In-cell structural insights into fungal ER stress responses

By combining cryo-focused ion beam milling with cryo-electron tomography, this study reveals that ER stress in *S. cerevisiae* induces organelle remodeling and a modest increase in inactive 80S ribosomes bound to elongation factors, demonstrating that translational inhibition serves as a mechanism to alleviate ER stress in this yeast.

The Gist

Imagine a cell as a bustling, high-tech factory. Inside this factory, there is a massive, specialized department called the Endoplasmic Reticulum (ER). Think of the ER as the "Quality Control and Packaging" wing. Its main job is to take raw materials (proteins) and fold them into perfect shapes so they can be shipped out to do their jobs.

Usually, this department runs smoothly. But sometimes, things go wrong. Maybe the factory gets too hot, or the raw materials are defective. Suddenly, the ER is flooded with misshapen, broken products. This is called ER Stress. It's like a warehouse where the conveyor belts are jammed with crumpled boxes, and the workers are overwhelmed.

The Factory's Emergency Response

When the ER gets jammed, the cell has a built-in alarm system called the Unfolded Protein Response (UPR). In complex animals (like humans), this alarm has a very aggressive "Plan B": it immediately shuts down the entire factory's production line to stop making more broken products. It's like pulling the main power cord to prevent a fire.

However, scientists have always wondered: What does the yeast cell (a simple, single-celled organism) do? Yeast doesn't have that same "pull the power cord" switch. For a long time, we thought yeast just ignored the jam and kept working, or perhaps just made a few minor adjustments.

The New Discovery: A "Soft Pause" Button

In this new study, researchers used a super-powerful microscope (like a 3D X-ray that can see inside a frozen cell) to take a close-up look at yeast factories under stress. They didn't just look at the big picture; they zoomed in on the tiny machines making the proteins: the ribosomes.

Here is what they found, explained simply:

1. The Warehouse Expands
First, they saw that the ER department physically grew larger. It was like the factory walls were pushed out to create more space to handle the mess. They also saw the cell starting to clean up, using a "recycling truck" (autophagy) to haul away the broken parts.

2. The "Hibernation" Mode
The most exciting discovery was about the ribosomes (the assembly machines).

• Normal times: In a healthy yeast cell, almost all ribosomes are busy, humming along, building proteins. They are like a highway full of cars moving at full speed.
• Stress times: When the ER got jammed, the researchers saw something surprising. About 25% of the ribosomes suddenly stopped working. They didn't break; they just parked themselves in a "hibernation" mode.

Think of it like a traffic jam where 25% of the cars decide to pull over and turn off their engines to save fuel, rather than crashing into the pile-up ahead.

3. The "Sleeping" Machines
The researchers even saw what was holding these ribosomes in hibernation. They found specific "brakes" (proteins called eIF5A, eEF2, and eEF3) clamped onto the machines, keeping them still. This is a clever way to slow down production without shutting the whole factory down. It's a "soft pause" button rather than a hard "off" switch.

4. The Same Rule Applies Everywhere
They checked both the ribosomes floating in the main factory floor (cytosol) and the ones attached to the ER walls. Both groups slowed down at the same time. This means the yeast cell isn't just stopping the delivery trucks; it's slowing down the entire production line to match the ER's reduced capacity.

Why Does This Matter?

For a long time, scientists thought yeast was too simple to have a sophisticated way to handle stress. They thought it just relied on making more helpers to fix the mess.

This study shows that yeast is actually quite smart. When the ER gets overwhelmed, the yeast cell doesn't panic and shut down completely. Instead, it gently slows down the assembly line, putting a quarter of its machines into a "sleep" mode. This gives the ER time to catch up, clean the mess, and expand its capacity without getting completely overwhelmed.

In a nutshell:
When a yeast factory gets too much work to handle, it doesn't just scream and stop everything. It quietly tells 25% of its workers to take a coffee break (hibernate), allowing the rest of the team to clear the backlog and keep the factory running safely. This is a subtle, elegant way of managing stress that we didn't know existed before.

Technical Summary

1. Problem Statement

The Endoplasmic Reticulum (ER) is responsible for folding approximately 30% of the cellular proteome. When misfolded proteins accumulate, the cell activates the Unfolded Protein Response (UPR) to restore homeostasis.

• The Gap: In metazoans (humans/mammals), the UPR reduces the protein folding load primarily by inhibiting mRNA translation initiation (via PERK/eIF2α phosphorylation) and degrading ER-targeted mRNAs (RIDD). However, Saccharomyces cerevisiae (budding yeast) lacks both the PERK pathway and the RIDD mechanism.
• The Question: It has been widely assumed that translational control plays a negligible role in the yeast UPR. This study investigates whether S. cerevisiae employs alternative, perhaps subtle, mechanisms of translational inhibition during ER stress and how this affects the structural state of ribosomes in their native cellular environment.

2. Methodology

The authors employed a high-resolution, in situ structural biology approach to visualize the translational machinery within intact yeast cells under stress.

• Stress Induction: ER stress was chemically induced in S. cerevisiae using:
  • Dithiothreitol (DTT): A reducing agent preventing disulfide bond formation (treated for 45 min and 4 hours).
  • Tunicamycin (Tm): A glycosylation inhibitor (treated for 3 hours).
  • Validation: Stress was confirmed via RT-PCR for HAC1 mRNA splicing and fluorescence microscopy of Ire1p puncta formation.
• Sample Preparation:
  • Cells were plunge-frozen in liquid ethane.
  • Cryo-FIB Milling: Focused Ion Beam milling was used to thin vitrified cells into lamellae (~100–200 nm thick) suitable for electron microscopy. Both manual and automated (AutoLamella) milling strategies were utilized.
• Data Acquisition:
  • Cryo-Electron Tomography (Cryo-ET): Tilt-series were collected on 200 kV (Talos Arctica) and 300 kV (Titan Krios) microscopes.
  • Magnifications: Two resolutions were targeted:
    • Intermediate (~6.3–7.1 Å/pix): For organelle morphology and volume quantification.
    • High (~2.17 Å/pix): For ribosome subtomogram averaging and translational state classification.
• Image Processing & Analysis:
  • Template Matching: Ribosomes were localized using a yeast 80S ribosome template.
  • Subtomogram Averaging (STA): Performed using RELION and M.
  • Classification: 3D classification without alignment was used to segregate ribosomes into distinct translational states (elongating vs. hibernating) and locations (cytosolic vs. ER-bound).
  • Membrane Segmentation: Automated and manual segmentation of ER and vacuolar membranes to quantify volume changes and ribosome localization.

3. Key Contributions

• First In Situ Structural View of Yeast UPR: Provides the first high-resolution structural map of the yeast translational landscape during ER stress, moving beyond biochemical assays to direct visualization.
• Discovery of a Novel Ribosome State (Dec3): Identified a previously uncharacterized decoding state (Dec3) where the elongation factor eIF5A binds to the ribosome during the decoding step, suggesting a role for eIF5A in stabilizing the P-site tRNA earlier in the elongation cycle than previously thought.
• eEF3 in Hibernation: Visualized the binding of eEF3 (a yeast-specific elongation factor) to inactive, hibernating ribosomes, suggesting a new role for eEF3 in ribosome dormancy.
• Quantification of Global vs. Local Inhibition: Demonstrated that translation inhibition in yeast is a global phenomenon affecting both cytosolic and ER-bound ribosomes, rather than being restricted to the ER membrane.

4. Key Results

A. Morphological Changes

• ER Expansion: Upon 4 hours of DTT stress, the cortical ER volume expanded approximately 7.4-fold, accompanied by the formation of parallel membrane sheets. This confirms the UPR's role in increasing folding capacity.
• Autophagy Activation: Vacuoles showed distinct morphological changes, including membrane invaginations and the uptake of ribosomes into autophagic bodies, indicating active recycling of the translation machinery.

B. Translational State Distribution

• Baseline (Non-stressed): ~95% of ribosomes were in active elongation states. Seven distinct states were resolved:
  • Six elongating states (Dec1, Dec2, Dec3, Pre, Rot2+, TL).
  • One inactive/hibernating state (Hib).
• Stress Response:
  • Increase in Hibernation: ER stress caused a significant increase in the relative abundance of inactive 80S ribosomes (hibernating state) from ~5% (control) to ~25% after 4 hours of stress.
  • Decrease in Elongation: This increase occurred at the expense of major elongating states (specifically Dec2 and Pre).
  • Global Effect: The increase in hibernation was observed in both the cytosol and at the ER membrane, indicating that the reduction in co-translational protein import is a consequence of global translation inhibition, not an ER-specific block.

C. Structural Insights into Factors

• eIF5A: Found bound to the ribosome in the Dec3 state (stabilizing P-site tRNA during decoding) and in the Hibernating state.
• eEF2: Observed bound to hibernating ribosomes.
• eEF3: A distinct density bridging the 40S and 60S subunits on hibernating ribosomes was identified and fitted as eEF3, suggesting it acts as a hibernation factor in yeast.
• ER-Translocon: Ribosomes bound to the ER translocon (Sec61 complex) were successfully resolved, showing interactions with Sec61p loops and Sss1p.

D. Vacuolar Ribosomes

• Ribosomes found inside vacuoles (autophagic bodies) after long-term stress were predominantly in the hibernating state, suggesting that autophagy may selectively degrade or sequester dormant ribosomes to reduce the folding load.

5. Significance

• Revising the Yeast UPR Model: The study challenges the dogma that translational control is negligible in yeast. It demonstrates that S. cerevisiae does inhibit translation during ER stress, albeit more modestly (~25% hibernation) compared to the near-complete shutdown seen in mammalian cells.
• Mechanism of Inhibition: The data suggests that yeast relies on a combination of general stress response pathways (e.g., Gcn2p/PKA activation) and UPR transcriptional programs to modulate translation initiation, rather than the specific PERK/eIF2α or RIDD pathways found in metazoans.
• Factor Function: The structural visualization of eIF5A and eEF3 on hibernating ribosomes provides new mechanistic hypotheses for how yeast regulates ribosome dormancy and recycling.
• Methodological Benchmark: The work showcases the power of Cryo-FIB/Cryo-ET in resolving dynamic cellular processes and macromolecular complexes in their native context, setting a standard for future in situ structural biology studies in fungi.