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Keywords:

  • BRI1;
  • endosome;
  • ESCRT;
  • FLS2;
  • GNOM;
  • PIN;
  • plants;
  • retromer;
  • trans-Golgi network

Abstract

  1. Top of page
  2. Abstract
  3. General Organization of the Endosomal System in Eukaryotes
  4. Plant Endosomal Recycling Functions
  5. Endosomal Sorting for Vacuolar Degradation
  6. Endosomes in Receptor-Mediated Signaling
  7. Endosomes as Transport Carriers for Vacuolar Proteins
  8. Conclusions and Perspectives – How Many Different Endosomes Coexist in Plant Cells?
  9. Acknowledgments
  10. References

Plant endosomes are highly dynamic organelles that are involved in the constitutive recycling of plasma membrane cargo and the trafficking of polarized plasma membrane proteins such as auxin carriers. In addition, recent studies have shown that surface receptors such as the plant defense-related FLS2 receptor and the brassinosteroid receptor BRI1 appear to signal from endosomes upon ligand binding and internalization. In yeast and mammals, endosomes are also known to recycle vacuolar cargo receptors back to the trans Golgi network and sort membrane proteins for degradation in the vacuole/lysosome. Some of these sorting mechanisms are mediated by the retromer and endosomal sorting complex required for transport (ESCRT) complexes. Plants contain orthologs of all major retromer and ESCRT complex subunits, but they have also evolved variations in endosomal functions connected to plant-specific features such as the diversity of vacuolar transport pathways. This review focuses on recent studies in plants dealing with the regulation of endosomal recycling functions, architecture and formation of multivesicular bodies, ligand-mediated endocytosis and receptor signaling from endosomes as well as novel endosomal markers and the function of endosomes in the transport and processing of soluble vacuolar proteins.


General Organization of the Endosomal System in Eukaryotes

  1. Top of page
  2. Abstract
  3. General Organization of the Endosomal System in Eukaryotes
  4. Plant Endosomal Recycling Functions
  5. Endosomal Sorting for Vacuolar Degradation
  6. Endosomes in Receptor-Mediated Signaling
  7. Endosomes as Transport Carriers for Vacuolar Proteins
  8. Conclusions and Perspectives – How Many Different Endosomes Coexist in Plant Cells?
  9. Acknowledgments
  10. References

A cell contains different types of endosomes that differ in their functions, architecture and biochemical composition. Usually, endosomes are classified based on their main functions into four classes: early, recycling, intermediate and late endosomes, but the distinction between them is not equally clear in all eukaryotic cells. In general, it can be said that the first endosomal compartment that receives endocytosed cargo from the plasma membrane is the early endosome. Early and recycling endosomes recycled endocytosed plasma membrane cargo back to the plasma membrane. These types of endosomes are believed to mature into intermediate and late endosomes (often called multivesicular bodies or MVBs), which play two major sorting functions: the recycling of vacuolar cargo receptors back to the trans Golgi network (TGN) and the sorting of membrane proteins for degradation. Membrane proteins targeted for degradation are sequestered into internal vesicles that arise from invaginations of the endosomal membrane. When the late endosomes or MVBs fuse with the lysosome/vacuole, the endosomal vesicles are released into the vacuolar lumen and degraded by hydrolases (1). In addition, late endosomes also mediate the transport of newly synthesized vacuolar proteins from the Golgi to the vacuole. Thus, endosomes are key sorting stations that contribute to regulate the protein composition of the plasma membrane, the TGN and the vacuoles/lysosomes.

The endosomal system in plants

As in animal and yeast cells, endosomes in plants traffic both biosynthetic and endocytic cargo. Several studies using membrane styryl FM is a commercial designation from Molecular Probes (Invitrogen) (FM) dyes such as FM4-64, filipin-labeled plant 3-β-hydroxysterols, fluid-phase endocytosis markers such as Lucifer Yellow and fluorescently-labeled Rab guanosine triphosphatases (GTPases) and plasma membrane proteins have unambiguously demonstrated the extraordinary dynamics of the endocytic pathway in plant cells (2–9) (Figure 1).

image

Figure 1. Working model depicting some of the characterized endosomal trafficking pathways in plants. The TGN or an immediate TGN-derived compartment acts as early endosome receiving plasma membrane cargo internalized by endocytosis. At least two different recycling pathways have been discovered in plants for auxin carriers. PIN proteins are recycled by a mechanism that requires the BFA-sensitive ARF-GEF GNOM, whereas AUX1 is recycled by a BFA-insensitive GNOM-independent mechanism. MVBs arise from the fusion of Golgi-derived vesicles carrying newly synthesized vacuolar proteins and, likely, from early/recycling endosomal compartments. Two very important sorting processes take place in MVBs: (i) the recycling of vacuolar cargo receptors mediated by the retromer complex and (ii) the sorting of plasma membrane protein into internal vesicles by the ESCRT machinery. Fusion of MVBs with the vacuole leads to the release of soluble vacuolar proteins and MVB vesicles into the lumen of the vacuole.

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Recent studies have also shown the central role of endocytosis and endosomes in key plant processes such as embryo differentiation (8,10–13), gravitropism (14,15), epidermis differentiation (16,17), guard cell movement (18,19), cell wall remodeling (20,21), the regulation of auxin transport (8,11,22–24) and defense responses against pathogens (25).

Whereas MVBs have been repeatedly observed in plant cells, other types of endosomes such as the typical tubulo–vesicular early endosomes found in mammals have not been described in plants. Recent studies based on FM4-64 uptake experiments and colocalization with TGN markers indicate that the TGN itself or an immediate TGN derivative acts as an early endosome, receiving recently endocytosed material from the plasma membrane (26,27). Further evidence supporting the TGN as an early compartment in the plant endocytic pathway comes from earlier electron microscopy studies tracking cationized ferritin in protoplasts. In these studies, ferritin deposits were first seen inside the cells in Golgi stacks, TGN and a compartment called ‘partially coated reticulum’, which probably correspond to TGN membrane detached from the Golgi stacks (28,29).

Although plants possess the common basic molecular machinery that regulates membrane traffic in other eukaryotes, they have evolved molecular and structural specializations related to plant-specific cellular processes. For example, plants are thought to have specialized mechanisms that allow individual cells to maintain a diversity of vacuolar trafficking pathways (30) and more than one type of vacuole (31–33).

A good example of functional divergence from conserved eukaryotic functions in plants are the endosomal Rab GTPases. Rab GTPases are proteins that co-ordinate important steps in intracellular trafficking, such as vesicle formation, transport and fusion. In mammalian cells, Rab11 and Rab4 GTPases are typically associated with recycling endosomes. Whereas Rab11 orthologs constitute the largest subgroup of plant Rab GTPases (34,35) and have been shown to localize to TGN and endosomes (36–39), no Rab4 orthologs have been identified in plants (40).

The Rab5 members of the Rab GTPase family regulate early endosomal functions in mammals. The Arabidopsis genome encodes three Rab5-like proteins: RHA1/AtRabF2a and ARA7/AtRabF2b and a plant-specific protein named ARA6/AtRabF1. However, all plant Rab5-like proteins localize to late endosomes or MVBs but not to early endosomes (41,42). In fact, RHA1/AtRabF2a and ARA7/AtRabF2b have been shown to mediate the transport of proteins from the Golgi to the vacuole (42–44), which is consistent with the role of late endosomes/prevacuolar compartment in the delivery of newly synthesized vacuolar proteins.

Interestingly, the three Arabidopsis Rab5 members are activated by the same guanine nucleotide exchange factor (GEF), VPS9a. In a vps9a null mutant, embryo development is arrested at the torpedo stage (10). The overexpression of a GTP-fixed form of ARA7/AtRabF2b, but not of ARA6/AtRabF1, is able to suppress the mutant phenotype in a vps9a mutant, indicating that the two types of Rab5 proteins have different functions in plants (10).

Plant Endosomal Recycling Functions

  1. Top of page
  2. Abstract
  3. General Organization of the Endosomal System in Eukaryotes
  4. Plant Endosomal Recycling Functions
  5. Endosomal Sorting for Vacuolar Degradation
  6. Endosomes in Receptor-Mediated Signaling
  7. Endosomes as Transport Carriers for Vacuolar Proteins
  8. Conclusions and Perspectives – How Many Different Endosomes Coexist in Plant Cells?
  9. Acknowledgments
  10. References

Endosome-to-plasma membrane recycling – GNOM-dependent and GNOM-independent pathways

Constitutive cycling, a specialized process that mediates cyclic membrane traffic between endosomes and the plasma membrane, allows rapid changes in plasma membrane composition by having a pool of readily synthesized molecules available in early/recycling endosomal compartments (45). In mammalian cells, constitutive recycling is tightly regulated by hormones such as insulin or vasopressin, which can change the relative rates of endocytosis and recycling and control the concentration of plasma membrane proteins, including channels and receptors. Interestingly, hormones have also been shown to affect endocytosis and endosomal constitutive recycling in plants. The phytohormone auxin has a general inhibitory effect on endocytosis (23), whereas abscisic acid, a plant hormone involved in drought signaling, triggers the internalization and recycling of the KAT1 K+ channel, which is important in the regulation of stomatal opening (19). Other proteins shown to cycle rapidly between the plasma membrane and endosomes are the water channel plasma membrane intrinsic protein 2 (PIP2a), a plasma membrane H+ adenosine triphosphatase (ATPase) and the auxin efflux carrier PIN-FORMED1 (PIN1) (2,3,24,46).

PIN proteins seem to be constitutively recycled in a pathway dependent on GNOM (8), a GEF for the ADP ribosylation factor (ARF) GTPases. This proposed role of GNOM is quite unusual within the ARF-GEF Golgi BFA resistance factor (GBF) subfamily to which GNOM belongs. In yeast and mammals, members of the ARF-GEF Golgi BFA resistance factor (GBF) subfamily function in the formation of coat protein (COP)I vesicles at the Golgi, and this function is also conserved in the closest GNOM Arabidopsis homolog GNOM-LIKE1 (GNL1). Intriguingly, GNOM can functionally substitute for GNL1 but not vice versa (47). This suggests that in addition to its conserved role in Golgi trafficking, GNOM has evolved additional plant-specific functions in the endosome-to-plasma membrane recycling of certain plasma membrane proteins (8,47,48).

GNOM has been hypothesized to localize to recycling endosomes and to mediate the constitutive recycling of PIN proteins by a Brefeldin A (BFA)-sensitive mechanism (8). PIN proteins show polarized distribution (either in the basal or in the apical part of the cell), consistent with their function in polar auxin transport. A given PIN protein may localize to either basal or apical membranes depending on the cell type or developmental stages. Recently, it has been shown that the dynamic polar localization of PIN proteins depends on ARF-GEF-mediated transcytosis (the vesicular transport of macromolecules from one side of a cell to the other). Both PIN1 and PIN2 basal localization seems to be GNOM dependent (49). BFA treatment induces a basal-to-apical localization shift of PIN1 and PIN2 in wild-type plants but not in those plants expressing the engineered BFA-resistant GNOMM696L version. Apicalization of PIN1 and PIN2, but not of nonpolar plasma membrane proteins, was also observed in partial loss-of-function gnom mutants (gnomR5and van7) (49).

Other endosomal components such as the retromer complex, which in yeast and animals mediates endosome-to-TGN recycling, also seem to affect the recycling and plasma membrane localization of PIN proteins (11,50). It is important to note that the polar distribution of the PIN proteins do not seem to depend only on endosomal trafficking but also in the sterol composition of the plasma membrane (51) and PIN phosphorylation state (52,53). In addition, light seems to affect the localization of PIN proteins because PIN1 and PIN2 are internalized from the plasma membrane and delivered to the vacuolar lumen when plants are kept in the dark (54).

Unfortunately, not much is known about what other plasma membrane proteins require GNOM for their recycling from endosomes. We do know, however, that GNOM does not mediate the recycling of all plasma membrane proteins. For example, the auxin influx carrier AUXIN-RESISTANT (AUX1) seems to be recycled from endosomes to the plasma membrane in a BFA-insensitive GNOM-independent manner (55).

Endosome-to-TGN retrieval – the retromer

The retromer complex mediates endosome-to-TGN recycling of a number of vacuolar cargo receptors such as the cation-independent mannose 6-phosphatase receptor in mammals and the vacuolar hydrolase receptor Vps10p in yeast (56) as well as the Drosophila Wntless protein, which is required for Wingless (a Wnt protein) secretion. It has been recently postulated that Wntless and Wingless travel together to the plasma membrane; upon dissociation from Wingless, Wntless is then endocytosed and returned to the Golgi by a retromer-dependent mechanism (57). In all these examples, the retromer recycling functions seem to be important for assuring steady supply of biosynthetic cargo receptors at the TGN.

In yeast, the retromer consists of two subcomplexes: the cargo recognition Vps35p–Vps26p–Vps29p heterotrimer and a subcomplex of two sorting nexins, Vps5p and Vps17p, which are responsible for membrane deformation (58,59). Sorting nexin 1 (SNX1) and the closely related SNX2 are the mammalian orthologs of yeast Vsp5p. Higher eukaryotes seem to lack VPS17 orthologs, and it has been postulated that other sorting nexins can play its role. It is assumed that the retromer mediates the formation of recycling vesicles, but in contrast to other vesicle coats such as COPI, COPII and clathrin, retromer recruitment does not seem to require a GTPase (60). The Arabidopsis genome contains three genes encoding VPS35 proteins (At2g17790, At1g75850 and At3g51310 designated VPS35a, VPS35b and VPS35c, respectively), two genes encoding VPS26 proteins (At5g53530 and At4g27690 designated VPS26a and VPS26b, respectively) and one gene encoding VPS29 (At3g47810) (11).

Arabidopsis VPS35c, VPS26a and VPS29 have been shown to localize to Rab5-positive MVBs together with the vacuolar sorting receptor VSR1/BP80 (11,61,62). Arabidopsis vps35 mutants exhibit dwarfism, early leaf senescence, missorting of vacuolar seed storage proteins to the extracellular space and increased accumulation of VSR receptors (63). In addition, VPS35c coimmunoprecipitates with VSR1 (61). Similar missorting of seed storage proteins has also been reported in the vps29/mag1 retromer mutant (64). In addition, vps29 mutants show abnormal embryo development, seedlings with multiple cotyledons and reduced primary root growth with fewer lateral roots and altered gravitropic response (11). A similar but less severe root phenotype was also reported for the Arabidopsis retromer snx1 mutant (50). Some of these phenotypic alterations are consistent with a role of the retromer in the subcellular localization of auxin carriers and the subsequent alteration of auxin transport. As mentioned above, the retromer has been postulated to affect the constitutive recycling of PIN proteins. Both PIN1 and PIN2 proteins show plasma membrane localization in root cells of the vps29 mutant, but in contrast to wild-type plants, both PIN proteins also accumulate in large intracellular compartments (11). In addition, PIN1 failed to undergo repolarization in some vps29 mutant root cells. Interestingly, these localization defects are not likely because of general alterations in the endomembrane system because other plasma membrane proteins such as the auxin transporter AUX1, the low-temperature-inducible protein 6b (LTi6b) and PIP2a show normal localization in the vps29 mutant. Moreover, it has been postulated that PIN1 would first traffic through GNOM-positive endosomes and later to SNX1 multivesicular endosomes where it would be recycled back to the plasma membrane (11). These results raise the question whether the plant retromer could have evolved additional functions in the endosome-to-plasma membrane recycling.

Endosomal Sorting for Vacuolar Degradation

  1. Top of page
  2. Abstract
  3. General Organization of the Endosomal System in Eukaryotes
  4. Plant Endosomal Recycling Functions
  5. Endosomal Sorting for Vacuolar Degradation
  6. Endosomes in Receptor-Mediated Signaling
  7. Endosomes as Transport Carriers for Vacuolar Proteins
  8. Conclusions and Perspectives – How Many Different Endosomes Coexist in Plant Cells?
  9. Acknowledgments
  10. References

Sorting of plasma membrane proteins into endocytic vesicles and subsequently into the internal vesicles of MVBs are crucial steps in the downregulation of receptors involved in development, signaling and cell differentiation as well as in general turnover of plasma membrane proteins. The endosomal invagination process is unique because, unlike most vesiculation processes characterized to date, the vesiculating membrane buds away from the cytoplasm. This invagination process requires the concentration of membrane proteins into specific membrane domains and the initiation of budding into the endosomal lumen (Figure 2). In yeast and animal cells, ubiquitination of receptors is now well established as a signal for sorting proteins into internal vesicles of MVBs (65). When the ubiquitinated receptors reach the intermediate/late endosome, flat clathrin coats highly enriched in hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) form on the endosomal membranes (66). However, it seems clear that additional mechanisms for endosomal sorting exist because non-ubiquitinated transmembrane proteins, such as yeast Sna3, are also sorted into MVB internal vesicles (67).

image

Figure 2. Simplified model of the main proteins and protein complexes that mediate the ESCRT-related formation of MVB luminal vesicles. After the recruitment of the ESCRT complexes to the endosomal membranes by interactions with phospholipids and the ubiquitin moiety in endocytosed receptors, the AAA ATPase Vps4p/SKD1 assembles in a complex with Vta1p/LIP5 and upon ATP hydrolysis releases the ESCRT complexes from the endosomal membrane. Other proteins such as BRO1 and Doa4 are involved in deubiquitination of endosomal cargo. Although the ESCRT machinery seems to be involved in the formation of MVB vesicles, other processes such as lipid sorting and membrane deformation are also required to complete MVB vesiculation. The proteins and protein complexes depicted in color have been studied in plants.

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Four multisubunit complexes called Vps27–Hse1 (also called endosomal sorting complex required for transport, ESCRT-0), ESCRT-I, ESCRT-II and ESCRT-III are also required for protein sorting into MVB internal vesicles in yeast and mammalian cells (68). Once the ESCRT complexes are assembled onto the endosomal membrane, yeast Vps4p and its mammalian homolog SKD1, which are members of the AAA ATPase family (69–71), are required for endosomal membrane invagination, presumably by releasing the ESCRT coats from the membrane (Figure 2). The binding of Vps4p/SKD1 to the membrane is regulated by its ATPase cycle, being membrane associated in its ATP-bound form and cytoplasmic in its ADP-bound form (Figure 2). Vps4p/SKD1 is a crucial player in the MVB pathway. Alterations in Vps4p/SKD1 functions, either in null vps4 mutants or in cells overexpressing ATPase-deficient forms of Vps4p/SKD1, lead to the formation of aberrant endosomes and thereby to the disruption of endosomal trafficking pathways (71,72).

Putative homologs of all the main ESCRT and ESCRT-related proteins have been identified in plants (73,74). However, only the endosomal functions of a few of these proteins, such as the ESCRT-I component ELCH and the ESCRT-related proteins SKD1, LIP5/Vta1p and Did2p/CHMP1, have been studied to date (Figure 2).

Arabidopsis ELCH is the functional homolog of the ESCRT-I subunit Vps23p in yeast and TSG101 in animals, and like its counterparts, the ELCH protein has ubiquitin-binding capacity and forms a complex with other Arabidopsis ESCRT-I subunits. As in animal cells, mutations in ELCH/TSG101 lead to multinucleated cells in plants because of cytokinesis defects (74).

Arabidopsis SKD1 has recently shown to localize to the cytoplasm and to endosomes. The overexpression of SKD1E232Q, which shows no ATPase activity in vitro, results in alterations in the endomembrane system and leads to a lethal phenotype in plants (41). The inducible expression of the same ATPase-deficient form of SKD1 in root cells causes the formation of enlarged MVBs with a reduced number of internal vesicles but does not affect the diameter of the internal vesicles/budding profiles (Figure 3). Interestingly, enlarged MVBs with fewer internal vesicles are also seen in mammalian (75) and plant cells (5,41) treated with wortmannin, a drug that specifically inhibits phosphatidylinositol 3 (PI3)-kinase activity. In mammalian and yeast cells, phosphatidylinositol 3-phosphate (PI3P) is highly enriched on luminal MVB vesicles (76), and its synthesis in endosomes is mediated by the class III PI3 kinase VPS34 (75). In plants, most of the PI3P has been shown to localize to ARA7/RabF2b-positive endosomes (77,78), and tobacco BY2 cells and Arabidopsis roots treated with wortmannin show distorted MVBs with less luminal vesicles (5,41). However, the wortmannin-treated plant MVBs contain larger luminal vesicles than the enlarged MVBs in the SKD1E232Q-expressing cells, suggesting that wortmannin is affecting early steps in the vesiculation process, upstream of SKD1 action, when the membrane budding areas are initially defined.

image

Figure 3. Function of SKD1 in plants. Ethanol-induced expression of GFP–SKD1 (A and C) and GFP–SKD1E232Q (B and D) in Arabidopsis root meristematic cells (A and B) and epidermal cells of the root elongation zone (C and D) incubated with FM4-64. Arrows indicate enlarged GFP–SKD1E232Q-positive compartments. Electron micrographs of MVBs in cells expressing GFP–SKD1 (E) and GFP–SKD1E232Q (F). The MVBs found in GFP–SKD1E232Q-expressing cells are enlarged and contain fewer vesicles (black arrows) and more numerous inward budding profiles (white arrows) compared with the MVBs in control cells. Bars = 10 μm in (A–D), 200 nm in (E and F). Reprinted from Haas et al. (41) (Copyright © 2007 American Society of Plant Biologists).

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In both yeast and Arabidopsis, Vps4p/SKD1 shows a strong interaction with Vta1p/LIP5, which results in an increased ATPase activity (41,79). However, it is not clear how important the role of LIP5 is in vivo because an Arabidopsis lip5 null mutant does not exhibit phenotypic alterations (41).

Another Vps4p/SKD1-interacting protein in yeast and mammals is Did2p/CHMP1. In plants, two putative DID2/CHMP1 homologues, Sal1 in Zea mays (16) and NbCHMP1 in Nicotiana benthamiana (80), have been described. The maize SAL1 protein has been shown to localize to MVBs. Interestingly, the sal1 mutant in maize shows alterations in aleurone (epidermal endosperm layer) differentiation, likely because of alterations in the recycling of plasma membrane proteins that are key regulators of aleurone differentiation, such as CRINKLY4 (CR4) and DEFECTIVE KERNEL1 (DEK1) (17).

Endosomes in Receptor-Mediated Signaling

  1. Top of page
  2. Abstract
  3. General Organization of the Endosomal System in Eukaryotes
  4. Plant Endosomal Recycling Functions
  5. Endosomal Sorting for Vacuolar Degradation
  6. Endosomes in Receptor-Mediated Signaling
  7. Endosomes as Transport Carriers for Vacuolar Proteins
  8. Conclusions and Perspectives – How Many Different Endosomes Coexist in Plant Cells?
  9. Acknowledgments
  10. References

The endosomal machinery that controls sorting of endocytosed receptors and other plasma membrane proteins is essential for signaling mechanisms to take place properly. In many cases, activated receptors have to be internalized by endocytosis and delivered to endosomes to find other factors of the downstream signaling machinery. One of the best studied examples in mammals is the epidermal growth factor receptor (EGFR), which is a plasma membrane receptor tyrosine kinase that controls cell proliferation and differentiation processes. Ligand-induced activation of EGFR results in rapid internalization by endocytosis and delivery to endosomes where the EGFR downstream signaling components are located. In fact, EGFR interacts on endosomes with Grb2 to initiate and regulate mitogen-activated protein kinase signaling (81). In addition, endosomes also regulate signal termination from activated receptors either by favoring ligand–receptor dissociation or by sequestering activated receptors into internal vesicles, which make the receptors inaccessible to cytoplasmic signaling factors and sort them for lysosomal degradation.

Receptor-like kinases (RLK) are well represented in plants with over 600 members in Arabidopsis and 1100 in rice (82). Some of them are implicated in plant development and growth, whereas others play a role in plant defense. The Arabidopsis FLAGELLIN-SENSING 2 (FLS2) receptor kinase responds to infections by bacterial pathogens. Upon recognition of flagellin or the flagellin peptide derivative flg22, FLS2 undergoes ligand-mediated endocytosis (25) and initiates the generation of reactive oxygen species, protein phosphorylation, mitogen-activated protein kinase signaling and transcriptional gene regulation. Interestingly, a point mutation in the threonine residue 867, a putative phosphorylation site in FLS2, hampered both FLS2 internalization and FLS2 response, suggesting that both processes are interconnected (25).

Another plant RLK is the BRASSINOSTEROID INSENSITIVE 1 (BRI1) protein, which acts as receptor for brassinosteroids, steroidal plant hormones that promote cell expansion and division. BRI1–green fluorescent protein (GFP) localizes to the plasma membrane and to endosomal compartments, but in contrast to FLS2–GFP, BRI1–GFP does not change its localization upon addition of its ligand brassinolide (83). Brassinolide binds the BRI1 extracellular domain, activates the BRI1 kinase and releases the inhibitory BKI1 protein from the plasma membrane (84). Interestingly, the endosomal BRI1 pool is not associated with BKI1, suggesting that BRI1 may be active on endosomes. In fact, treatment with the drug BFA, which leads to the accumulation of BRI1 in endosome-derived compartments, causes enhancement of brassinosteroid signaling in both cultured cells and Arabidopsis seedlings (83). This suggests that BRI1 signals from endosomes although its internalization does not depend on ligand binding.

Endosomes as Transport Carriers for Vacuolar Proteins

  1. Top of page
  2. Abstract
  3. General Organization of the Endosomal System in Eukaryotes
  4. Plant Endosomal Recycling Functions
  5. Endosomal Sorting for Vacuolar Degradation
  6. Endosomes in Receptor-Mediated Signaling
  7. Endosomes as Transport Carriers for Vacuolar Proteins
  8. Conclusions and Perspectives – How Many Different Endosomes Coexist in Plant Cells?
  9. Acknowledgments
  10. References

Late endosomes or MVBs, often called prevacuolar compartments by plant cell biologists, are also involved in the trafficking from the Golgi to the vacuole. This function is particularly important in plant cells in which vacuoles are diverse and essential for cell viability. The critical role of late endosomes/MVBs in transport of vacuolar cargo is also supported by the observation that plant cells with mutations in MVB-localized proteins such as the Rab5-like GTPases and retromer subunits all show vacuolar transport defects (42–44,63,64).

MVBs in developing embryos of Arabidopsis arise, at least partially, from the fusion of Golgi-derived vesicles (Figure 4) and carry both vacuolar storage proteins, such as the 2S albumins and 12S globulins, and their processing proteases. However, endosomes do not act just as passive carriers for vacuolar proteins; they also provide the appropriate environment for protease activation. In fact, the initial proteolytic processing of the 2S albumins occurs inside the MVBs before the storage proteins reach the vacuoles (85). In addition, the proteolytic activity of the endosomes seems to be important for the efficient fusion of endosomes with vacuoles (12).

image

Figure 4. Formation and architecture of MVBs in Arabidopsis embryo cells during the deposition of storage proteins in the vacuole visualized by electron tomography. A) Tomographic model of a cluster the Golgi stacks and their associated TGNs surrounded by dense vesicles (DV) carrying vacuolar storage proteins, clathrin-coated vesicles (CCV) and MVBs of different sizes. B–D) Details of MVB compartments with different number of internal vesicles (arrows) and storage protein aggregates (stars). In these tomographic models, the MVB limiting membrane has been made translucent to visualize the structures in the MVB lumen. These MVBs have been postulated to arise, at least partially, from the fusion of Golgi-derived vesicles carrying vacuolar storage proteins and processing proteases. Bars = 200 nm in (A), 100 nm in (B–D). NCV, non-coated vesicle. Reprinted from Otegui et al. (85). (Copyright © 2006 American Society of Plant Biologists).

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Another Arabidopsis endosomal protein that is important for vacuolar trafficking is KATAMARI2 (KAM2)/GRAVITROPISM DEFECTIVE 2 (GRV2). The kam2/grv2 mutants show secretion of seed storage proteins to the extracellular space and altered vacuolar morphology (12). The GRV2/KAM2 protein has sequence similarity to receptor-mediated endocytosis 8 (RME-8), a DnaJ domain protein that localizes to endosomes and regulates endocytosis in Caenorhabditis elegans and Drosophila melanogaster. The GRV2/KAM2 protein seems to localize to a wortmannin-sensitive compartment but does no colocalize with the MVB marker ARA7/RABF2b or ARA6/RABF1 (12,13). Interestingly, Haupt et al. (7) found that the GRV2 ortholog in tobacco interacts with the TGB2 movement protein of potato mop-top virus, suggesting that endosomal recycling of viral movement proteins may be an important step in viral spreading.

Conclusions and Perspectives – How Many Different Endosomes Coexist in Plant Cells?

  1. Top of page
  2. Abstract
  3. General Organization of the Endosomal System in Eukaryotes
  4. Plant Endosomal Recycling Functions
  5. Endosomal Sorting for Vacuolar Degradation
  6. Endosomes in Receptor-Mediated Signaling
  7. Endosomes as Transport Carriers for Vacuolar Proteins
  8. Conclusions and Perspectives – How Many Different Endosomes Coexist in Plant Cells?
  9. Acknowledgments
  10. References

Although a number of endosomal markers and proteins with well-defined functions have been studied, it is not completely understood how the plant endosomal system is organized. The Arabidopsis Rab5 GTPases, the retromer and some of the ESCRT components all seem to localize to structurally similar multivesicular endosomes, but it is not clear if all plant MVBs represent a homogenous endosome population. For example, the RHA1/RabF2a–ARA7/RabF2b-positive endosomes seem to represent earlier compartments in the endosomal pathway because they take up FM4-64 before the ARA6/RabF1-positive endosomes do. In addition, GNOM appears to be important for functional integrity of RHA1/RabF2a–ARA7/RabF2b-positive endosomes but not for ARA6/RabF1-positive endosomes (86). However, GNOM does not seem to colocalize with ARA7/RabF2b (62). There is compelling evidence suggesting that the TGN or a TGN-derived compartment may receive the endocytic vesicles and thus act as an early endosome (26,27), but our current knowledge on plant recycling endosomes is very limited. Although it is clear that plant cells undergo constitutive endosomal recycling of plasma membrane proteins and that GNOM mediates the recycling of a subset of plasma membrane proteins, the structural architecture and diversity of plant recycling endosomes are not known. The identification of proteins that define different stages in endosome maturation and different endosomal subpopulations is clearly needed to understand the organization of plant endosomal system.

This is a very exciting time for plant cell biology research, and future work on plant endosomal functions will shed light on questions such as follows: What plant receptors signal from endosomes and how is their endocytic/endosomal trafficking regulated? How are the different endosomal recycling pathways (GNOM dependent and GNOM independent) regulated? Have conserved endosomal proteins and protein complexes such as ESCRTs and retromer evolved specific functions in plants? What are the cargo proteins for the ESCRT sorting machinery in plants? How many different endosomes/endosomal functions coexist in plant cells?

References

  1. Top of page
  2. Abstract
  3. General Organization of the Endosomal System in Eukaryotes
  4. Plant Endosomal Recycling Functions
  5. Endosomal Sorting for Vacuolar Degradation
  6. Endosomes in Receptor-Mediated Signaling
  7. Endosomes as Transport Carriers for Vacuolar Proteins
  8. Conclusions and Perspectives – How Many Different Endosomes Coexist in Plant Cells?
  9. Acknowledgments
  10. References