*2.1.2.1 Retrograde transport and COPI*

Of the 12 ARF isoforms, ARF1 is targeted to the Golgi and post-Golgi structures in plant cells. *Arabidopsis* ARF1 has been shown to be involved in different trafficking pathways including ER-Golgi traffic, vacuolar trafficking, and endocytosis and/ or recycling [51, 52]. Arfs are divided into three classes and express six isoforms, namely, Arf1 to Arf 6 (with Arf2 being absent in human) in the mammalian system.

#### **Figure 3.**

*Model of membrane trafficking to the vacuole in plant cells. The vacuolar trafficking pathway involves three trafficking routes in* Arabidopsis *(i) depending on the sequential action of RAB5 and RAB7, (ii) AP-3 dependent but RAB5- and RAB7- independent pathway, and (iii) RAB5-dependent and AP-3-independent route (modified from Ebine et al. [46]).*

ARF1 manages ER-to-Golgi transport and Golgi-derived transport to the plasma membrane, depending on the COPI vesicle coat protein components [53, 54]. A large number of ARF in plants suggest the possibility for highly regulated vesicle trafficking [53]. Similar to other small GTPases, ARF GTPases cycle between an active state, when associated with GTP (membrane-bound form), and an inactive state when bound to GDP (predominantly cytosolic form).

In its GDP form, ARF1 is present in the cytosol and is recruited to the surface of Golgi membranes by a GEF. A SEC7-type GEF stimulates the binding of GTP to ARF1. This progression can be inhibited by the fungal metabolite Brefeldin A (BFA) in mammalian cells [55]. In the same system, the GDP-bound form of ARF1 interacts with p24 cytosolic tails [56]. The cytosolic ARF1 activation initiates COPI biogenesis. The GTP-bound form of ARF1 interacts with coatomer, which can also interact directly with the p24 cytosolic tails. In this manner, the p24 cytosolic tail can interact both with ARF1 and coatomer [56]. The conformational change of ARF1 occurs by the GTP/GDP exchange that may cause its dissociation from p24 cytosolic tails [56].

COPI vesicles mediate different transport steps, including ER-to-Golgi intermediate compartment transport, Golgi transport, and/or intra-Golgi transport (anterograde transport and/or retrograde transport) [57, 58]. Two types of COPIcoated vesicles form containing anterograde or retrograde cargo (KDEL receptor), and low amounts of Golgi enzymes have been reported to exist at the Golgi apparatus level [59]. COPI proteins are involved in transport along the endocytic pathway [60]. During the selective transport of vesicles, the coat proteins must distinguish between cargo and resident proteins of the donor organelle. *Arabidopsis* has single genes for γ-COP and δ-COP and multiple genes for the other COPI subunits [61]. COPI coatomer forms a coat around vesicles budding from the Golgi. Two different sizes of COPI (COPIa and COPIb) vesicles have been identified by multiparameter electron tomography analysis in *Arabidopsis* [62]. COPIa coats are retrograde transport vesicles, and COPIb vesicles are restricted to medial- and trans-cisternae and are involved for retrograde transport within the Golgi stack. The multiple copies of COPI in plants suggest the presence of different classes of COPI vesicles. The protein complex COPI coatomer is composed of seven subunits (α, β, β', γ, δ, ε, and ζ-COP). COPI represents approximately 0.2% of soluble cytosolic protein indicating their roles as unassembled precursors of COPI vesicles [63].

In intracellular transport, cargo transmembrane protein sorting at each step depends on the specific interaction of certain signals in their cytoplasmic tails with the correct coat proteins [64]. In yeast and mammalian cells, the cytosolic dilysine motif is essential for the ER localization of type I membrane proteins [65]. The two lysine residues must be in the −3, −4 (KKXX) or − 3, −5 (KXKXX) positions relative to the carboxy (C) terminus [65]. For ER localization, the lysine residue at the −3 position is the most critical residue [66]. In mammals, lysine residue mutations within the KKXX motif lead to the expression of reporter proteins at the cell surface [65]. In contrast, the same mutation leads to vacuolar transfer in yeast [67]. The p24 proteins have been suggested to function in Golgi-to-ER retrograde transport, as they contain cargo receptors on their luminal side and coatomer and/or ARF1 receptors on their cytoplasmic side in mammalian cells [68] COPI is necessary for recycling p24 proteins to the ER from the Golgi apparatus [69].

In general p24 proteins are only found in the ER. The binding of p24 proteins to COPI is mediated by dilysine motifs at the −3 and − 4 positions of p24 [69]. Up to 11 different p24 family members proteins have been identified in *Arabidopsis*. The p24 proteins appear to bind COPI with higher affinity than COPII. In the cytosolic tail of the *Arabidopsis* p24 (Atp24), the dilysine motif is important both for binding of coatomer subunits and ARF1 [70].

**29**

to a younger cisternae.

**2.2 Vacuolar trafficking**

*Endomembrane Trafficking in Plants*

*DOI: http://dx.doi.org/10.5772/intechopen.91642*

proteins in retrograde transport [69].

transport and (ii) cisternal progression/maturation.

*2.1.2.2 Intra-Golgi transport*

ARF1 has been shown to localize to Golgi and endosomes and regulate cell proliferation, cell elongation, and fertility, whereas ARF6 is associated with plasma membrane and important for actin remodeling and receptor endocytosis in plant cells [54]. The ARF6 overexpression was shown in breast cancer cells and also involved ERK signaling during invasion. On the other hand, the use of ARF1

Low expression level of ARF1 (Q71L) mutant in tobacco mesophyll protoplasts has been reported to lead to wtp24 accumulation in the Golgi apparatus [69]. These studies verify the COPI-recycling mechanism can efficiently function in plants. COPI-binding dilysine motif-deficient p24 mutants are transported to the PVC and vacuole [69]. It was observed that p25 may function as an anchor for the p24

Two different models for intra-Golgi transport were suggested: (i) vesicular

Between two different models, the direction of COPI vesicles is a critical distinguishing factor. (i) The *vesicular transport model* assumes that anterograde cargo is transported between static cisternae by coordinated budding and fusion reactions of anterograde-directed COPI vesicles [72]. Retrograde-directed COPI vesicles antagonize the continuous loss of material at the trans-Golgi. Therefore, two different COPI vesicles are involved for this model, one mediating anterograde transport and the other mediating retrograde transport. (ii) In the *cisternal progression/maturation model*, Golgi cisternae are stable compartments. In anterograde COPII vesicles, secretory cargoes are transported from one cisterna to the next, which finally disassemble at the trans-Golgi. Anterograde cargo would not leave the

lumen, and resident Golgi proteins are maintained in the cisternae [72].

The COPI vesicles contain Golgi enzymes at a concentration that is up to 10 times higher than that found in the cisternae in animal cells [73]. The cisternal progression/maturation model does not clarify the presence of anterograde cargo within COPI vesicles or different anterograde cargo transportation rates in animal cells [74]. The "cisternal progression/maturation" model is the most widely accepted model for distinct and essential trafficking tasks in the Golgi. The stack of Golgi cisternae involves the historical record of progression from entry at the cis-face to exit at the trans-face [75]. The cargo molecules stay within a given cisternae as it passes, across a regular of seven locations within the Golgi stack on its way to the *trans*-face, and exit from the Golgi by transport carriers. In the cisternal progression, the newly arrived cargo in the Golgi exited with exponential kinetics rather than exhibiting a discrete lag or transit time [76]. Conserved oligomeric Golgi (COG) complex proteins accelerate the tethering of the vesicles to the target cisternae [77]. Resident Golgi proteins are assumed to recycle from older to younger cisternae. In retrograde COPI vesicles, transmembrane Golgi proteins may recycle. Peripheral Golgi proteins may recycle by dissociating from a given cisternae and then bind and combine

Plants have a complex vacuolar transport system different from that of mammalian systems by assigning evolutionarily conserved machinery to unique trafficking pathways. These pathways provide a fundamental basis for plant development at the cellular and higher-ordered levels [78]. Plants have evolved unique and complex vacuolar trafficking pathways compared with non-plant systems (**Figure 3**) [46].

protein as a prognostic marker for gastric cancer has been reported [71].

#### *Endomembrane Trafficking in Plants DOI: http://dx.doi.org/10.5772/intechopen.91642*

*Electrodialysis*

cytosolic tails [56].

ARF1 manages ER-to-Golgi transport and Golgi-derived transport to the plasma

membrane, depending on the COPI vesicle coat protein components [53, 54]. A large number of ARF in plants suggest the possibility for highly regulated vesicle trafficking [53]. Similar to other small GTPases, ARF GTPases cycle between an active state, when associated with GTP (membrane-bound form), and an inactive

In its GDP form, ARF1 is present in the cytosol and is recruited to the surface of Golgi membranes by a GEF. A SEC7-type GEF stimulates the binding of GTP to ARF1. This progression can be inhibited by the fungal metabolite Brefeldin A (BFA) in mammalian cells [55]. In the same system, the GDP-bound form of ARF1 interacts with p24 cytosolic tails [56]. The cytosolic ARF1 activation initiates COPI biogenesis. The GTP-bound form of ARF1 interacts with coatomer, which can also interact directly with the p24 cytosolic tails. In this manner, the p24 cytosolic tail can interact both with ARF1 and coatomer [56]. The conformational change of ARF1 occurs by the GTP/GDP exchange that may cause its dissociation from p24

COPI vesicles mediate different transport steps, including ER-to-Golgi intermediate compartment transport, Golgi transport, and/or intra-Golgi transport (anterograde transport and/or retrograde transport) [57, 58]. Two types of COPIcoated vesicles form containing anterograde or retrograde cargo (KDEL receptor), and low amounts of Golgi enzymes have been reported to exist at the Golgi apparatus level [59]. COPI proteins are involved in transport along the endocytic pathway [60]. During the selective transport of vesicles, the coat proteins must distinguish between cargo and resident proteins of the donor organelle. *Arabidopsis* has single genes for γ-COP and δ-COP and multiple genes for the other COPI subunits [61]. COPI coatomer forms a coat around vesicles budding from the Golgi. Two different sizes of COPI (COPIa and COPIb) vesicles have been identified by multiparameter electron tomography analysis in *Arabidopsis* [62]. COPIa coats are retrograde transport vesicles, and COPIb vesicles are restricted to medial- and trans-cisternae and are involved for retrograde transport within the Golgi stack. The multiple copies of COPI in plants suggest the presence of different classes of COPI vesicles. The protein complex COPI coatomer is composed of seven subunits (α, β, β', γ, δ, ε, and ζ-COP). COPI represents approximately 0.2% of soluble cytosolic protein indicat-

state when bound to GDP (predominantly cytosolic form).

ing their roles as unassembled precursors of COPI vesicles [63].

recycling p24 proteins to the ER from the Golgi apparatus [69].

of coatomer subunits and ARF1 [70].

In intracellular transport, cargo transmembrane protein sorting at each step depends on the specific interaction of certain signals in their cytoplasmic tails with the correct coat proteins [64]. In yeast and mammalian cells, the cytosolic dilysine motif is essential for the ER localization of type I membrane proteins [65]. The two lysine residues must be in the −3, −4 (KKXX) or − 3, −5 (KXKXX) positions relative to the carboxy (C) terminus [65]. For ER localization, the lysine residue at the −3 position is the most critical residue [66]. In mammals, lysine residue mutations within the KKXX motif lead to the expression of reporter proteins at the cell surface [65]. In contrast, the same mutation leads to vacuolar transfer in yeast [67]. The p24 proteins have been suggested to function in Golgi-to-ER retrograde transport, as they contain cargo receptors on their luminal side and coatomer and/or ARF1 receptors on their cytoplasmic side in mammalian cells [68] COPI is necessary for

In general p24 proteins are only found in the ER. The binding of p24 proteins to COPI is mediated by dilysine motifs at the −3 and − 4 positions of p24 [69]. Up to 11 different p24 family members proteins have been identified in *Arabidopsis*. The p24 proteins appear to bind COPI with higher affinity than COPII. In the cytosolic tail of the *Arabidopsis* p24 (Atp24), the dilysine motif is important both for binding

**28**

ARF1 has been shown to localize to Golgi and endosomes and regulate cell proliferation, cell elongation, and fertility, whereas ARF6 is associated with plasma membrane and important for actin remodeling and receptor endocytosis in plant cells [54]. The ARF6 overexpression was shown in breast cancer cells and also involved ERK signaling during invasion. On the other hand, the use of ARF1 protein as a prognostic marker for gastric cancer has been reported [71].

Low expression level of ARF1 (Q71L) mutant in tobacco mesophyll protoplasts has been reported to lead to wtp24 accumulation in the Golgi apparatus [69]. These studies verify the COPI-recycling mechanism can efficiently function in plants. COPI-binding dilysine motif-deficient p24 mutants are transported to the PVC and vacuole [69]. It was observed that p25 may function as an anchor for the p24 proteins in retrograde transport [69].

#### *2.1.2.2 Intra-Golgi transport*

Two different models for intra-Golgi transport were suggested: (i) vesicular transport and (ii) cisternal progression/maturation.

Between two different models, the direction of COPI vesicles is a critical distinguishing factor. (i) The *vesicular transport model* assumes that anterograde cargo is transported between static cisternae by coordinated budding and fusion reactions of anterograde-directed COPI vesicles [72]. Retrograde-directed COPI vesicles antagonize the continuous loss of material at the trans-Golgi. Therefore, two different COPI vesicles are involved for this model, one mediating anterograde transport and the other mediating retrograde transport. (ii) In the *cisternal progression/maturation model*, Golgi cisternae are stable compartments. In anterograde COPII vesicles, secretory cargoes are transported from one cisterna to the next, which finally disassemble at the trans-Golgi. Anterograde cargo would not leave the lumen, and resident Golgi proteins are maintained in the cisternae [72].

The COPI vesicles contain Golgi enzymes at a concentration that is up to 10 times higher than that found in the cisternae in animal cells [73]. The cisternal progression/maturation model does not clarify the presence of anterograde cargo within COPI vesicles or different anterograde cargo transportation rates in animal cells [74].

The "cisternal progression/maturation" model is the most widely accepted model for distinct and essential trafficking tasks in the Golgi. The stack of Golgi cisternae involves the historical record of progression from entry at the cis-face to exit at the trans-face [75]. The cargo molecules stay within a given cisternae as it passes, across a regular of seven locations within the Golgi stack on its way to the *trans*-face, and exit from the Golgi by transport carriers. In the cisternal progression, the newly arrived cargo in the Golgi exited with exponential kinetics rather than exhibiting a discrete lag or transit time [76]. Conserved oligomeric Golgi (COG) complex proteins accelerate the tethering of the vesicles to the target cisternae [77]. Resident Golgi proteins are assumed to recycle from older to younger cisternae. In retrograde COPI vesicles, transmembrane Golgi proteins may recycle. Peripheral Golgi proteins may recycle by dissociating from a given cisternae and then bind and combine to a younger cisternae.

#### **2.2 Vacuolar trafficking**

Plants have a complex vacuolar transport system different from that of mammalian systems by assigning evolutionarily conserved machinery to unique trafficking pathways. These pathways provide a fundamental basis for plant development at the cellular and higher-ordered levels [78]. Plants have evolved unique and complex vacuolar trafficking pathways compared with non-plant systems (**Figure 3**) [46].

#### *Electrodialysis*

The diverse functions of plant vacuoles are fulfilled through tight regulation of trafficking to and from the vacuoles, which involves evolutionarily conserved machinery components including Rab GTPases [79]. However, the basic framework of the Rab GTPase action is well conserved in eukaryotic cells [80]. Recent comparative genomic studies suggest that each eukaryotic lineage has acquired a unique repertoire of Rab GTPases during evolution [81] Autophagy-related and Golgi-independent transport from the ER to the vacuole is another example of such trafficking pathway [82]. This pathway also involves an exocyst subcomplex, although Rab and SNARE molecules associated with this pathway have not been identified thus far.

#### *2.2.1 RAB GTPases*

RAB GTPases constitute the largest family of small GTPases; 57 members are encoded in the *Arabidopsis* genome [83]. Based on their similarity to animal RAB GTPases, RAB GTPases are grouped into eight clades, i.e., RAB1/RABD, RAB2/ RABB, RAB5/RABF, RAB6/RABH, RAB7/ RABG, RAB8/RABE, RAB11/RABA, and RAB18/RABC in angiosperms [84]. Plants also harbor a unique set of Rab GTPases partly characterized by diversification of the RAB5 group acting in endosomal/ vacuolar trafficking pathways [85]. RAB7 is also proposed to regulate vacuolar traffic in plants [86]. In animal cells, a sequential action of RAB5 and RAB7 mediated by the effector complex HOPS [18] and a guanine nucleotide exchange factor for RAB7 consisting of SAND1/Mon1 and CCZ1 is responsible for the maturation from early to late endosomal compartments (**Figure 4**) [86].

Future studies on the molecular mechanisms of these plant-specific vacuolar trafficking pathways will reveal how plants have used unique vacuolar trafficking routes and how plants have developed their unique vacuolar trafficking pathways during evolution. The tethering of transport vesicles to the target membranes is mediated by the interaction between RAB GTPases and specific sets of tethering factors, many of which have been shown to be RAB effectors, which bind to specific RABs at the GTP-bound active state in yeast and animal systems [80]. After the tethering of transport vesicles by the tethering factors, soluble N-ethylmaleimidesensitive-factor attachment protein receptors lead to the membrane fusion [80]. Tethering factors comprise long coiled-coil proteins and protein complexes called

#### **Figure 4.**

*RAB and the other proteins in intracellular trafficking (modified from Malaria Parasite Metabolic Pathways [47]).*

**31**

**Figure 5.**

*Endomembrane Trafficking in Plants*

proteins are not conserved [84].

*2.3.1 Clathrin-mediated pathway*

**2.3 The endocytic pathway**

*DOI: http://dx.doi.org/10.5772/intechopen.91642*

tethering complexes. Each tethering step is mediated by a specific tethering complexes such as COG (functioning in retrograde trafficking within the Golgi), HOPS-CORVET (tethering with the lysosome/vacuole), and exocyst (functioning in the last step of the secretory pathway). In plants, homologous genes encoding these tethering complex proteins are also found, whereas some fibrous coiled-coil

Endocytosis in plant cells has an essential role for basic cellular functions and communication with the environment. Through the formation of closed membrane vesicles (60–120 nm), the uptake of extracellular molecules or the internalization of plasma membrane lipids and proteins is achieved [37]. During the life cycle of the plant, endosomes have vital importance for different processes including lateral organ differentiation, hormone signal transduction, root hair formation, embryo patterning, and plant immunity [36, 87–92]. Transportation of various cargo molecules involved in a broad range of physiological processes from the plasma membrane into the cytoplasm is achieved by this pathway. **Figure 5** shows the general

As in animal cells, endocytosis in plant cells is mediated by (i) *clathrin-mediated*

In plants, the major endocytic mechanism depends on the coat protein clathrin. This pathway starts at the plasma membrane by clathrin-coated vesicle formation. CME is important for different physiological processes involving cell signaling, cell adhesion, nutrient uptake, developmental regulation, etc. The pathway starts by clathrin-coated vesicle formation at the plasma membrane; in the cytosolic parts of different transmembrane cargo molecules, the clathrin coat binds to specific binding sites. The recruitment of the pioneer proteins to the plasma membrane is ensured by the cargo molecules and enhanced by the initiation of an endocytic pathway. Clathrin involved in a variety of other processes such as the salt stress response, the defense response, cryptogein-induced signaling, and cytokinesis [94–96].

*The general organization of the endosomal trafficking system in plants. Current models of retromer localization and function place the retromer recycling complex in the TGN, in the MVB, or both [36].*

organization of the endosomal trafficking system in plants [36].

(CME) and (ii) *clathrin-independent pathways* (CIE) [93].

#### *Endomembrane Trafficking in Plants DOI: http://dx.doi.org/10.5772/intechopen.91642*

tethering complexes. Each tethering step is mediated by a specific tethering complexes such as COG (functioning in retrograde trafficking within the Golgi), HOPS-CORVET (tethering with the lysosome/vacuole), and exocyst (functioning in the last step of the secretory pathway). In plants, homologous genes encoding these tethering complex proteins are also found, whereas some fibrous coiled-coil proteins are not conserved [84].
