Proximity Labeling Uncovers Novel Roles for Pef1 in Yeast Autophagy and DNA Repair

Study Background and Research Question

Cyclin-dependent kinase 5 (Cdk5) is a highly conserved noncanonical kinase, with essential roles in terminal differentiation and disease mechanisms across eukaryotes. In fission yeast (Schizosaccharomyces pombe), the ortholog Pef1 has been previously linked to lifespan regulation and autophagy suppression. However, the precise molecular context of Pef1 activity—its interactome and function during cellular stress—remained poorly defined. Traditional affinity purification methods are limited in capturing transient or condition-specific protein associations. To address these gaps, the study by Zhang et al. (bioRxiv preprint) investigated the Pef1 proximal proteome using a live-cell, enzyme-mediated biotinylation technique.

Key Innovation from the Reference Study

The central methodological advance in this study is the adaptation of APEX2-biotin phenol proximity labeling for use in S. pombe. This approach leverages the peroxidase activity of APEX2 to catalyze the deposition of biotin-tyramide (biotin phenol) on proteins within a defined spatial radius, enabling the capture of both stable and transient interactors. Notably, the protocol was optimized for yeast by including a brief cell wall digestion and inducing autophagy through nutrient deprivation, thus making it possible to interrogate the Pef1 interactome under physiologically relevant conditions.

Methods and Experimental Design Insights

The study utilized a two-step workflow. First, S. pombe strains expressing Pef1 fused to APEX2 were generated. Proximity labeling was initiated by treating cells with biotin-phenol (biotin-tyramide) and hydrogen peroxide, which APEX2 uses to catalyze biotinylation of nearby proteins. To ensure efficient labeling, cells underwent partial cell wall digestion and were incubated in glucose- and nitrogen-free medium to induce autophagy where appropriate. Following labeling, biotinylated proteins were affinity-purified using streptavidin-coated beads and subsequently identified by mass spectrometry.

Protocol Parameters

• Cell wall digestion: A brief enzymatic treatment to increase cell permeability, facilitating entry of biotin-tyramide.
• APEX2-biotinylation induction: Addition of biotin-tyramide and hydrogen peroxide for a short, controlled duration to minimize off-target labeling.
• Autophagy induction: Glucose and nitrogen starvation, mimicking physiological stress conditions relevant to Pef1 function.
• Protein capture: Streptavidin-based affinity purification for downstream mass spectrometry analysis.

These optimized parameters allowed for efficient and condition-specific labeling of proximal proteins in living yeast cells, advancing the application of enzyme-mediated signal amplification in eukaryotic model organisms.

Core Findings and Why They Matter

The proximity labeling approach identified a total of 255 high-confidence Pef1 neighbors in actively growing cells and 177 during autophagy. Among these, a novel interaction between Pef1 and Rad24—a DNA damage response protein—was discovered and validated through reciprocal proximity labeling and co-immunoprecipitation. Functionally, genetic deletion of pef1 partially rescued the DNA damage sensitivity of rad24-deficient cells, indicating that Pef1 modulates the DNA damage response pathway.

Gene ontology analysis revealed that many Pef1-proximal proteins participate in autophagosome expansion, actin dynamics, and vesicle-mediated transport—processes fundamental to cellular adaptation under nutrient stress. The overlap of certain interactors between growth and starvation conditions suggests that Pef1 orchestrates both core and stress-responsive molecular networks. These findings expand the known repertoire of Cdk5 ortholog functions, hinting at conserved regulatory axes between cell cycle kinases, autophagy, and genome maintenance.

Comparison with Existing Internal Articles

Several internal analyses have highlighted the versatility of biotin-tyramide as a tyramide signal amplification reagent for proximity labeling and spatial proteomics. For instance, "Biotin-tyramide (A8011): Data-Driven Solutions for Signal..." underscores the reagent's role in enabling high-sensitivity and reproducible label deposition, which directly parallels the enzyme-mediated approach employed in the reference study. Similarly, "Biotin-tyramide: Amplifying Precision in Biological Imaging" and "Advancing Nuclear Niche Mapping and Signal..." discuss how tyramide-based labeling achieves spatially precise detection, a core requirement for mapping dynamic interactomes such as Pef1's. The reference study exemplifies these principles in a live-cell, condition-specific context, reinforcing the reagent's relevance across model systems and workflow designs.

Limitations and Transferability

While the study demonstrates robust proximity labeling in fission yeast, several limitations remain. The need for partial cell wall digestion may complicate direct translation to other organisms with more complex barriers. Additionally, the temporal resolution is constrained by the kinetics of labeling and stress induction. Although the approach captures transient and weak interactions missed by affinity purification, it cannot distinguish direct binding from spatial proximity alone. Transferability to higher eukaryotes is feasible, as APEX2 and biotin-tyramide chemistries have been adapted to mammalian systems, but protocol optimization will be necessary to account for cell-type specificities and subcellular targeting. The workflow's dependence on horseradish peroxidase (HRP) catalysis and biotin phenol reactivity also warrants careful titration of reagents to balance sensitivity with background labeling.

Research Support Resources

For researchers aiming to replicate or extend proximity labeling workflows in yeast or mammalian cells, Biotin-tyramide (SKU A8011) from APExBIO offers a high-purity, DMSO- and ethanol-soluble biotinylation reagent compatible with both HRP and APEX2-mediated signal amplification. Used as described in the reference study, this reagent supports sensitive and spatially resolved detection in applications including immunohistochemistry, in situ hybridization, and proximity labeling. Protocol optimization for cell wall permeability and labeling duration is recommended to ensure maximal yield and specificity in diverse biological systems.