Proximity labelling of the BAK macropore uncovers a new role for SLC35A4-MP in mitochondrial dynamics

This study employs TurboID proximity labeling to characterize the dynamic BAK proximal proteome during mitochondrial apoptosis, revealing that the microprotein SLC35A4-MP modulates OPA1 processing and accelerates mitochondrial fragmentation, while the MICOS complex destabilizes as the apoptotic pore forms.

The Gist

Imagine your cells are bustling cities, and inside every building (cell) are tiny power plants called mitochondria. These power plants don't just generate energy; they also hold a "kill switch" that can shut down the entire city if things go wrong. This is called apoptosis (programmed cell death).

For a long time, scientists knew how the "kill switch" was flipped, but they didn't fully understand the messy construction work happening inside the power plant right before it shuts down. Specifically, they didn't know how the inner walls of the power plant (the inner mitochondrial membrane) crumble to release dangerous "debris" (DNA) that can cause inflammation.

This paper is like a team of detectives using a high-tech "glow-in-the-dark" camera to watch exactly what happens inside the power plant during this demolition. Here is the story of their discovery, broken down simply:

1. The "Glow-in-the-Dark" Spy (TurboID)

The researchers needed to see which proteins hang out near the "kill switch" (a protein called BAK) as the cell starts to die.

• The Analogy: Imagine the BAK protein is a VIP at a party. The scientists attached a special "glow-in-the-dark" marker (called TurboID) to the VIP.
• The Trick: This marker is like a super-fast spray paint. It instantly paints a tiny dot on anyone standing close to the VIP. By taking photos at different times, they could see who moved closer to the VIP as the party (the cell) started to fall apart.

2. The Collapse of the "Support Beams" (MICOS)

As the cell started dying, the scientists noticed something interesting. A group of proteins called MICOS, which act like the steel support beams holding the inner walls of the power plant together, started to fall apart and drift away from the VIP.

• The Analogy: Think of the power plant as a cathedral with beautiful, folded stained-glass windows (cristae). The MICOS complex is the scaffolding holding those windows in place. As the cell decides to die, the scaffolding is deliberately taken down so the windows can break and release their contents.

3. The Discovery of the "Tiny Foreman" (SLC35A4-MP)

While watching the chaos, the scientists found a very small, previously overlooked protein hanging out right next to the VIP. They named it SLC35A4-MP.

• What is it? It's a "microprotein"—basically a tiny, 103-piece Lego structure. It's so small it was almost missed!
• The Job: This tiny foreman seems to be in charge of a specific task: managing the "glue" that holds the power plant's walls together. That glue is a protein called OPA1.

4. The "Glue" Problem

The scientists realized that SLC35A4-MP acts like a regulator for the OPA1 glue.

• Normal Situation: OPA1 comes in two forms: "Long" (strong glue) and "Short" (weak glue). You need a balance to keep the walls stable but flexible.
• The Discovery: When SLC35A4-MP is present, it keeps the "Long" glue strong. But when the cell is stressed, SLC35A4-MP moves away, and the "Long" glue gets chopped up into "Short" glue.
• The Result: Without the "Long" glue, the power plant walls become rigid and slow to break.

5. The "Traffic Jam" Effect

To test this, the scientists removed the tiny foreman (SLC35A4-MP) from the cells.

• The Analogy: Imagine a city trying to evacuate during a fire. Normally, the doors open quickly, and everyone gets out. But without the tiny foreman, the doors get stuck. The power plant tries to break apart (fragment), but it moves in slow motion.
• The Consequence: The cell still dies, but it takes longer. Because the "debris" (DNA) is released slowly, it might trigger a different kind of alarm in the body—like a false fire alarm that causes inflammation instead of a clean shutdown.

Why Does This Matter?

This research is a big deal for a few reasons:

1. New Players: It shows that even tiny, "micro" proteins play huge roles in life-and-death decisions for our cells.
2. Inflammation: If the cell dies too slowly or messily, it releases DNA that the body mistakes for a virus or bacteria. This can lead to chronic inflammation, which is linked to diseases like Lupus and Parkinson's.
3. Fine-Tuning: The cell isn't just a "on/off" switch; it's a dimmer switch. This tiny foreman helps the cell decide how fast to shut down, ensuring the process is clean and doesn't accidentally set off the body's immune system.

In a nutshell: The scientists used a glowing camera to find a tiny, previously unknown protein that acts as a traffic controller for a cell's self-destruct sequence. Without this tiny controller, the cell's self-destruct mechanism gets stuck in "slow motion," potentially causing unnecessary inflammation in the body.

Technical Summary

1. Problem Statement

Mitochondrial outer membrane permeabilization (MOMP) by the pro-apoptotic proteins BAK and BAX is a critical checkpoint in intrinsic apoptosis. This process leads to the formation of "macropores" that facilitate the herniation of the inner mitochondrial membrane (IMM) into the cytosol, releasing mitochondrial DNA (mtDNA). While the release of mtDNA can trigger inflammatory responses (e.g., in Systemic Lupus Erythematosus or Parkinson's disease), the specific molecular mechanisms governing the dynamic remodeling of the IMM and the protein complexes involved during this herniation event remain poorly understood. Specifically, it is unclear how integral IMM proteins, such as those in the MICOS complex and OPA1, are reorganized or recruited during the stress of IMM herniation.

2. Methodology

The authors employed a multi-faceted approach combining proximity labeling proteomics, genetic manipulation, and live-cell imaging:

• Proximity Labeling (TurboID): The study utilized a TurboID-BAK fusion protein stably expressed in Mcl1⁻/⁻Bak⁻/⁻Bax⁻/⁻ mouse embryonic fibroblasts (MEFs). TurboID is a fast-acting biotin ligase that biotinylates proteins within a ~10 nm radius within 10 minutes.
  • Experimental Design: Cells were treated with the BH3 mimetic ABT-737 to induce BAK activation. To isolate the herniation phase from downstream caspase execution, a pan-caspase inhibitor (QVD-OPh) was used.
  • Time-Course Sampling: Samples were collected at 0 h (steady state), 1 h (pre-MOMP), 2 h (post-MOMP), and 4 h (post-herniation) to capture temporal changes in the BAK proximal proteome.
• Mass Spectrometry (MS):
  • Proximity Proteomics: Biotinylated proteins were enriched via streptavidin beads and analyzed via LC-MS/MS (DIA mode) to identify proteins moving into proximity with the BAK pore.
  • Affinity Enrichment MS (AE-MS): To characterize the interactome of the microprotein SLC35A4-MP, FLAG-tagged SLC35A4-MP was expressed in U2OS cells, immunoprecipitated, and analyzed by MS.
  • Whole-Cell Proteomics: Label-free proteomics was performed on Wild-Type (WT) vs. SLC35A4-MP Knockout (KO) cells to assess global proteomic changes.
• Genetic Manipulation:
  • CRISPR/Cas9 was used to generate SLC35A4-MP KO U2OS cells.
  • Inducible re-expression systems (doxycycline-inducible SLC35A4-MP-FLAG) were used for rescue experiments.
• Imaging and Structural Analysis:
  • Live-Cell Microscopy: Cells expressing TOM20-HaloTag were imaged to track mitochondrial fragmentation and mtDNA release in real-time.
  • Electron Microscopy (TEM): Used to assess cristae architecture and mitochondrial morphology.
  • Blue-Native (BN)-PAGE & Western Blotting: Used to analyze the assembly status of the MICOS complex and OPA1 isoform processing (L-OPA1 vs. S-OPA1).

3. Key Contributions

• Temporal Mapping of the BAK Proximal Proteome: The study provides the first comprehensive, time-resolved map of proteins interacting with the BAK macropore during the specific window of IMM herniation.
• Discovery of SLC35A4-MP's Role: Identification of SLC35A4-MP, a 103-amino acid microprotein encoded by an upstream open reading frame (uORF), as a novel regulator of mitochondrial dynamics during stress.
• Mechanistic Insight into OPA1 Regulation: Elucidation of a direct functional link between SLC35A4-MP and the processing of OPA1 isoforms, specifically the regulation of the long isoform (L-OPA1 'a').
• MICOS Dynamics: Demonstration that the MICOS complex (Mitochondrial Contact Site and Cristae Organising System) destabilizes and loses proximity to the BAK pore as apoptosis proceeds.

4. Key Results

A. Dynamics of the BAK Proximal Proteome

• Steady State: The BAK pore is proximal to proteins involved in mitochondrial membrane organization, fission, and transport.
• During Apoptosis:
  • Loss of MICOS: There is a significant temporal decline in the enrichment of MICOS complex subunits (MIC60, MIC27, MIC19, etc.) and SAM complex proteins near the BAK pore. BN-PAGE confirmed the disassembly of the MICOS complex within 1.5 hours of apoptosis induction.
  • Recruitment of IMM Proteins: Four specific IMM proteins showed increased proximity to the BAK pore over time: Abcb8 (Mitosur), Atad3, Pnlpla8, and SLC35A4-MP.

B. Characterization of SLC35A4-MP

• Localization: SLC35A4-MP localizes to the IMM and forms distinct foci, colocalizing with TOM20.
• Steady-State Phenotype: Loss of SLC35A4-MP (KO) did not alter mitochondrial ultrastructure, cristae architecture, or the global steady-state proteome.
• Interactome: SLC35A4-MP stably interacts with the MICOS-MIB complex and OPA1 at steady state.
• Effect on OPA1 Processing:
  • In SLC35A4-MP KO cells, there is a specific reduction in the L-OPA1 'a' isoform and a corresponding increase in the S-OPA1 'e' isoform.
  • This leads to an accumulation of soluble OPA1 complexes.
  • Re-expression of SLC35A4-MP restores the L-OPA1 'a' isoform levels.

C. Impact on Mitochondrial Dynamics

• Delayed Fragmentation: Live-cell imaging revealed that SLC35A4-MP KO cells exhibit a delayed mitochondrial fragmentation response to apoptotic stimuli (ABT-737/S63845) compared to WT.
• General Stress Response: This delay is not specific to apoptosis; SLC35A4-MP KO mitochondria also resist fragmentation induced by CCCP (uncoupler) and H₂O₂ (oxidative stress).
• Herniation: Despite the delay, SLC35A4-MP KO cells eventually undergo IMM herniation and mtDNA release, indicating SLC35A4-MP fine-tunes the kinetics of the response rather than being an absolute requirement for the event.

5. Significance

• Novel Regulatory Mechanism: The study identifies SLC35A4-MP as a critical "fine-tuner" of mitochondrial dynamics. It suggests that SLC35A4-MP protects the L-OPA1 'a' isoform from proteolytic cleavage, thereby maintaining a more flexible mitochondrial network that can rapidly fragment under stress.
• Microprotein Function: It highlights the importance of uORF-encoded microproteins in regulating complex cellular processes like apoptosis and mitochondrial quality control, a field often overlooked in traditional proteomics.
• Inflammatory Implications: By modulating the speed of mitochondrial fragmentation and mtDNA release, SLC35A4-MP may influence the immunogenicity of cell death. Slower fragmentation could alter the timing of mtDNA exposure to cytosolic sensors, potentially impacting the severity of inflammatory diseases linked to mtDNA leakage (e.g., SLE, neurodegeneration).
• Therapeutic Potential: Understanding the SLC35A4-MP/OPA1 axis offers new targets for modulating mitochondrial dynamics in diseases characterized by aberrant apoptosis or mitochondrial stress.

In conclusion, this paper utilizes advanced proximity labeling to map the dynamic proteome of the BAK pore, revealing that the microprotein SLC35A4-MP acts as a crucial regulator of OPA1 processing and mitochondrial fragmentation kinetics during apoptotic stress.