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Evidence for penetration of diphtheria toxin to the cytosol through a prelysosomal membrane
Abstract:
To kill mammalian cells, diphtheria toxin must be endocytosed and encounter a low pH within intracellular vesicles. The low pH initiates penetration of the catalytically active A fragment of the toxin through a membrane and into the cytosol where the A fragment arrests protein synthesis. To investigate whether penetration occurred through a prelysosomal or a lysosomal membrane, we studied the effect of low temperature on the entry of the toxin into the cytosolic and lysosomal compartments. The toxin arrested protein synthesis at 15 degrees C, indicating entry into the cytosol; however, access to lysosomes was apparently blocked at 15 degrees C, suggesting that the toxin had encountered a low pH before reaching lysosomes and had penetrated a prelysosomal membrane. To further investigate the possibility of prelysosomal acidification, we measured the time required for the toxin to encounter a low pH after endocytosis. Acidification occurred within 3 to 4 min after the toxin was internalized into vesicles. This interval is consistent with prelysosomal acidification since the entry of endocytosed ligands into secondary lysosomes usually takes more than 3 to 4 min.
Insights
Diphtheria toxin enters mammalian cells and arrests protein synthesis at low temperatures, suggesting prelysosomal membrane penetration. This study investigates the toxin
Area of Science:
- Cell Biology
- Molecular Toxicology
- Membrane Transport
Background:
- Diphtheria toxin requires endocytosis and low pH for cell entry and toxicity.
- The catalytically active A fragment penetrates the vesicle membrane to reach the cytosol.
- Understanding the specific membrane penetration site (prelysosomal vs. lysosomal) is crucial.
Purpose of the Study:
- To determine whether diphtheria toxin penetrates a prelysosomal or lysosomal membrane.
- To investigate the effect of low temperature on toxin entry into cellular compartments.
Main Methods:
- Studied the effect of 15°C on diphtheria toxin's ability to arrest protein synthesis (cytosolic entry).
- Assessed toxin access to lysosomes at low temperatures.
- Measured the time for the toxin to encounter a low pH after endocytosis.
Main Results:
- Diphtheria toxin arrested protein synthesis at 15°C, confirming cytosolic entry.
- Lysosomal access was blocked at 15°C, indicating penetration before reaching lysosomes.
- Low pH was encountered 3-4 minutes post-endocytosis, consistent with prelysosomal acidification.
Conclusions:
- Diphtheria toxin likely penetrates a prelysosomal membrane.
- Prelysosomal acidification plays a critical role in diphtheria toxin entry into the cytosol.
- Low temperature selectively inhibits lysosomal trafficking but not early endosomal penetration.