Ubiquitin interacts with the Tollip C2 and CUE domains and inhibits binding of Tollip to phosphoinositides - PubMed (original) (raw)

Ubiquitin interacts with the Tollip C2 and CUE domains and inhibits binding of Tollip to phosphoinositides

Sharmistha Mitra et al. J Biol Chem. 2013.

Abstract

A large number of cellular signaling processes are directed through internalization, via endocytosis, of polyubiquitinated cargo proteins. Tollip is an adaptor protein that facilitates endosomal cargo sorting for lysosomal degradation. Tollip preferentially binds phosphatidylinositol 3-phosphate (PtdIns(3)P) via its C2 domain, an association that may be required for endosomal membrane targeting. Here, we show that Tollip binds ubiquitin through its C2 and CUE domains and that its association with the C2 domain inhibits PtdIns(3)P binding. NMR analysis demonstrates that the C2 and CUE domains bind to overlapping sites on ubiquitin, suggesting that two ubiquitin molecules associate with Tollip simultaneously. Hydrodynamic studies reveal that ubiquitin forms heterodimers with the CUE domain, indicating that the association disrupts the dimeric state of the CUE domain. We propose that, in the absence of polyubiquitinated cargo, the dual binding of ubiquitin partitions Tollip into membrane-bound and membrane-free states, a function that contributes to the engagement of Tollip in both membrane trafficking and cytosolic pathways.

Keywords: Endosomes; Membrane Trafficking; Nuclear Magnetic Resonance; Phosphoinositides; Ubiquitin.

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Figures

FIGURE 1.

FIGURE 1.

Ubiquitin inhibits the PtdIns(3)P binding of Tollip. Lipid-protein overlay assay of the indicated proteins with immobilized PtdIns(3)P (A) or PtdIns(4,5)P2 (B). To test ubiquitin function, GST-Tollip was preincubated with ubiquitin at the indicated molar ratios for 1 h at room temperature. GST and GST-ubiquitin were employed as negative controls. C, representative SPR sensorgram for the binding of GST-Tollip to PtdIns(3)P-containing liposomes. Various indicated concentrations of GST-Tollip were flown over immobilized PtdIns(3)P liposomes. D, kinetics of ubiquitin-mediated inhibition of Tollip-PtdIns(3)P association. Tollip was preincubated with ubiquitin at the indicated concentrations and association of Tollip to immobilized PtdIns(3)P liposomes was followed using SPR.

FIGURE 2.

FIGURE 2.

The Tollip C2 domain is an ubiquitin-binding domain. A, lipid-protein overlay assay of the indicated proteins with immobilized PtdIns(3)P. To test ubiquitin function, the Tollip C2 and CUE domains were preincubated with ubiquitin at the indicated molar ratios for 1 h at room temperature. B, lipid-protein overlay assay of the Vam7p PX domain with immobilized PtdIns(3)P and PKCα and PKCβ II C2 domains with immobilized PtdSer in the absence and presence of ubiquitin. C, representative SPR sensorgram for binding of the Tollip C2 domain to immobilized ubiquitin. Various concentrations of Tollip C2 were flown over His-tagged ubiquitin attached on an NTA sensor chip. D, identification of the ubiquitin residues involved in Tollip C2 domain binding. 15N-Labeled ubiquitin was subjected to HSQC analysis in the absence (black) and presence (red) of the Tollip C2 domain. Perturbed ubiquitin resonances are boxed. E, histogram identifying ubiquitin critical residues for Tollip C2 domain recognition. The colored dashed lines represent significant changes, based on the magnitude of their associated chemical shifts changes: red (Δδaverage + 1.5 × S.D.) > orange (Δδaverage + 1 × S.D.) > yellow (Δδaverage). F, two different views of ubiquitin showing the residues involved in Tollip C2 domain binding and color-coded according to the scales defined in E.

FIGURE 3.

FIGURE 3.

Identification of ubiquitin residues involved in Tollip CUE domain binding. A, 15N-labeled ubiquitin was subjected to HSQC analysis in the absence (black) and presence (red) of the Tollip CUE domain. Perturbed ubiquitin resonances are boxed. B, histogram identifying ubiquitin critical residues in Tollip CUE domain recognition. Chemical shift perturbations are classified as indicated in the legend to Fig. 2. C, two different views of ubiquitin showing the residues involved in Tollip CUE domain binding and color-coded according to the scales defined in B. D, representative SPR sensorgram for the binding of GST-Tollip to immobilized His-ubiquitin. Various indicated concentrations of GST-Tollip were flown over immobilized His-ubiquitin.

FIGURE 4.

FIGURE 4.

Identification of the Tollip CUE domain residues involved in ubiquitin binding. A, 15N-labeled Tollip CUE domain was subjected to HSQC analysis in the absence (black) and presence (red) of ubiquitin. Perturbed resonances of the Tollip CUE domain are boxed. B, the histogram shows normalized chemical shift perturbations in the backbone amides of the CUE domain induced by ubiquitin. Chemical shift perturbations are classified as indicated in the legend to Fig. 2. C, residues that exhibit significant chemical shift perturbations in A are labeled on the modeled Tollip CUE domain surface and color-coded according to the scales defined in B. D, representative SPR sensorgram for the binding of Tollip CUE domain to immobilized ubiquitin. Various concentrations of the Tollip CUE were flown over His-ubiquitin attached on an nitrilotriacetic acid sensor chip.

FIGURE 5.

FIGURE 5.

Hydrodynamic properties of the Tollip CUE-ubiquitin complex. A, representative sedimentation velocity analysis of the Tollip CUE-ubiquitin complex. Sedimentation coefficient distribution of free Tollip CUE (_s_app = 1.64; green line), free ubiquitin (_s_app = 1.18; red line), and Tollip CUE-ubiquitin complex (_s_app = 1.73; black line). B, representative gel filtration analysis of ubiquitin (red), Tollip CUE domain (green), and ubiquitin:Tollip CUE domain (1:1 molar ratio; black) using a Superdex 75 column. Fractions of each of the peaks were analyzed using SDS-PAGE (top). C, summary of the results obtained from sedimentation velocity ultracentrifugation and analytical gel filtration analyses.

FIGURE 6.

FIGURE 6.

A proposed model for the regulation of the endosomal membrane-associated Tollip. Tollip cycles between ubiquitin-free and -bound states in the absence of cargo proteins. Endosomal membrane binding of Tollip is mediated by the interaction of its C2 domain with PtdIns(3)P. The CUE domain mediates Tollip dimerization and this event and PtdIns(3)P binding is negatively regulated by ubiquitin. Of note, other ubiquitin-independent regions in Tollip (e.g. TBD) may also contribute to Tollip oligomerization.

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References

    1. Husnjak K., Dikic I. (2012) Ubiquitin-binding proteins. Decoders of ubiquitin-mediated cellular functions. Ann. Rev. Biochem. 81, 291–322 - PubMed
    1. Ikeda F., Crosetto N., Dikic I. (2010) What determines the specificity and outcomes of ubiquitin signaling? Cell 143, 677–681 - PubMed
    1. Kaiser S. E., Riley B. E., Shaler T. A., Trevino R. S., Becker C. H., Schulman H., Kopito R. R. (2011) Protein standard absolute quantification (PSAQ) method for the measurement of cellular ubiquitin pools. Nat. Methods 8, 691–696 - PMC - PubMed
    1. Dikic I., Wakatsuki S., Walters K. J. (2009) Ubiquitin-binding domains. From structures to functions. Nat. Rev. Mol. Cell Biol. 10, 659–671 - PMC - PubMed
    1. Lange O. F., Lakomek N. A., Farès C., Schröder G. F., Walter K. F., Becker S., Meiler J., Grubmüller H., Griesinger C., de Groot B. L. (2008) Recognition dynamics up to microseconds revealed from an RDC-derived ubiquitin ensemble in solution. Science 320, 1471–1475 - PubMed

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