The discovery of ubiquitin-dependent endocytosis

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The link between ubiquitin and endocytosis in yeast was discovered almost simultaneously in different laboratories, using three approaches: 1) analysis of the downregulation of Ste6p, the ATP-binding cassette (ABC)-transporter for secretion of the pheromone a-factor [64], 2) demonstration that the endocytic signal identified in a C-terminal truncated form of Ste2p, the receptor for a-factor, was an ubiquitylation signal [65], and 3) cloning of a gene involved in the downregulation of amino acid permeases, which  was found to encode a ubiquitin protein ligase [66].

In a pioneering study, Kölling and coworkers observed the accumulation in plasma membrane fractions prepared from a mutant with impaired endocytic internalization, of ubiquitylated forms of Ste6p [64]. These forms were less abundant in a mutant lacking two ubiquitin-conjugating enzymes, Ubc4p and Ubc5p, and this mutant was protected from Ste6p degradation. This suggested that a ubiquitylation event might precede and be required for Ste6p internalization. Secondly, extensive work was devoted to determining the endocytic signals in the a-factor receptor, Ste2p, by tracing a-factor internalization by truncated or mutated receptors. A non-classical signal, SINNDAKSS, was found to be necessary and sufficient for the ligand-induced endocytosis of a C-terminally truncated Ste2p. Within this sequence, the Lys residue was found to be critical [67]. These observations were clarified with the understanding that this residue is the target for a-factor-induced ubiquitylation of the truncated receptor, which is required for internalization [65]. The third line of evidence for a link between ubiquitin and endocytosis in yeast was obtained by genetic analysis of the ammonium-induced downregulation of amino acid permeases (reviewed in [68]). The discovery that one gene involved in this process, NPI1, encodes a ubiquitin-protein ligase of the HECT family, Rsp5p (Fig. 1) [66], and that a human homolog of this enzyme, Nedd4, is critical for downregulation of the epithelial sodium channel ENaC, provided major insights into the emerging field of ubiquitin-dependent endocytosis (reviewed in [2, 21, 50]).

Rsp5p was then demonstrated to be involved in constitutive ubiquitylation of the uracil permease, Fur4p [69], and ammonium-induced ubiquitylation of the general amino acid, Gap1p [70]. Impairments affecting Rsp5p, or mutations of the two target lysines in the two permeases inhibit their ubiquitylation, thereby preventing internalization [69-72]. The fusion of ubiquitin in-frame at the N-terminus of a mutant form of uracil permease lacking the two target lysines trapped at the plasma membrane results in the partial restoration of permease internalization [5]. Similarly, the fusion in-frame of ubiquitin restores some internalization of variant forms of a-factor and a-factor receptors lacking their own ubiquitylation signals [73, 74]. Moreover, the fusion in-frame of ubiquitin to a stable plasma membrane protein, the [H⁺] ATPase, leads to the internalization and subsequent vacuolar degradation of this protein [73]. These data, together with the observation that endocytic cargoes accumulate in ubiquitylated forms in mutants defective for the internalization step of endocytosis, demonstrate that ubiquitylation is required for, and precedes the internalization of many plasma membrane proteins, and that ubiquitin (monoubiquitin) constitutes an internalization signal in itself.

It soon became clear that ubiquitylation is a prerequisite for the internalization of most known plasma membrane substrates of endocytosis, and that Rsp5p, the only HECT ligase of the Nedd4 family in yeast, is the only ubiquitin protein ligase involved in this posttranslational modification [1, 21, 50, 75]. The only exception reported to date in this apparent general requirement for ubiquitin in yeast internalization is the ligand-induced internalization of the a-factor receptor, Ste3p. Whereas constitutive Ste3p endocytosis displays typical ubiquitin-dependence [74, 76], Ste3p ligand-dependent internalization and recycling proceeds normally after conservative Lys to Arg mutations at all possible ubiquitin acceptor sites [77]. Ligand-dependent Ste3p endocytosis has been shown to involve a specific signal, NPFSTD, recognized by the protein Sla1p, linked to the actin cytoskeleton, which has been suggested to act as an endocytic adaptor [78]. It should be noted that a di-Leu motif is required for endocytosis of the Gap1p permease [79]. Point mutations in the di-Leu signal, or small deletions in this region, impair endocytosis but not ubiquitylation of the Gap1p permease, suggesting that this region of the protein is involved in endocytosis, at a stage downstream from ubiquitylation [70, 79]. No endocytic adaptor has yet been identified in this case.