Non-26S Proteasome Proteolytic Role of Ubiquitin in Plant Endocytosis and Endosomal Trafficking


  • Miaomiao Tian,

    1. State Key Laboratory of Plant Genomics, National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, the Chinese Academy of Sciences, Beijing 100101, China
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  • Qi Xie

    Corresponding author
    1. State Key Laboratory of Plant Genomics, National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, the Chinese Academy of Sciences, Beijing 100101, China
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The 76 amino acid protein ubiquitin (Ub) is highly conserved in all eukaryotic species. It plays important roles in many cellular processes by covalently attaching to the target proteins. The best known function of Ub is marking substrate proteins for degradation by the 26S proteasome. In fact, other consequences of ubiquitination have been discovered in yeast and mammals, such as membrane trafficking, DNA repair, chromatin modification, and protein kinase activation. The common mechanism underlying these processes is that Ub serves as a signal to sort proteins to the vacuoles or lysosomes for degradation as opposed to 26S proteasome-dependent degradation. To date, several reports have indicated that a similar function of Ub also exists in plants. This review focuses on a summary and analysis of the recent research progress on Ub acting as a signal to mediate endocytosis and endosomal trafficking in plants.

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[ Qi Xie (Corresponding author)]


Ubiquitin (Ub) was first isolated from calf sweetbread in 1975, but was later found in almost all tissues of virtually all eukaryotic organisms. Hence, the Latin word “ubique”, which means “everywhere”, was given to this protein. Indeed, the sequence of 76 amino acids (a.a.) encoding Ub differs very slightly in all species, making it a very highly conserved small protein. This small protein acts as a post-translational modification, and regulates many crucial processes in cells. The best known function of Ub is the regulation of protein turnover through proteasome-mediated protein degradation, which was first described by Aaron Ciechanover, Avram Hershko and Irwin Rose (Hershko 1983). The discoveries made by these three scientists fundamentally changed our way of thinking about protein degradation, and the scientists were awarded the Nobel Prize in Chemistry in 2004.

The function of the ubiquitin-proteasome system (UPS) in different organisms, as well as its central role in mediating different cellular processes, has recently been well reviewed (Bader and Steller 2009; Vierstra 2009; Jiang et al. 2010; Santner and Estelle 2010; Barberon et al. 2011; Schlossarek and Carrier 2011). The core mechanism of the UPS is the covalent attachment of a K48 polyubiquitin chain on the substrates, which can then be recognized and degraded by the 26S proteasome. However, recent research carried out mostly in mammals yielded the surprising finding that Ub, or polyubiquitin chains, can also serve as a signal to the lysosomal degradation route or to the autophagy pathway (Floyd et al. 2012), rather than only to 26S proteasome degradation (Clague and Urbe 2010). In endolysosomal degradation, specific plasma membrane receptors utilize Ub as an internalization signal for endocytosis into the cytoplasm. The internalized ubiquitinated proteins go through membrane trafficking, and then arrive at lysosomes for their degradation. In plants, endocytosis and endosomal trafficking have been a focus of interest in the scientific community in recent years. Several studies have indicated that ubiquitinated proteins are associated with these processes. Here, we focus on the recent progress made toward defining the role of Ub as a signal to mediate endocytosis and endosomal trafficking in plants.

Ubiquitination and Ub Chains

Ubiquitination in plants

Ubiquitination is a complex cascade process which is governed by three categories of enzymes, including E1 (Ub-activating enzyme), E2 (Ub-conjugating enzyme) and E3 (Ub ligase). In the first step, E1 activates Ub by forming a high-energy thioester bond between its Cys residue and a carboxy-terminal Gly of the Ub molecule. This is an adenosine triphosphate (ATP)-dependent reaction. Next, the activated Ub is transferred to a Cys residue of the E2 enzyme. This Ub-E2 intermediate serves as the Ub donor, which is then co-opted by the E3 which mediates the transfer of Ub to a Lys ɛ-amino in the target protein. The Ub chain formed on the target protein can be disassembled by a deubiquitination enzyme (DUB), which then releases the target protein and Ub. The released free Ub molecules can be used for another round of protein ubiquitination (Ciechanover et al. 1984; Hershko 1988; Hochstrasser 2009; Vierstra 2009).

In the Arabidopsis genome, there are 16 genes encoding Ub, two genes encoding E1s, 45 genes encoding E2s, and more than 1 400 genes encoding E3s. Thirty-seven of the 45 E2s contain a core ubiquitin conjugating (UBC) domain of 140 a.a., while the other eight E2s lack an active Cys residue in the UBC domain. Based on these eight E2s lacking an active Cys residue in the UBC domain, they are called ubiquitin-conjugating enzyme variants (UEV) or E2-like. More than 1 400 E3s can be classified into four subfamilies by their subunit composition: homology to the E6AP C-terminus (HECT), really interesting new gene (RING), U-box, and cullin-RING ligase (CRLs). The HECT, RING, and/or U-box E3 ligases are single polypeptides which function as E3 ligase alone, while the cullin-RING ligases are multisubunit E3s which contain a cullin, a RING-box1 and a variable target recognition module coordinated together to act as E3 ligase. The HECT domain E3 ligases contain a HECT domain, and the RING or U-box type E3s contain a RING domain or modified RING domain. The HECT domain E3s have a Cys residue which can accept Ub from E2s and transfer it to the target proteins. In contrast, the RING or U-box type E3s lack this active Cys residue, so they act as a scaffold to bind Ub-E2s and target protein, and facilitate the Ub transfer from E2s to target protein directly. The CRLs are further divided into four subtypes: the S-phase kinase-associated protein (SKP1)-cullin 1 (CUL1)-F-box (SCF) E3 ligase, the bric-a-brac-tramtrack-broad complex (BTB) E3 ligase, the DNA damage-binding (DDB) E3 ligase and the anaphase-promoting complex (APC) E3 ligase. As for the deubiquitination enzymes, there are only 32 genes encoding DUB proteins in Arabidopsis (Bachmair et al. 2001; Vierstra 2003; Smalle and Vierstra 2004; Vierstra 2009).

Types of Ub chains

Target proteins can be modified by Ub monomers or by a polyubiquitin chain. When one or several (usually no more than four) Ub monomers are attached to one or more Lys ɛ-NH2 of a target protein through their Gly76 residue, this is defined as monoubiquitination or multi-monoubiquitination, respectively. Alternatively, polyubiquitin chains can also be utilized to modify the target proteins. Different types of polyubiquitin chains can be found: these chains can in fact be built by the formation of an isopeptide bond between the Gly76 residue of one Ub and the ɛ-NH2 group of one of seven potential Lys residues (K6, K11, K27, K29, K33, K48, or K63) in the preceding Ub molecule (Adhikari and Chen 2009; Ye and Rape 2009). The type of polyubiquitin chain determines the fate of the target proteins. It is a common theory that proteins targeted with K48-linked polyubiquitin chains usually participate in 26S-proteasome degradation. K11-linked polyubiquitin chains have recently been reported to participate in this type of degradation as well (Matsumoto et al. 2010). Monoubiquitin, multi-monoubiquitin, and K63-linked polyubiquitin chains have always been associated with non-26S proteasome degradation processes. For example, monoubiquitination or multi-monoubiquitination have been involved in proteins endocytosis, membrane trafficking, and histone modification. K63-linked polyubiquitin chains have been involved in membrane trafficking, signal transduction and DNA repair. Unfortunately, there are still only a few known functions of K6-, K27-, K29- and K33-linked polyubiquitin chains involved in biological processes (Ikeda and Dikic 2008; Li and Ye 2008; Hochstrasser 2009) (Figure 1).

Figure 1.

Different types of ubiquitin (Ub) chain modified substrates.
Monoubiquitination or multi-monoubiquitination is usually involved in endocytosis, membrane trafficking, and histone modification. K48- or K11-linked polyubiquitin chain is usually participated in proteasomal degradation. K63-linked polyubiquitin chain is involved in membrane trafficking, signal transduction, and DNA repair. However, the K6, K27-, K29-, and K33-linked polyubiquitin chains' function is still unknown.

Membrane Trafficking in Plants

Some highly organized membrane structures or compartments, such as the endoplasmic reticulum (ER), the Golgi apparatus, the endosomes, the vacuole, and the cell plate, form a very complex endomembrane system in plants. In this system, proteins, lipids, and polysaccharides are ceaselessly exchanged from one compartment to another through vesicle trafficking (Worden et al. 2012). There are two major trafficking avenues that exist in cells: the secretory pathway and the endocytic pathway (Battey et al. 1999; Jurgens 2004). In the secretory pathway, proteins are first synthesized in the ER, then transported to the Golgi apparatus, and finally reach the plasma membrane or the vacuole through the late endosomes. In the endocytic pathway, the cargo located in the plasma membrane is first internalized into the vesicles, and is then transported to the early endosomes, the late endosomes, and finally reaches the vacuole (Matsuoka and Bednarek 1998; Hadlington and Denecke 2000; Qiu 2012). The plant secretory pathway has been very well studied in the past few years; however, plant scientists only became interested in the endocytic pathway in more recent years. This is one of the reasons why research on plant endosomal trafficking is lagging far behind its counterpart in mammals and yeast. Below, we will discuss the recent progress in research on endocytosis and endosomal trafficking in plants.

Endocytosis in plants

In the past four decades, scientists could not detect endocytosis in plant cells because of the turgor pressure and the presence of the cell wall. However, with the development of new microscope technologies as well as the discovery of new amphiphilic styryl dyes such as FM4-64, endocytosis could be finally detected in plant cells, and has since become a hot research topic (Holstein 2002; Robinson et al. 2008). Recent work indicates that the endocytosis pathway is highly conserved in eukaryotic cells. In animal cells, the molecular mechanisms underlying endocytosis can be simply classified into two groups, clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE) (Hansen and Nichols 2009; McMahon and Boucrot 2011). In plants, it has recently been demonstrated that CME plays a major role (Fujimoto et al. 2010; Chen et al. 2011). CME initiates in specific regions of the plant plasma membrane where cargo is recruited, internalized, and packaged. In animals or yeast, CME is dependent on the formation of clathrin-coated pits (CCPs). CCPs are usually coated with proteins which including clathrin, the AP2-complex, and other accessory adaptor proteins. The cargo is then internalized to form clathrin-coated vesicles (Jurgens 2004; Chen et al. 2011). Although orthologues for clathrin, AP-2 complex proteins and accessory adaptor proteins have been identified in plants, and their functions may be different from their animal and yeast counterparts. For example, in Arabidopsis, an μA-adaptin protein which showed a high similarity to the μ2 subunits of the AP-2 complex and was therefore expected to associate with the plasma membrane, was instead found to be located in the Golgi/trans-Golgi-located tubular-vesicle (TGN) organelle (Happel et al. 2004). This indicated that a different, plant-specific, μA-adaptin protein, or a different AP complex may mediate the plant CME pathway. Similarly, the conserved domains of the accessory adaptors proteins, such as epsin N-terminal homology, AP180 N-terminal homology, Eps15 homology domain, and Src homology 3 were all found in the Arabidopsis genome, but there is little evidence linking them with the CME pathway (Holstein 2002; Legendre-Guillemin et al. 2004; Chen et al. 2011).

In mammalian cells, many cell surface transmembrane receptors undergo endocytosis to transfer environmental signals inside cells after binding their ligands. Most of these mammalian receptors harbor a tyrosine kinase activity, which is used to propagate the signal inside the cell, and are therefore referred to as receptor tyrosine kinases (RTKs). In plants, transmembrane receptors also exist, but unlike animal RTKs, they harbor a serine/threonine kinase activity. In Arabidopsis, the two well-studied leucine-rich repeat receptors kinases Flagellin-sensitive 2 (FLS2) and Brassinoteroid-insensitive 1 (BRI1) have both been associated with the endocytic pathway (Robatzek et al. 2006; Geldner et al. 2007; Robert et al. 2008). In fact, it has been shown that FLS2 and BRI1 are both plasma membrane receptors, and that they can be internalized into the cell through endocytosis (Robatzek et al. 2006; Robert et al. 2008). Robatzek et al. (2006) found that a functional fusion protein of FLS2-green fluorescent protein (GFP) resides at the cell membrane in most tissues in Arabidopsis. Stimulation with the flagellin epitope flg22, the ligand of FLS2, induces the FLS2 transfer to intracellular mobile vesicles. These vesicles are Brefeldin A (BFA, an inhibitor of post-Golgis-derived vesicles trafficking)-insensitive and Wortmannin (an inhibitor of protein sorting to the vacuole)-sensitive, providing evidence that the FLS2 internalized vesicles indeed arise from a Wortmannin-sensitive endocytic process (Robatzek et al. 2006). They also show that phosphorylation of FLS2 may affect its endocytosis. For example, site mutagenesis of Thr 867 in FLS2, a potential phosphorylation site, directly impaired FLS2 endocytosis (Robatzek et al. 2006). In Arabidopsis, brassinosteroid (BR) perception is mediated by two leucine-rich repeat receptor-like kinases, the aforementioned BRI1 and BRI1-associated receptor kinase 1 (BAK1). The transmembrane receptor BRI1 directly binds to brassinolide, while BAK1 interacts with BRI1 to form an endosome-localized heterodimer complex, which initiates BR signaling transduction (Song et al. 2009). BRI1 always forms a homodimer in the plasma membrane, whereas BRI1 and BAK1 preferentially form a heterodimer in the endosomes. It was found that BAK1 could accelerate BRI1 endocytosis, and that this process was also BFA insensitive (Russinova et al. 2004). Recently, Irani et al. (2012) developed a bioactive fluorescent BR analog (Alexa Fluor 647-castasterone (AFCS)). They used BRI1–AFCS complexes to reveal that BRI1 endocytosis is a clathrin-dependent process (Irani et al. 2012).

In Arabidopsis, another protein, the auxin efflux carriers PIN-FORMED 2 (PIN2) (Kitakura et al. 2011), was also found to be internalized. The proteins of the PIN family have been demonstrated to be localized polarly in the plasma membrane in Arabidopsis, as well as in other plants. This polarization is necessary to mediate a directional auxin transport (Krecek et al. 2009; Forestan and Varotto 2012; Pattison and Catala 2012). Moreover, the establishment and maintenance of PIN polarized localization in the cellular membranes have been shown to be necessary for plant embryonic and postembryonic development (Feraru et al. 2011). Genetic research and pharmacological interference of clathrin indicate that the endocytosis of PIN proteins is clathrin dependent (Dhonukshe et al. 2007).

Endosomal trafficking in plants

The endocytic plasma membrane cargo is delivered to early endosomes (EEs) for subsequent protein sorting. However, EEs not only sort endocytic cargo, but also sort secretory proteins in plant cells. These endocytic cargoes can either be recycled back to the plasma membrane or delivered to vacuoles through late endosomes. In plants, TGN structures act as early endosomes, and they are distinct from mammals’ typical tubulo-vesicle early endosomes (Lam et al. 2007; Otegui and Spitzer 2008). It has been shown that multivesicular bodies (MVBs; also known as prevacuolar compartments) function as late endosomes in plants (Scheuring et al. 2011). Moreover, plants have also evolved other conserved and specialized complexes, such as the ADP-ribosylation factor (ARF) machinery, and the retromer and endosomal sorting complexes required for transport (ESCRTs), to mediate their endosomal trafficking (Reyes et al. 2011).

The ARF machinery participates in vesicle budding. It consists of two classes of small GTPases belonging to the Ras superfamily: ARFs and RABs (Ras-related proteins in brain). ARFs act on the budding vesicles, while RABs function in targeting and/or tethering the transport vesicle to the acceptor compartment. In vesicular trafficking, the final step is membrane fusion. This process requires another family of membrane proteins called soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). SNAREs are categorized into vesicle-associated SNAREs (v-SNAREs) and target membrane-associated SNAREs (t-SNAREs) on the basis of their subcellular localization. v-SNAREs correspond to Q-SNAREs, which depend on a conserved glutamine (Q) residue in the center of the SNARE domain, while t-SNAREs correspond to R-SNAREs, which depend on a conserved arginine (R) residue in the center of the SNAREs (Saito and Ueda 2009). In Arabidopsis, it has recently been demonstrated that ARFs mediate vesicle budding (Naramoto et al. 2010), while RABs and SNAREs mediate membrane trafficking (Ebine et al. 2011). Furthermore, researchers identified the coated GNOM endosomes, which belongs to the ARF-GEFs (guanine-nucleotide exchange factors on ADP-ribosylation factor GTPases) family. These endosomes function as recycling endosomes, which carry plasma membrane proteins from the endosomes back to the plasma membrane (Richter et al. 2007; Otegui and Spitzer 2008; Richter et al. 2011). The retromer is thought to be located in the TGN and/or the MVB, and to mediate endosome-to-TGN recycling of vacuolar cargo in plants (Otegui and Spitzer 2008; Schellmann and Pimpl 2009; Reyes et al. 2011). It consists of two subcomplexes: a large heterotrimer composed of VPS26 (encoded by two genes, VPS26a and VPS26b in Arabidopsis), VPS29, and VPS35 (encoded by VPS35a, VPS35b and VPS35c in Arabidopsis), and a small heterodimer formed by three sorting proteins of the nexin family (SNX1, SNX2a, and SNX2b) (Oliviusson et al. 2006; Reyes et al. 2011).

Endosomal sorting complexes required for transport is another endosome-associated complex, and is responsible for sorting proteins in MVBs. In yeast and mammals, it consists of four different protein complexes, which include ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III. This highly-conserved complex is required for MVB biogenesis, cytokinesis, and the HIV budding pathway (Wollert et al. 2009). However, these three different biological processes adopt one or more ESCRT complexes to perform their special function (Schmidt and Teis 2012). Based on the presence of a Ub-binding domain in specific subunits of ESCRT-0, ESCRT-I, and ESCRT-II, Ub has been hypothesized to serve as a signal to mediate the interaction between ESCRTs and ubiquitinated cargo (Raiborg and Stenmark 2009; Shields and Piper 2011). In yeast, the first example of ubiquitination as a signal for protein sorting was the finding that monoubiquitinated pCPS (carboxypeptidase S) is recognized by the UEV domain of VPS23p in ESCRT-I and ESCRT-I, which directs it to MVB vesicles (Katzmann et al. 2001). Proteins sharing homology with ESCRT-I, ESCRT-II, and ESCRT-III, but not with ESCRT-0, have been found in Arabidopsis (Spitzer et al. 2006). However, their cargo, which should be ubiquitinated and degraded in the vacuole, has not yet been found.

Ubiquitin-Mediated Membrane Trafficking and Sorting in Plants

Learning from other organisms

The first evidence of Ub-mediated lysosome or vacuolar degradation, but not proteasomal degradation, comes from a study conducted on Ste2p, Ste6p, Pdr5, and uracil permease in yeast. Ste2p is a G protein-coupled α-factor receptor which is located in the plasma membrane. The binding of α-factor to Ste2p induces ubiquitination of the Ste2p and leads to Ste2p endocytosis (Hicke and Riezman 1996). Thus, Ub acts as a signal for the endocytosis of Ste2p. Ste6p belongs to the ABC-transporter family, and is necessary for the secretion of the yeast mating pheromone α-factor. Although this protein is usually localized in the Golgi apparatus in normal cells, it can accumulate in the plasma membrane and has been shown to be ubiquitinated when endocytosis is disrupted in cells (Kolling and Hollenberg 1994). Pdr5 is a short-lived ATP binding cassette protein, and it has also been shown to be ubiquitinated in the plasma membrane. Ub-modified Pdr5 could be detected in both the wild-type and in conditional endocytosis mutants cells (Egner and Kuchler 1996). Uracil permease also localizes to yeast plasma membrane, and is involved in cell surface ubiquitination. Galan and Haguenauer-Tsapis (1997) first clarified that monoubiquitination was sufficient for endocytosis of this protein, and showed that K63-linked Ub chains stimulate this process. A similar situation was also found in PEST-like sequence (polypeptide sequences which are enriched in proline (P), glutamic acid (E), serine (S), and threonine (T)) within Ste3p (Roth and Davis 2000).

In mammalian cells, Ub-mediated membrane trafficking has also been found in recent years, but the mechanism is less clear (Mukhopadhyay and Riezman 2007). Ubiquitin-modified proteins associated with membrane trafficking could be classified into receptors and transmembrane proteins in mammalian cells. The above-mentioned RTK receptors play important roles in a variety of cellular processes, and have been shown to be ubiquitinated in a ligand-dependent fashion. Indeed, the monoubiquitin-modified RTKs are signals for their endocytosis and degradation in the lysosome (Haglund et al. 2003). However, for other receptor types such as the transforming growth factor-β (TGF-β) receptor, the mechanism of Ub-mediated internalization is far more complicated. For example, when the TGF-β receptor binds to Smad7-Smurf2 E3 ligase, it is rapidly degraded (Kavsak et al. 2000). However, when this receptor associates with the Smad anchor, it is internalized (Di Guglielmo et al. 2003). The other examples come from transmembrane proteins. Epithelial sodium channel (ENaC) is an important human plasma membrane protein which regulates salt homoeostasis and blood pressure. It can be multiple monoubiquitinated by a HECT E3 ligase (Nedd4-2). This multi-ubiquitin is further recognized by clathrin, which induces ENaC endocytosis and subsequent trafficking (Wiemuth et al. 2007). In addition, the Drosophila gene neutralized (Neur) is a RING-type E3 ligase, and it is required for internalization of D1 in the developing eye through ubiquitination (Lai et al. 2001). Major histocompatibility complex (MHC) class I molecules are another family of transmembrane proteins which display peptides from endogenous and viral proteins for immunosurveillance by cytotoxic T lymphocytes. MHC class I proteins can be modified by the K63-linked Ub chain through a viral Ub-E3 ligase (K3). The ubiquitinated MHC class I was further internalized and degraded by lysosomal pathway (Duncan et al. 2006).

In plants

In plants, Ub-mediated membrane trafficking is also complicated. Some plasma membrane proteins have been demonstrated to be ubiquitinated and to be subjected to membrane trafficking. However, their ubiquitination seems to be linked not to membrane trafficking, but to proteasome-dependent degradation. For example, Abas et al. (2006) have indicated that PIN2 could be ubiquitinated, and that this ubiquitination mediates PIN2 proteasome-dependent degradation. FLS2, a plasma membrane receptor which can be polyubiquitinated by two U-box type E3 ligases PUB12 and PUB13, is also degraded by the proteasome (Robatzek et al. 2006; Lu et al. 2011). BRI1-9, a structurally-defective protein of BRI1 which is retained in the ER, can be ubiquitinated by the ER membrane-localized Ub ligase Hrd1 (Su et al. 2011). However, ubiquitinated BRI1-9 is thought to be a target of endoplasmic reticulum-associated degradation (ERAD), which is also a type of Ub proteasome-mediated degradation (Liu et al. 2011; Su et al. 2011; Cui et al. 2012).

However, there are other evidences indicating that Ub-mediated membrane trafficking is also associated with vacuolar degradation in plants. Three examples come from the study of iron transporter 1 (ITR1) (Barberon et al. 2011), plasma membrane ATPase (PMA) (Herberth et al. 2012) and high boron requiring 1 (BOR1) (Kasai et al. 2011) in Arabidopsis (Figure 2). ITR1 is essential for the uptake of iron from the soil into the Arabidopsis root (Vert et al. 2002). This protein is localized in the trans-Golgi network/early endosomes of root hair cells, and is monoubiquitinated on several cytosol-exposed residues in vivo. K154R and K179R mutations in ITR1 trigger its stabilization in the plasma membrane. Furthermore, iron-deficient roots treated with concanamycin A, an inhibitor of plant vacuolar lytic activity, display IRT1 protein accumulation in their vacuoles (Barberon et al. 2011). These results suggest that ITR1 endocytosis is monoubiquitin-dependent and that monoubiquitination triggers ITR1 degradation in vacuoles. Another example of Ub-mediated endocytosis comes from studies on PMA. Herberth et al. (2012) constructed an artificial fusion protein PMA-enhanced green fluorescent protein (EGFP)-Ub to detect Ub-mediated endocytosis, based on the principle that Ub might act as an endocytic signal. Their results indicate that a single Ub is sufficient for PMA-EGFP endocytosis, because Ub chain formation did not affect sorting of this artificial protein. When they constructed a different fusion protein in which Lys residues were mutated to arginine, the protein was also mainly found in the vacuole of the transfected protoplasts. These two examples show that Ub modification is involved in protein endocytosis, but that the internalized ubiquitinated proteins go through vacuolar degradation and not proteasomal degradation.

Figure 2.

Ubiquitin-mediated vacuolar degradation of iron transporter 1 (ITR1), plasma membrane ATPase (PMA), and high boron requiring 1 (BOR1).
ITR1 and PMA endocytosis are ubiquitin-dependent; the internalized ubiquitin-modified ITR1 and PMA are first transferred to the trans-Golgi-located tubular-vesicle/early endosomes (TGN/EE), which later maturates to multivesicular bodies (MVBs). The MVB fused to the vacuole and released the intraluminal vesicles containing proteins, such as ITR1 and PMA, for degradation. Ubiquitin-modified BOR1 mainly participated in sorting to vacuoles, but did not participate BOR1 endocytosis. BOR1 endocytosis is dependent on boron.

In contrast, ubiquitination of BOR1 has little effect on its endocytosis, but helps in sorting it to the vacuole. BOR1 is one of the boron-transporting proteins which regulate boron uptake in roots and boron transport into shoots under boron-limiting conditions (Takano et al. 2008). The endocytosis of BOR1 is boron dependent but ubiquitination independent (Takano et al. 2005; Kasai et al. 2011). However, boron could induce mono- or diubiquitination of BOR1, which is crucial for the sorting of internalized BOR1 to MVB for degradation in vacuoles (Kasai et al. 2011).

E3 ligases play a pivotal role in ubiquitination and determine the target specificity. Their roles in Ub-dependent membrane protein trafficking are also important. In Arabidopsis, KEEP ON GOING (KEG) is a RING-type E3 ligase which localizes to TGN/EE. The most known function of KEG is to mediate abscisic acid signaling through the Ub-mediated degradation of the ABI5 (ABA insensitive 5) transcription factor (Stone et al. 2006). Recent work has indicated that KEG interacts with enhanced disease resistance 1 (EDR1), and that the two proteins co-accumulate in TGN/EE to regulate powdery mildew infection, programmed cell death under both abiotic and biotic stress. These two proteins function together to regulate endocytic trafficking and/or the formation of signaling complexes on TGN/EE vesicles during stress responses (Gu and Innes 2011). However, very little is known about the role of ubiquitinated KEG in endosomal trafficking. Moreover, endosomes located in E3 ligases are not well identified in plants.

Conclusions and Perspectives

Although Ub-dependent 26S proteasomal degradation has been well studied in plants in recent years, research on Ub as a signal to govern endocytosis and trafficking has just started to emerge. In yeast and animals, three major types of Ub chain formation – monoubiquitination, multi-monoubiquitination, and K63-linked polyubiquitination – have been demonstrated to modify trafficking proteins. The question that still needs to be answered is how these types of ubiquitination chains are connected to endocytosis and endosomal trafficking. The challenge ahead for plants is not only the detection of more ubiquitinated trafficking proteins, but also the clarification of the functions of modified Ub chains of trafficking proteins. Many subcellular structures, such as different organelles, are similar in plants, yeast, and animals, and thus the communication between cells and between different organelles inside single cells should pose similar regulatory questions. In addition, plants should have evolved even more complex interactions due to their growth environment. Thus, we predict that plants should have a complex system for the formation of specific Ub chains and for Ub-mediated endocytosis and endosomal trafficking. This notion is supported by a number of studies that were summarized in this review, and also by the presence of homolog genes in the Arabidopsis genome. As the field of Ub-mediated 26S proteasomal-independent degradation is developing quickly in yeast and in mammals, there is no doubt that exciting discoveries will be made in the near future in all organisms, including plants.

(Co-Editor: Giovanna Serino)


This research was supported by a grant from the National Basic Research Program of China (973 Program, 2011CB915402), and from the National Science Foundation of China (CNSF 31030047/90717006).