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Recognition of the 300-kDa mannose 6-phosphate receptor cytoplasmic domain by 47-kDa tail-interacting protein
J G Orsel1, P M Sincock, J P Krise
1Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305-5307, USA.
Abstract:
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.
Insights
Tail-interacting protein (TIP47) binds cation-independent mannose 6-phosphate receptors (CI-MPRs) via a membrane-proximal region. This interaction is conformation-dependent and crucial for receptor transport from endosomes to the Golgi complex.
Area of Science:
- Cell biology
- Molecular biology
- Protein trafficking
Background:
- Tail-interacting 47-kDa protein (TIP47) is essential for the transport of cation-dependent (CD) and cation-independent (CI) mannose 6-phosphate receptors (MPRs) from endosomes to the Golgi.
- TIP47 recognizes a specific signal in the CD-MPR.
Purpose of the Study:
- To elucidate the structural basis of TIP47 interaction with the CI-MPR cytoplasmic domain.
- To identify the specific residues and conformational requirements for TIP47 binding to CI-MPR.
Main Methods:
- Competition binding experiments were used to map TIP47 interaction sites.
- Analysis of CI-MPR cytoplasmic domain fragments to determine binding affinity.
Main Results:
- TIP47 binding to CI-MPR is conformation-dependent and requires membrane-proximal residues (48-74).
- Residues 48-74 are essential but not sufficient for high-affinity binding; the C-terminal region (75-163) influences presentation.
- TIP47 binding site overlaps with AP-2 clathrin adaptor binding site (residues 24-29), but not AP-1 binding site (residues 160-161).
- TIP47 likely recognizes a loop structure involving PPAPRPG sequence and hydrophobic residues.
Conclusions:
- TIP47 associates with the membrane-proximal region of the CI-MPR cytoplasmic domain in a conformation-dependent manner.
- This interaction is distinct from AP-1 binding but shares a region with AP-2 binding, suggesting a role in cargo selection and transport.
- These findings provide a structural framework for understanding TIP47-CI-MPR association in protein trafficking.