Recognition of the 300-kDa mannose 6-phosphate receptor cytoplasmic domain by 47-kDa tail-interacting protein

AUTOR(ES)
FONTE

The National Academy of Sciences

RESUMO

Tail-interacting 47-kDa protein (TIP47) binds the cytoplasmic domains of the cation-dependent (CD) and cation-independent (CI) mannose 6-phosphate receptors (MPRs) and is required for their transport from endosomes to the Golgi complex. TIP47 recognizes a phenylalanine-tryptophan signal in the CD-MPR. We show here that TIP47 interaction with the 163-residue CI-MPR cytoplasmic domain is highly conformation dependent and requires CI-MPR residues that are proximal to the membrane. CI-MPR cytoplasmic domain residues 1–47 are dispensable, whereas residues 48–74 are essential for high-affinity binding. However, residues 48–74 are not sufficient for high-affinity binding; residues 75–163 alone display weak affinity for TIP47, yet they contribute to the presentation of residues 48–74 in the intact protein. Independent competition binding experiments confirm that TIP47 interacts with the membrane-proximal portion of the CI-MPR cytoplasmic domain. TIP47 binding is competed by the binding of the AP-2 clathrin adaptor at (and near) residues 24–29 but not by AP-1 binding at (and near) residues 160–161. Finally, TIP47 appears to recognize a putative loop generated by the sequence PPAPRPG and other hydrophobic residues in the membrane-proximal domain. Although crystallography will be needed to define the precise interaction interface, these data provide an initial structural basis for TIP47–CI-MPR association.

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