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Related Experiment Videos

Multiple folate transport systems in L1210 cells.

J Fan1, K S Vitols, F M Huennekens

  • 1Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA 92037.

Advances in Enzyme Regulation
|January 1, 1992
PubMed
Summary

Researchers synthesized biotin derivatives to purify folate transporters from L1210 cells. They identified distinct characteristics of the microM and nM folate transporters, revealing the microM transporter is integral and non-glycosylated, while the nM transporter is glycosylated and membrane-anchored.

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Background:

  • Folate transporters are crucial for cellular nutrient uptake.
  • L1210 murine leukemia cells express distinct folate transporters with varying affinities.
  • Understanding transporter structure and membrane association is key to their function.

Purpose of the Study:

  • To synthesize and utilize biotinylated folate and methotrexate derivatives for single-step purification of folate transporters.
  • To characterize the structural and membrane association properties of the microM and nM folate transporters in L1210 cells.

Main Methods:

  • Synthesis and purification of biotin-SS-MTX and biotin-SS-folate.
  • Affinity purification using streptavidin-agarose beads.
  • Protein characterization by SDS-PAGE, HPLC, mass spectrometry, and amino acid analysis.

Related Experiment Videos

  • Enzymatic treatments (PNGase F, PI-PLC) to assess glycosylation and membrane anchoring.

    • Main Results:

    • Successfully purified the 43 kDa microM transporter using biotin-SS-MTX.
    • Purified both 43 kDa microM and 39 kDa nM transporters using biotin-SS-folate.
    • The microM transporter is non-glycosylated and integral.
    • The nM transporter is heavily glycosylated and GPI-anchored.
    • Blocked N-termini prevented Edman sequencing for both transporters.

    Conclusions:

    • Biotinylated folate and methotrexate derivatives are effective tools for folate transporter purification.
    • The L1210 microM transporter is a non-glycosylated integral membrane protein.
    • The L1210 nM transporter is a heavily glycosylated protein anchored via a glycosylphosphatidylinositol moiety.