Purification and analysis of TIP47 function in Rab9-dependent mannose 6-phosphate receptor trafficking

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Alondra Schweizer Burguete, Ulf Sivars, Suzanne Pfeffer

    Methods in Enzymology
    |February 14, 2006
    PubMed

    Insights

    Tail interacting protein of 47 kDa (TIP47) is crucial for receptor transport. A specific mutation enhances TIP47 binding, stabilizing receptors in cells.

    Area of Science:

    • Cell biology
    • Molecular transport mechanisms
    • Protein-protein interactions

    Background:

    • Tail interacting protein of 47 kDa (TIP47) is a cytosolic protein.
    • It plays a key role in the transport of mannose 6-phosphate receptors (MPRs) from endosomes to the trans-Golgi network.
    • Rab9 GTPase recruits TIP47 to late endosomes, facilitating MPR binding.

    Purpose of the Study:

    • To investigate the role of TIP47 in MPR transport.
    • To characterize a TIP47 mutation (F236C) that enhances binding affinity to MPRs.
    • To describe methods for purifying TIP47 and monitoring its function in living cells.

    Main Methods:

    • Purification of native and recombinant TIP47 proteins.
    • Development and application of assays to monitor MPR transport in living cells.
    • Characterization of TIP47-MPR binding interactions.

    Main Results:

    • A mutation (F236C) in TIP47 was found to confer enhanced binding affinity to MPR cytoplasmic domains.
    • This mutation stabilizes MPRs within living cells.
    • Protocols for TIP47 purification and functional assays were established.

    Conclusions:

    • TIP47 is essential for efficient MPR transport.
    • The F236C mutation provides a tool to study TIP47 function and MPR stabilization.
    • The described methods facilitate further research into endosomal transport pathways.

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