the signal peptide). The signal is recognized by the signal recognition particle as it emerges from the ribosome exit tunnel, and the complex that includes the mRNA, ribosome, and signal recognition particle is targeted to the ER (18). As an outcome of this process, mRNAs that encode proteins destined for the ER and the secretory pathway are associated with this compartment (7). The second mode for mRNA localization occurs prior to translation and in many cases prevents initiation of protein synthesis. Sequences or structural elements of the mRNA are bound by RNA-binding proteins, and these interact with transport factors, which direct the mRNA to its destination (5, 35, 42). Genome-wide studies indicate that localization by either mode is a broad phenomenon that encompasses many mRNAs and various cellular destinations (6, 21, 32, 38). Interestingly, we along with others have recently shown that noncoding regions may also be involved in localization of ER-associated mRNAs (1, 26, 38), demonstrating that these two modes are not mutually exclusive.Most of the mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Various in vitro and in vivo assays led to the widely accepted notion that import may occur posttranslationally, i.e., after the protein is fully synthesized in the cytosol (33). However, mounting evidence also supports a cotranslational import of proteins into the mitochondria. Specifically, polysomes were shown to be associated with the mitochondrial surface, and these translated a distinct set of proteins (12, 19, 20). Moreover, isolated mitochondria are associated with many different mRNAs that encode mitochondrial proteins (28, 46). Elements from both the coding region (the mitochondrial targeting signal [MTS]) and the 3′ untranslated region (UTR) were shown to be important for targeting of some of these mRNAs (4, 29). One model for localization suggests association of the nascent peptide chain (specifically, the N-terminal MTS) with receptors on the mitochondria, coupled to cotranslational insertion of the protein (24). As an outcome of this cotranslational mechanism, polysomal mRNAs become associated with the mitochondria, analogously to what is observed in the ER. However, experimental support for this hypothesis is currently lacking.Recently, Saint-Georges et al. (41) have shown a role for Puf3p in localization of many mRNAs to the mitochondria of Saccharomyces cerevisiae. Puf3p is associated with the mitochondria outer membrane (11), and its role is mediated through interaction with UTRs. This may suggest a translation-independent mode of action. Intriguingly, however, most Puf3 targets appeared to be mislocalized also after treatment with the translation inhibitor cycloheximide (CHX), suggesting that an active translation process is required for their asymmetric localization (41). Moreover, a large number of mRNAs that are not Puf3 targets appeared to be affected from treatments with the translation inhibitors puromycin and cycloheximide (41), further supporting the existence of an additional, translation-dependent mode of mRNA targeting to the mitochondria.The translocase of the mitochondrial outer membrane (TOM complex) is a multiprotein machinery which mediates the import of the vast majority of proteins into the mitochondria (36, 39). Its core protein (Tom40) forms a β-barrel structure and serves as the main component of the import pore. Tom20 is a peripheral component of the TOM complex that functions as a primary receptor for mitochondrial precursor proteins (15). It was hypothesized that protein receptors interact with the incoming polypeptide while it is translated, and this leads to a local increase in mRNA concentration (24). An open question is whether the TOM complex, through Tom20, interacts with polypeptides while they are translated and thereby leads to higher local concentrations of mRNAs near the mitochondria. To address this issue, we analyzed the effects of TOM20 deletion on mRNA association with mitochondrial fractions and the role of the MTS on mRNA localization. We also tested the interactions between Tom20 and Puf3. We found that Tom20 is involved in mitochondrial association of many mRNAs by a process that requires the MTS. Tom20 deletion affects the localization of Puf3p, and a strain with deletions of both Tom20 and Puf3 exhibits a growth defect under conditions that require mitochondrial optimal function.