Molecular Basis of Oligoubiquitin‐Dependent Internalization of Membrane Proteins in Mammalian Cells

Abstract

Ubiquitination induced down-regulation of cell surface proteins by internalization and lysosomal targeting plays a fundamental role in cell physiology and pathogenesis of diseases. The molecular basis of a single ubiquitin (Ub) as an autonomous endocytic signal, the widely accepted mechanism, however, remains elusive in higher eukaryotes. Using Ub containing reporter proteins without signalling abilities, we present evidence that only multiple Ub moieties, linked either covalently or assembled as oligomers with an intact interface for recognition by Ub-interacting motifs (UIMs), are recognized by the endocytic machinery in vivo and associate with a subset of Ub-binding clathrin adaptors in vitro. Genetic and pharmacological approaches show that internalization of plasma membrane proteins harbouring multiple Ub moieties is clathrin-dependent, but caveolinindependent. Functional assays demonstrate the cargodependent involvement of eps15/15R and epsin, UIM containing clathrin adaptors, in the endocytosis of model proteins, CD4 and the activated b 2 -adrenergic receptor complex, containing polymeric or oligomeric Ub. These results provide a paradigm for the clathrinmediated uptake of ubiquitinated membrane proteins in mammalian cells, requiring the assembly of multiple UIM-Ub interactions to overcome the low affinity binding of mono-Ub to UIM.

Polyubiquitination of Prolactin Receptor Stimulates Its Internalization, Postinternalization Sorting, and Degradation via the Lysosomal Pathway

“…These differences underscore the fact that different receptors may require different types of ubiquitin linkages for their downregulation. For example, whereas monoubiquitination is required for downregulation of the (14,33,36,44), and some model chimerical receptors (2,3). Intriguingly, K29-linked chains have been proven to regulate the degradation of the regulator of the Notch pathway, Deltex (7).…”

“…Lysosomes were labeled with FITC-dextran (50 g/ml; molecular mass, 10 kDa) as described previously for CD4 or IFNAR1 (3,24). Cells expressing HA-PRLr were allowed to internalize anti-HA antibody complexed with FITC-conjugated goat anti-mouse Fab for 1 h in DMEM and were chased for 4 h. During the last 45 min, cells were labeled with tetramethyl rhodamine isocyanate-transferrin (TRITC-transferrin) (10 g/ml) to visualize recycling endosomes.…”

“…pH v measurement. To monitor the endocytic trafficking of internalized PRLr, the vesicular pH (pH v ) of cargo-containing vesicles was determined by fluorescence ratio image analysis (FRIA) essentially as described for CD4 or IFNAR1 (3,24). Cell surface PRLr were labeled with anti-HA primary antibody (Covance MMS101R) and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary Fab (Jackson ImmunoResearch Laboratories, West Grove, PA) by incubating primary (1/500 dilution) and secondary (1/500 dilution) antibodies together routinely for 1 h at 37°C in the presence of 50 ng of PRL (WT or antagonist)/ml.…”

“…Each individual ubiquitin moiety harbors seven lysine residues, allowing for the formation of ubiquitin chains linked through its internal lysine residues such as K48 and K63, etc. While the overall role of ubiquitination in mediating the internalization of many yeast receptors (6, 21) and mammalian receptors (such as the nerve growth factor receptor TrkA [16], major histocompatibility complex class II proteins [14], growth hormone receptor [42], model fusion proteins [3], and many others) has been reported previously, the requirements of ubiquitination specificity differ substantially between the various receptors.We have recently demonstrated that linkages of both K48 and K63 types were required for efficient internalization of the alpha/beta interferon receptor chain IFNAR1 (24). We pro-* Corresponding author.…”

Ubiquitin-Dependent Sorting in Endocytosis

“…In experimental settings, namely, where Ub is permanently attached cargo, a single Ub is sufficient for delivery to lysosomes (Shih et al 2000;Raiborg et al 2002;Stringer and Piper 2011). However, as an internalization signal, a single Ub is exceptionally weak and Ub is likely operational only in chains (Barriere et al 2006;Hawryluk et al 2006;Bertelsen et al 2011). For multivesicular body (MVB) sorting, multiple Ubs are the favored signal over mono-Ub (Huang et al 2006.…”