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Identification of a cold-sensitive step in the mechanism of modeccin action
Abstract:
Modeccin is a toxic lectin that arrests protein synthesis in mammalian cells by catalytically inactivating 60 S ribosomes. To interact with 60 S ribosomes, the catalytic subunit of modeccin must pass through a membrane and enter the cytosol. Two known steps in the mechanism of modeccin action are the receptor-mediated internalization of the toxin into vesicles and a second step that requires a low pH within the vesicles. We report here another step required for modeccin to arrest protein synthesis, identified because this step was blocked at 15 degrees C. Modeccin traveling from cell surface receptors to the cytosol at 37 degrees C passed the low pH step within vesicles in a minimum time of 15 min after endocytosis and reached the cold-sensitive step 15 min later. There was no effect on protein synthesis until about 45 min after modeccin had passed the cold-sensitive step, suggesting that the toxin was still within vesicles at the time of the cold-sensitive event. The low temperature at which modeccin failed to reach the cytosol correlated with an apparent low temperature block in the transfer of endocytosed modeccin to lysosomes. The possibility is discussed that modeccin does not penetrate to the cytosol directly from endocytic vesicles.
Insights
Modeccin, a toxic lectin, requires a cold-sensitive step to enter the cytosol and inhibit protein synthesis. This step appears linked to endocytosed toxin transport, not direct vesicle penetration.
Area of Science:
- Cell Biology
- Molecular Toxicology
- Protein Synthesis Inhibition
Background:
- Modeccin is a toxic lectin that inhibits protein synthesis by inactivating 60S ribosomes.
- Entry into the cytosol requires passage through membranes after receptor-mediated endocytosis.
- Known steps involve vesicle internalization and a low pH requirement.
Purpose of the Study:
- To identify additional steps in modeccin's mechanism of action.
- To investigate the role of temperature sensitivity in modeccin's cellular entry.
Main Methods:
- Utilized temperature shifts (15°C vs. 37°C) to identify cold-sensitive steps.
- Tracked modeccin's intracellular transport post-endocytosis.
- Monitored protein synthesis inhibition as an endpoint.
Main Results:
- A cold-sensitive step, blocked at 15°C, was identified after the low pH requirement.
- This cold-sensitive step occurred approximately 30 minutes post-endocytosis.
- Inhibition of protein synthesis was delayed until 45 minutes after the cold-sensitive step, suggesting continued vesicle confinement.
Conclusions:
- Modeccin's journey to the cytosol involves a critical cold-sensitive step.
- This step correlates with the transport of endocytosed modeccin, potentially to lysosomes.
- Findings suggest modeccin may not directly exit endocytic vesicles to reach the cytosol.