SRI-41315: A Detailed Overview of eRF1 Degradation Pathways

Cells regulate protein synthesis to ensure proper translation termination, with eukaryotic release factor 1 (eRF1) playing a key role in this process. Understanding eRF1 degradation pathways provides insight into broader gene expression regulation. Examining these mechanisms highlights their impact on translation termination efficiency and cellular homeostasis.

Structural Features

The structure of eRF1 is fundamental to its function in translation termination, with distinct domains enabling stop codon recognition and polypeptide release. eRF1 consists of three domains, each with specialized roles. The N-terminal (N) domain recognizes stop codons, containing conserved motifs that interact with the ribosomal decoding center. Structural studies show this domain mimics tRNA, allowing it to engage the ribosome similarly to aminoacyl-tRNA.

The middle (M) domain coordinates the release of nascent polypeptide chains. It contains the universally conserved GGQ motif, essential for catalyzing peptide hydrolysis at the ribosomal peptidyl transferase center. Mutagenesis studies confirm alterations in this motif impair termination efficiency. The M domain also interacts with eRF3, a GTPase that enhances eRF1 activity by promoting ribosomal binding and release. Conformational changes in the M domain, induced by eRF3 and GTP hydrolysis, are necessary for efficient termination.

The C-terminal (C) domain contributes to ribosome binding and stability, ensuring proper positioning within the translation machinery. This domain, though less conserved across species, retains structural elements facilitating interactions with ribosomal components and regulatory factors. Nuclear magnetic resonance spectroscopy highlights its role in stabilizing eRF1’s architecture and preventing premature dissociation from the ribosome. Post-translational modifications such as phosphorylation and ubiquitination in this region suggest a regulatory function influencing eRF1 turnover and activity.

Mechanism Of eRF1 Degradation

eRF1 degradation is regulated through ubiquitin-proteasome and autophagy-lysosome pathways, ensuring dynamic control of its levels. Ubiquitination marks eRF1 for proteasomal degradation, with specific E3 ubiquitin ligases facilitating ubiquitin attachment. This modification alters eRF1’s stability and interaction with ribosomal complexes, directing it toward proteasomal processing. Proteomic studies identify candidate E3 ligases, including the anaphase-promoting complex (APC) and cullin-RING ligases, as potential regulators of eRF1 turnover. These ligases selectively target eRF1 under conditions requiring translation termination adjustments, such as cellular stress or nutrient fluctuations.

eRF1 ubiquitination is influenced by sequence-specific degrons and conformational cues exposing lysine residues for ubiquitin conjugation. Modifications in the C-terminal domain affect ubiquitination efficiency, with phosphorylation acting as a priming mechanism for degradation. Inhibiting specific kinases or phosphatases alters eRF1 turnover rates, highlighting phosphorylation as a key determinant of degradation kinetics. Interactions with regulatory proteins such as eRF3 also influence eRF1’s susceptibility to degradation, as eRF3 binding can shield or expose degradation signals depending on its nucleotide-bound state.

Beyond proteasomal degradation, eRF1 can be eliminated through autophagy, particularly during prolonged translational stalling or ribosome-associated quality control activation. Autophagy-related degradation often involves eRF1 incorporation into ribonucleoprotein aggregates, leading to lysosomal clearance. This pathway is more pronounced under stress conditions such as amino acid starvation or oxidative damage. Autophagy inhibitors cause eRF1-containing aggregates to accumulate, confirming autophagic degradation as a complementary mechanism for maintaining translation termination fidelity.

Influence On Protein Termination Pathways

eRF1 degradation directly impacts translation termination efficiency and fidelity. Properly controlled eRF1 levels ensure precise termination, preventing disruptions in ribosome recycling. Reduced eRF1 availability slows termination, increasing the likelihood of stop codon readthrough, which alters protein isoform expression and affects cellular adaptations.

Changes in eRF1 degradation also influence competition between termination and mRNA surveillance mechanisms such as nonsense-mediated decay (NMD). When termination efficiency declines, premature termination codons become more susceptible to NMD activation, leading to selective degradation of aberrant transcripts. This balance prevents defective protein accumulation. However, excessive eRF1 degradation can exacerbate transcript loss, while stabilization can suppress NMD, allowing premature stop codon-containing transcripts to persist.

Altered eRF1 turnover affects cellular responses to environmental and metabolic changes. Cells adjust eRF1 degradation to regulate translation rates during nutrient deprivation or oxidative stress. This mechanism enables selective translation of stress-responsive proteins while limiting non-essential polypeptide production. Disrupting eRF1 degradation pathways impacts cellular fitness, particularly in rapidly dividing cells requiring precise translation control. Certain cancer cells exhibit modified eRF1 stability, influencing their ability to bypass normal termination constraints and sustain oncogenic protein expression.

Observations In Cell-Based Assays

Cell-based assays provide insights into eRF1 regulation and degradation. Fluorescence-based reporter systems track eRF1 dynamics, with tagged eRF1 variants allowing real-time visualization of turnover. Time-lapse microscopy reveals eRF1 stability decreases under proteotoxic stress, suggesting active modulation in response to environmental cues.

Western blot analyses of cell lysates treated with proteasome inhibitors, such as MG132 and bortezomib, show significant accumulation of ubiquitinated eRF1, confirming the ubiquitin-proteasome system as a primary degradation pathway. Co-immunoprecipitation assays indicate eRF1 interacts with distinct E3 ubiquitin ligases in a context-dependent manner, with certain ligases preferentially targeting eRF1 under nutrient-starved conditions. Cycloheximide chase experiments demonstrate eRF1 has a shorter half-life in actively dividing cells, while its stability increases in quiescent or differentiated cells, linking eRF1 turnover to cell cycle progression.