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A new method for detecting endocytosed proteins.

M S Bretscher1, R Lutter

  • 1Medical Research Council, Laboratory of Molecular Biology, Cambridge, UK.

The EMBO Journal
|December 20, 1988
PubMed
Summary
This summary is machine-generated.

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A novel reagent, DPSgt, enables cell surface protein labeling at 0°C. This method quantifies endocytosis by measuring resistance to reduction after cellular warming, revealing insights into receptor trafficking.

Area of Science:

  • Cell Biology
  • Biochemistry
  • Molecular Biology

Background:

  • Cell surface proteins are crucial for cellular functions.
  • Understanding protein trafficking, including endocytosis, is vital for cell biology.
  • Existing methods for studying endocytosis can be complex or limited.

Purpose of the Study:

  • To introduce a new reagent, DPSgt, for labeling cell surface proteins.
  • To establish a simple method for measuring protein endocytosis.
  • To estimate intracellular receptor pools and assess endocytic uptake.

Main Methods:

  • Development and synthesis of the DPSgt reagent.
  • Labeling of cell surface proteins at 0°C.
  • Disulfide bond cleavage with glutathione for label removal.

Related Experiment Videos

  • Differential reduction assays after temperature shift to measure endocytosis.
  • Quantification of intracellular transferrin and LDL receptors in K562 cells and fibroblasts.

    • Main Results:

    • DPSgt is a water-soluble reagent with a reactive ester, radioiodinatable tyrosine, and a cleavable disulfide bond.
    • Label removal is efficient at 0°C with glutathione.
    • Endocytosed proteins gain resistance to reduction upon warming to 37°C.
    • Intracellular receptors reside in non-reducing compartments.
    • Non-coated pit-mediated uptake of average cell surface proteins in K562 cells is minimal.

    Conclusions:

    • DPSgt provides a straightforward method for studying cell surface protein endocytosis.
    • The findings suggest that intracellular compartments involved in receptor trafficking are non-reducing.
    • The study quantifies receptor pools and provides insights into the mechanisms of cell surface protein uptake.