Hibernating ribosomes tether to mitochondria as an adaptive response to cellular stress during glucose depletion

Background

During nutrient starvation, cells have limited energy to spare and therefore they shut down most anabolic processes. Only proteins whose function will help the cells overcome starvation will be translated under such conditions [1].This leaves the cell with many inactive ribosomes, which are targeted for degradation [2, 3]. However, a recent study examining septin assemblies in the fission yeast Schizosaccharomyces pombe (S. pombe) noted that cytosolic ribosomes accumulate on mitochondria in glucose-starved cells, but the authors did not explore this curious finding further [4]. Gemin and colleagues have now made this phenomenon the main subject of their research and present their findings in this preprint. They illuminate the molecular details underlying the sequestration of inactive ribosomes to mitochondria using cryo-electron microscopy (EM) methods in conjunction with biochemical tools.

 

Key findings

1. Ribosomes enter a self-inhibition mode upon prolonged glucose depletion

• • After 7 days of glucose depletion of S. pombe, no active ribosomes could be observed by polysome profiling. In addition, no mRNA or tRNA was found to be associated with purified ribosomes – as analysed by single particle cryoEM – suggesting that indeed no protein synthesis was taking place.
  • The cryoEM structures of ribosomes from glucose-deprived yeast revealed that the P-site, where the peptide-bound tRNA usually interacts with the ribosome during protein synthesis, is blocked by a helix of the large ribosomal subunit. Furthermore, this conformational shift in the helix disrupts an interaction important for translation initiation.

2. Prolonged glucose depletion induces ribosomal tethering to the outer mitochondrial membrane (see Figure below)

• • Using cryo-electron tomography (cryoET), the research team could analyse the cellular structure of glucose-starved S. pombe. They saw that the mitochondrial network was fragmented, and that the resulting round mitochondria were richly decorated with ribosomes, as has been described before [4].
  • To show that ribosomal tethering to the mitochondria is a consequence of glucose-starvation and not mitochondrial fragmentation, the authors inhibited mitochondrial fragmentation in the yeast cells by deleting the mitochondrial fission factor Dnm1. Just like in wild-type cells, ribosomes in Ddnm1 cells associated with the outer mitochondrial membrane upon prolonged glucose depletion, despite the mitochondria being elongated.

3. Ribosomes are arranged into oligomers on the outer mitochondrial membrane and face the membrane with the small ribosomal subunit (see Figure below)

• • The authors analysed the orientation of the ribosomes on the mitochondrial membrane by mapping sub-tomogram averaged electron densities of ribosomes back to the electron tomograms of cell slices.
  • The ribosomes were attached to the membrane via the small ribosomal subunit, an orientation distinct from active ribosomes previously found on mitochondria [5, 6].
  • The ribosomes formed organised clusters on the mitochondrial membrane with the help of previously unknown binding sites mediating dimer, trimer, tetramer and pentamer formation between ribosomes.

4. Ribosomes interact with the outer mitochondrial membrane via the Cpc2/RACK1 subunit

• • Sub-tomogram averaging of the cryo-tomograms could not provide molecular details about the connection between ribosomes and the outer mitochondrial membrane, probably due to high flexibility. The researchers therefore fitted the single-particle cryoEM structures of purified ribosomes into the tomograms.
  • The fit placed the ribosome-associated protein Cpc2 in close proximity to the membrane. Indeed, a Dcpc2 deletion strain did not accumulate ribosomes on mitochondria upon prolonged glucose starvation, suggesting that Cpc2 is the mitochondria-interacting factor of ribosomes.

 

This figure shows how glucose-starved S. pombe cells were sectioned into thin slices by cryo-FIB (A), and how cryoET imaging of these revealed ribosome-decorated mitochondria. Through subtomogram averaging, the mitochondria-tethered ribosomes could be further resolved to determine the location of individual subunits within the ribosome (C). When these higher-resolution ribosomes were mapped back onto a 3D reconstruction of the mitochondrion, all of the ribosomes faced the mitochondrial membrane via their small subunit (coloured in yellow) (D). Figure adapted from the preprint.

 

Hypotheses for the function of hibernating ribosomes on mitochondria during glucose starvation:

1. Ribosomes tethered to fragmented mitochondria protect them from mitophagy and stabilize the mitochondrial potential, thereby averting cell death.
2. Mitochondria-bound ribosomes are waiting for the starvation to be over, at which time they can start to synthesise mitochondrial proteins, directly feeding them into mitochondria, which then will have plenty of resources to power the rest of the cell.

 

What I like about this preprint:

I really enjoyed reading this preprint because it examines such a fascinating phenomenon. I had never heard of ribosomes sitting on mitochondria before and found the cryoET images showing this rather impressive. I also liked that the preprint was not too long and was kept to the point. I hope that there will be more research on this topic and that we can find out more about what these ribosomes are doing on the mitochondria!

 

Questions and comments

1. Do you have any ideas as to what Cpc2’s binding partner in the outer mitochondrial membrane might be? Would it be possible to map translocation complexes of the outer membrane onto the tomograms of mitochondria to determine whether the ribosomes are in close proximity to these, supporting your hypothesis that the hibernating ribosomes are ready to restart mitochondrial protein synthesis upon nutrient repletion?
2. Mammalian mitochondria tend to fuse, rather than fragment, during starvation to protect themselves from mitophagy. Do you have an explanation for why pombe mitochondria do the opposite? Do other yeast species’ mitochondria behave the same as S. pombe’s during starvation?
3. In your preprint, especially in the abstract, it sounds like you propose that the sequestration of ribosomes to mitochondria may confer cell survival. However, in the corresponding data, Cpc2 deletion impacted cell survival even in EMM 2% glucose media (Figure 4B), where cells are not glucose-deprived, mitochondria are not fragmented and ribosomes are not sequestered to mitochondria (Figure S9). Therefore Cpc2’s interaction with the mitochondrial membrane and the associated ribosome sequestration may not be the factors that confer cell survival, but rather it may be another function of Cpc2 important specifically in the EMM medium but not under glucose-deprivation.

 

References

 

doi: https://doi.org/10.1242/prelights.35991

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