Cytosolic ribosomes on the surface of mitochondria

Moon

et al. 2023

JIPB

Protein biogenesis is a complex process, and complexity is greatly increased in eukaryotic cells through specific targeting of proteins to different organelles. To direct targeting, organellar proteins carry an organelle‐specific targeting signal for recognition by organelle‐specific import machinery. However, the situation is confusing for transmembrane domain (TMD)‐containing signal‐anchored (SA) proteins of various organelles because TMDs function as an endoplasmic reticulum (ER) targeting signal. Although ER targeting of SA proteins is well understood, how they are targeted to mitochondria and chloroplasts remains elusive. Here, we investigated how the targeting specificity of SA proteins is determined for specific targeting to mitochondria and chloroplasts. Mitochondrial targeting requires multiple motifs around and within TMDs: a basic residue and an arginine‐rich region flanking the N‐ and C‐termini of TMDs, respectively, and an aromatic residue in the C‐terminal side of the TMD that specify mitochondrial targeting in an additive manner. These motifs play a role in slowing down the elongation speed during translation, thereby ensuring mitochondrial targeting in a co‐translational manner. By contrast, the absence of any of these motifs individually or together causes at varying degrees chloroplast targeting that occurs in a post‐translational manner.

Section: Discussionsupporting

confidence: 87%

Translation rate underpins specific targeting of N‐terminal transmembrane proteins to mitochondria

Moon

et al. 2023

JIPB

“…To investigate which nuclear-encoded mitochondrial transcripts are likely to be located on the surface of mitochondria and hence participate in localized translation (Figure 1A), we established a new protocol for biochemical fractionation (Figure 1B) in zebrafish larvae. Although this approach largely adopts a strategy that was employed to obtain intact mitochondria with associated ribosomes in yeast 17 , it was optimized to obtain a membrane-bound fraction containing mitochondria and ER together with their associated ribosomes. Preservation of ER in this fraction allows us to perform more global analysis of membrane associated nuclear-encoded mitochondrial transcripts, as ER and mitochondria are highly interconnected in the cell.…”

Section: Resultsmentioning

confidence: 99%

“…Whereas, the localized translation that occurs at the surface of mitochondria may be directly coupled with the translocation of proteins into organelle. (B) Biochemical fractionation of 5dpf zebrafish larvae based on strategy to obtain intact mitochondria with associated ribosomes in yeast 17 . Isolation of mitochondria with associated ribosomes was performed using a ribosome-friendly buffer (IB-M) containing MgCl 2 , cycloheximide (CHX) and RNAse inhibitors.…”

Section: Resultsmentioning

confidence: 99%

“…As a consequence, numerous studies have indicated the presence of mRNAs encoding mitochondrial proteins, either on the mitochondrial surface or in its close proximity 9–13 . Moreover, mitochondria associated mRNAs have been proven to be active templates for protein synthesis 14 , whereas the cytosolic ribosomes observed at OMM 15,16 are able to interact with the TOM complex 17 . Thus, in living cells the translation of nuclear-encoded proteins can occur at the surface of mitochondria and might be directly coupled to their import into this organelle 14,18 .…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Profiling subcellular localization of nuclear-encoded mitochondrial gene products in zebrafish

Uszczyńska-Ratajczak

Sugunan

Kwiatkowska

et al. 2021

Preprint

Self Cite

Most mitochondrial proteins are encoded by nuclear genes, synthetized in the cytosol and targeted into the organelle. The import of some, but not all, nuclear-encoded mitochondrial proteins begins with translation of messenger RNAs (mRNAs) on the surface of mitochondria. To characterize the spatial organization of mitochondrial gene products in zebrafish (Danio rerio), we sequenced RNA from different cellular fractions. Our results confirmed the presence of nuclear-encoded mRNAs in the mitochondrial fraction, which in unperturbed conditions, are mainly transcripts encoding large proteins with specific properties, like transmembrane domains. To further explore the principles of mitochondrial protein compartmentalization in zebrafish, we quantified the transcriptomic changes for each subcellular fraction triggered by the chchd4a-/- mutation, causing the disorders in the mitochondrial protein import. Our results indicate that the proteostatic stress further restricts the population of transcripts on the mitochondrial surface, allowing only the largest and the most evolutionary conserved proteins to be synthetized there. We also show that many nuclear-encoded mitochondrial transcripts translated by the cytosolic ribosomes stay resistant to the global translation shutdown. Thus, vertebrates, in contrast to yeast, are not likely to employ localized translation to facilitate synthesis of mitochondrial proteins under proteostatic stress conditions.

The mitochondrial surface receptor Tom70 protects the cytosol against mitoprotein-induced stress

Backes

Bykov

Räschle

et al. 2020

Preprint

SummaryMost mitochondrial proteins are synthesized as precursors in the cytosol and post-translationally transported into mitochondria. The mitochondrial surface protein Tom70 acts at the interface of the cytosol and mitochondria. In vitro import experiments identified Tom70 as targeting receptor, particularly for hydrophobic carriers. Using in vivo methods and high content screens, we revisited the question of Tom70 function and considerably expanded the set of Tom70-dependent mitochondrial proteins. We demonstrate that the crucial activity of Tom70 is its ability to recruit cytosolic chaperones to the outer membrane. Indeed, tethering an unrelated chaperone-binding domain onto the mitochondrial surface complements most of the defects caused by Tom70 deletion. Tom70-mediated chaperone recruitment reduces the proteotoxicity of mitochondrial precursor proteins, in particular of hydrophobic inner membrane proteins. Thus, our work suggests that the predominant function of Tom70 is to tether cytosolic chaperones to the outer mitochondrial membrane, rather than to serve as a mitochondria-specifying targeting receptor.