Related Experiment Videos
Temporal separation of protein toxin translocation from processing events
1Laboratory of Molecular Biology, National Institute of Mental Health, Bethesda, Maryland 20892.
Abstract:
Intoxication of Vero cells by ricin, modeccin, diphtheria toxin (DT), and Pseudomonas exotoxin A requires: 1) binding to cell surface receptors; 2) transport to the cytoplasm; and 3) enzymatic inactivation of a component of the protein synthetic machinery. The kinetic profiles of all four toxins consist of a lag followed by the apparent first-order decrease in protein synthesis. Autoradiographic analysis of DT-intoxicated cell populations has demonstrated that two subpopulations of cells exist during the period of decreasing protein synthesis: one population synthesizing at control levels and the other synthesizing little or no protein (Hudson, T. H., and Neville, D. M., Jr. (1985) J. Biol. Chem. 260, 2675-2680). The present study correlates the autoradiographic data with the rates of protein synthesis decline in cells intoxicated with modeccin, ricin, Pseudomonas exotoxin A, as well DT. In all cases, the first time point which exhibits a decrease in protein synthetic activity also exhibits two subpopulations of cells, one synthesizing protein at control rates and the other synthesizing little or no protein. As the intoxication progresses, cells leave the control population by the rapid cessation of all protein synthesis. These experiments demonstrate that transport of all four toxins to the cytosol is the rate-limiting step during the pseudo first-order decline in protein synthesis. Furthermore, the final step in the transport process (translocation) must result in the release to the cytoplasm of a quantity of toxin sufficient to rapidly inactivate all protein synthesis in that cell. The probability of a translocation event occurring in any cell of the population is established during the lag and remains constant throughout the first-order decrease in protein synthesis. The requirement for acidification during the intoxication by DT, Pseudomonas exotoxin A, or modeccin is restricted to the lag period. Acidification is therefore necessary to establish the probability of translocation, but it is not directly involved in the actual translocation of these toxins. The pseudo first-order passage of DT intoxications through antitoxin and NH4Cl- or monensin-sensitive stages are shown to have the same cellular basis as the pseudo first-order decrease in protein synthesis. A kinetic model is presented which defines the DT intoxication process from one of its earliest events (endocytosis) to its penultimate event (translocation of toxin to the cytosol).(ABSTRACT TRUNCATED AT 400 WORDS)
Insights
The transport of ricin, modeccin, diphtheria toxin (DT), and Pseudomonas exotoxin A to the cytosol is the rate-limiting step in protein synthesis inhibition. Acidification is crucial for establishing translocation probability but not for the translocation itself.
Area of Science:
- Cell Biology
- Molecular Toxicology
- Biochemistry
Background:
- Cellular intoxication by toxins like ricin, modeccin, diphtheria toxin (DT), and Pseudomonas exotoxin A involves receptor binding, cytoplasmic transport, and inhibition of protein synthesis.
- Previous studies indicated two cell subpopulations during toxin intoxication: one active in protein synthesis and another inhibited.
Purpose of the Study:
- To correlate autoradiographic data with protein synthesis decline rates across multiple toxins.
- To elucidate the rate-limiting steps and cellular mechanisms involved in toxin-induced protein synthesis inhibition.
Main Methods:
- Intoxication of Vero cells with ricin, modeccin, DT, and Pseudomonas exotoxin A.
- Kinetic analysis of protein synthesis decline.
- Autoradiographic analysis to identify cell subpopulations with varying protein synthesis activity.
Main Results:
- All four toxins induced a lag phase followed by a pseudo first-order decrease in protein synthesis.
- Autoradiography revealed two distinct cell subpopulations during intoxication: actively synthesizing and inhibited cells.
- Cytosolic transport of toxins was identified as the rate-limiting step, with translocation releasing sufficient toxin to rapidly halt protein synthesis.
Conclusions:
- Cytosolic transport is the rate-limiting step for protein synthesis inhibition by these toxins.
- Acidification is required for establishing translocation probability during the lag phase, not for the translocation event itself.
- A kinetic model for DT intoxication, from endocytosis to translocation, was proposed, highlighting shared cellular bases with protein synthesis decline.