Ubiquitin on the Move: The Ubiquitin Modification System Plays Diverse Roles in the Regulation of Endoplasmic Reticulum- and Plasma Membrane-Localized Proteins

Ubiquitin ligation to other proteins modulates the activity, longevity, and/or localization of the target proteins in eukaryotic systems. As components of the ubiquitin pathway, plant hormone receptors determine the abundance of key transcriptional regulators of auxin, GA, and jasmonate response pathways (for review, see Kelley and Estelle, 2012). Ubiquitin has also been shown to alter the abundance, function, and localization of membrane- and endoplasmic reticulum (ER)-resident proteins. Other hormone signaling cascades, including the ethylene and brassinosteroid pathways, depend on the regulated proteolysis of membrane-associated receptor-like protein kinases. Significantly, the consequences of ubiquitin modification in some cases are independent of the proteasome. An appropriate general term, inclusive of all processes involving ubiquitin, is the ubiquitin modification system (UMS). This Update, in addition to reviewing general aspects of the UMS, specifically focuses on the roles of ubiquitin in the plant endomembrane system. Ubiquitin is a highly conserved 76-amino acid protein whose posttranslational covalent ligation to other proteins, including itself, serves myriad physiological functions. It was shown to be an essential protein in Saccharomyces cerevisiae, as deletion of all ubiquitin-encoding genes resulted in complete loss of viability (Finley et al., 1994). The breadth and depth of ubiquitin’s known physiological roles suggest that it is also essential in plants, although this has not been directly demonstrated. Initially, ubiquitin was identified in two independent areas of investigation: as a single ubiquitin covalently attached to Lys-119 of histone 2A in mammals (Böhm et al., 1980), and as a required component for the in vitro degradation of model proteins in a reticulocyte extract (for review, see Wilkinson, 2005; Ciechanover, 2009). Our knowledge of the in vivo functions of ubiquitin have greatly expanded subsequently to include the regulated proteolysis of transcription factors; internalization of plasma membrane (PM)-resident transporters and receptors; clearance of misfolded ER-localized proteins via endoplasmic reticulum-associated degradation (ERAD); and cell cycle progression, among others (Smalle and Vierstra, 2004; Deshaies and Joazeiro, 2009). Ubiquitin alters the longevity, localization, and/or activity of proteins via one or more covalent bonds between the C-terminal carboxyl group of ubiquitin and a nucleophilic moiety on the substrate protein (referred to here as the “target”). Canonically, ligation occurs at ɛ-amino groups of target lysyl residues or at the target’s N terminus. More recently, Ser, Thr, and Cys esters at the C terminus of ubiquitin have been reported in yeast (Saccharomyces cerevisiae) and mammalian cells (for review, see Wang et al., 2012), although the in vivo abundance of these linkages is still unclear. For some known ubiquitination targets, substitutions of Lys residues with nonconjugatable Arg residues result in reduced degradation, implicating Lys residues as the primary modification sites. On the other hand, N-terminal or Ser/Thr ubiquitination is made more likely when substitutions of all Lys residues have a minimal effect. Ubiquitin can also be polymerized, as its Lys residues serve as acceptor sites for additional ubiquitin molecules. A global proteomic study using mammalian cells found that while Lys-48 ubiquitin-ubiquitin chains are the most abundant, all seven ubiquitin Lys linkages were present (Kim et al., 2011). Similar global analyses in Arabidopsis (Arabidopsis thaliana) yield the same relative ubiquitin chain abundance pattern: Lys-48 chains are most abundant, followed by Lys-63, Lys-11, and then Lys-33 and Lys-29 in much lower abundance (Saracco et al., 2009). Linear polyubiquitin called M1 chains (in which the C terminus of one ubiquitin is attached to the N terminus of the next) are also possible, and in mammals, M1 chains play a role in the activation of Nuclear factor kappa-light-chain-enhancer of activated B cells, a transcription factor activated in response to a number of signals including extracellular cytokines (Emmerich et al., 2011). Site-specific substitution of ubiquitin Lys residues uncovered the role of Lys-63 chains in DNA repair and kinase activation in mammalian cells (Peng et al., 2003). Lys-63 chains are also implicated in vacuolar targeting (for review, see Lauwers et al., 2010). In vivo roles of these linkages remain to be defined in plants, as there are only a few intriguing results: mutations in RING Domain Ligase1-2 (RGLG1-2), which forms Lys-63 chains, suppress apical dominance in Arabidopsis (Yin et al., 2007), and recently, Lys-63 chains were found to play a role in PIN-FORMED2 (PIN2) degradation (Leitner et al., 2012). Attachment of ubiquitin to target proteins, including ubiquitin itself to form chains, typically requires a cascade of three enzymes: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme (UBC); and E3, the ubiquitin ligase. The cascade can be thought of as a protein pyramid. For example, in Arabidopsis, there are two highly related E1s, approximately 37 predicted E2s, and more than 1,000 predicted E3s (Smalle and Vierstra, 2004). While plant-specific gene duplication events surely contribute to this diversity, the pyramidal structure is present in yeast and mammals as well. However, it should be borne in mind that, to date, only a subset of predicted plant E2s and E3s have been demonstrated to have the predicted activity. E1 and E2 function sequentially, with E1 catalyzing activation of the C-terminal carboxyl group via adenylylation and subsequent thioester formation (Fig. 1) and then transferring the ubiquityl group to an E2 Cys, denoted as Ub∼E2. A subset of E3s called the HECT type (for homology to E6-AP C terminus) also carry thioester-linked ubiquitin prior to transfer to target proteins, but the majority of E3s catalyze ubiquitin transfer by serving as scaffolds for the target and the Ub∼E2, and allosterically activating ubiquitin transfer. Named for the E2-interacting domain shared among their respective family members, these scaffold ligases fall into two very general classes: U-box (in plants, termed plant U-box) and RING (for really interesting new gene) E3s (Yee and Goring, 2009). A hybrid of HECT and RING types has been reported in the RING-between-RING proteins. RING-between-RING proteins contain a functional RING domain, which interacts with the Ub∼E2, and additionally resemble the HECT-type E3s, with a Cys acceptor for activated ubiquitin prior to transfer to the target (Wenzel and Klevit, 2012). RING proteins are also found as subunits in multisubunit E3s, such as the well-characterized SCF-type complex (Hua and Vierstra, 2011; for review, see Kelley and Estelle, 2012) and the anaphase-promoting complex (Peters, 2006). Examples exist of targets with multiple E3s (Popov et al., 2010) and E3s with relaxed target specificities (Kostova et al., 2007). Adding another layer of complexity, some E3s (in conjunction with particular E2s) even specify different linkage types to certain targets (for review, see Ikeda and Dikic, 2008). The organization of E3s, particularly the discernment of accessory proteins, remains an active area of investigation. The ubiquitination enzyme cascade. A, The modification of eukaryotic intracellular proteins with the small protein ubiquitin (Ub; gray sphere) depends on a three-enzyme cascade. E1 (tan oval) activates ubiquitin at the expense of two ATP equivalents by forming a high-energy thioester bond between the E1 active-site Cys and the C terminus of ubiquitin. Ubiquitin is then passed to an active-site Cys on E2 (yellow octagon). Lastly, the Ub∼E3 thioester is brought into proximity with the desired protein target (red oval) by a scaffold protein called E3 (blue). Ubiquitin can be added to a variety of nucleophilic groups on the target protein, including the N terminus (Cα1-NH2), Lys residues (K-NH2), Ser and Thr residues (S/T-OH), and Cys residues (C-SH). B, Types of E3s. Multisubunit RING E3s, such as CULLIN RING Ligases (CRLs), recognize substrates with adaptor proteins (i.e. F-box proteins). Single-subunit RINGs like MDM2 consist of one polypeptide with binding sites for the E2 (the RING domain) and the target protein. The U-box is a modified RING domain lacking zinc-binding sites. HECT-type E3s form a Ub∼E3 thioester prior to ubiquitin being transferred to the target. APC, Anaphase-promoting complex. In addition to families of E2 and E3 enzymes, eukaryotes express proteins with ubiquitin-deconjugating and ubiquitin-binding abilities. The former serve to remove ubiquitin (D’Andrea and Pellman, 1998), while the latter interact with different types of ubiquitinated proteins (Andersen et al., 2005). Ubiquitin-binding proteins can mediate interaction between ubiquitin-modified targets and the proteasome (Fu et al., 2010), shuttle ubiquitinated proteins from one compartment to another, or serve to modulate the activities of others in the same complex (Hicke and Dunn, 2003). Alternative fates of ubiquitinated targets include proteolysis in the lytic compartment of the vacuole or the central cavity of the proteasome, nonproteolytic inhibition, and even protein activation (Fig. 2). Fates of ubiquitinated proteins. The fate of a ubiquitinated target protein, typically mediated by ubiquitin-binding proteins (UBP; dark green), depends on the number of ubiquitin moieties attached to the substrate, the type of ubiquitin-ubiquitin linkage, and whether the substrate protein is membrane associated. A, Monoubiquitinated integral membrane proteins are internalized, leading to activation or vacuolar degradation upon exit from the sorting endosome (SE). B, Polyubiquitinated integral membrane proteins can be degraded either by vacuoles or, via ERAD, the proteasome. C, Polyubiquitinated soluble proteins, in which ubiquitin moieties are linked via Lys-48, are proteasomally degraded; in contrast, Lys-63-linked substrates tend not to be proteolyzed but have been shown to play roles in DNA repair. D, Monoubiquitination and multiubiquitination of soluble proteins can bring about activation or inhibition of a protein’s activity. The complexity of the UMS presents a challenge to studying the consequences of ubiquitination. In the simple case of a cytosolic protein with a short half-life, the compounds MG132 (a proteasome inhibitor with some activity against Cys proteases [Woffenden et al., 1998]) and cycloheximide (CHX; an inhibitor of protein synthesis) will lead to the hyperaccumulation and rapid disappearance of the target, respectively. This simple reciprocal response will not be observed if, for instance, the target’s E3 turns over faster than the target, if the treatment is prolonged (proteasome inhibition artificially depletes the free ubiquitin pool, which in turn affects proteolysis and endocytosis), or if the target’s degradation depends on new protein synthesis. What do we know about the sequence specificity of ubiquitin attachment? Proteomic surveys in yeast and cultured mammalian cells have failed to detect a specific sequence around the ubiquitin Lys attachment site, although there is a slight enrichment of basic amino acids, Ala, and Gly one to six residues N terminal to the given Lys. Overall, accessibility of the lysyl amino group seems to be a key factor in determining whether ubiquitination will take place (Xu et al., 2010). The presence of membrane-localized E2 and E3 enzymes suggests that ubiquitination occurs near membrane surfaces. Following a general description of the biogenesis of membrane proteins, we discuss the role of ubiquitin in ER protein quality control and in regulating membrane protein abundance. In eukaryotes, the nuclear envelope, ER, Golgi, PM, and lysosomes form a continuum of lipid bilayer-enclosed compartments known collectively as the endomembrane system. Lumenal and/or integral membrane proteins follow a discrete pathway from the ER to their final destinations. Prior to entering the ER, a stretch of approximately 20 to 30 amino acids at the N terminus of the nascent protein is cotranslationally recognized by the signal recognition particle, which relocates the translating ribosome to the Secretion61 translocon on the ER, thus funneling the new protein into the ER lumen (for review, see von Heijne, 2007). Once inside the ER, secreted and membrane proteins are subjected to chaperone-assisted folding, protein disulfide isomerase-mediated disulfide synthesis, and N-glycosylation (for review, see Kleizen and Braakman, 2004). In yeast, nascent polypeptides are modified via N-glycosylation and subsequent glycan trimming, followed by association with the chaperone calnexin (Cnx), if soluble, or calreticulin (Crt), if membrane associated. Cnx-Crt recruits protein disulfide isomerase and binding immunoglobulin protein, a chaperone that aids in the folding of a nascent protein’s nonglycosylated domains. Properly folded proteins are then exported to the Golgi. Improperly folded proteins are recognized by UDP-Glc:glycoprotein glucosyltransferase, which reglucosylates them, thereby enabling additional rounds of Cnx-Crt-driven folding (for review, see Hong and Li, 2012). If a protein is properly folded, it is exported to the cis-Golgi, where it is further processed via modifications such as O-glycosylation, phosphorylation, and assembly into multisubunit complexes before being packaged into exocytic that with the (for review, see et al., 2011). As the ER is with multiple to that proteins their However, a of proteins to this and these substrates for in yeast and mammalian cells, the pathway is a by which misfolded or and ER proteins are into the and degraded by the proteasome (for review, see et al., 2005). are two E3 ligases with cytosolic RING known to in which and target and the of which target cytosolic (Kostova et al., 2007). requires an of accessory proteins to in target and recognition the adaptor and the soluble yeast and interacts with the membrane E2s and et al., 2009). on the E2s and as as the and the et al., 2006). folded proteins interact with these E3s via and glycan moieties that are added prior to binding and Li, 2012). While the is by that an protein response and 2010), it has been shown to physiological processes as well. are ER-localized which the in is proteasomally degraded ubiquitination by the E3 et al., et al., 2003). is also some for protein quality control pathways that at the and the (for review, see et al., et demonstrated that a form of the which is degraded by ERAD, at the at a a was and proteolyzed in a that a quality control at the In have uncovered plant of yeast and mammalian including the E3s and the E2s and and accessory proteins such as the and the chaperone et al., 2005). functional on plant from of mutations in the receptor-like kinase the brassinosteroid et al., 2008). 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