**1. Introduction**

Endomembrane trafficking plays a crucial role for maintaining fundamental cellular functions (signal transduction, cellular homeostasis, etc.) and in response to environmental stimuli. The membrane trafficking pathways start from the endoplasmic reticulum (ER) then go through the Golgi apparatus to different destinations including vacuoles/lysosomes, endosomes, and the plasma membrane (PM) [1].

In plant cells the membrane trafficking system comprises three major trafficking pathways: the biosynthetic secretory pathway, the endocytic pathway, and the vacuolar transport pathway. (i) *The biosynthetic secretory pathway* transports newly synthesized proteins from the endoplasmic reticulum to the plasma membrane

and/or the extracellular space. (ii) *The endocytic pathway* functions in the recycling of PM-localized and extracellular factors between the PM and the endosomal compartments. (iii) *The vacuolar transport pathway* drives the transportation of newly synthesized protein to the vacuole (**Figure 1**) [3].

Each trafficking pathway is mediated by the following steps: (i) budding of the transport vesicle from the donor membrane, which is mediated by ARF/SAR1 GTPase (and coat proteins in many cases); (ii) transport and targeting of the transport vesicle; (iii) tethering of the vesicle by tethering proteins under the regulation of RAB GTPase and fusion of the transport vesicle to the target membrane mediated by soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNARE); and (iv) recycling of the transport machinery components to the donor membrane (**Figure 2**).

In eukaryotic cells, small GTP-binding proteins involve the largest family of signaling proteins. The activation of small GTP-binding proteins (GTPases) is essential for vesicle formation from a donor membrane. Four main subfamilies have been identified in plants: (i) ADP-ribosylation factor (*ARF*)/secretion-associated RAS superfamily (*SAR*), (ii) *RAB*, (iii) Rho-like proteins in plants (*ROP*), and (iv) *RAN* [5–7]. Over the evolution of eukaryotic organisms, the conservation of GTPases explains their significance in cellular signaling processes [7–9]. Small GTPases serve as molecular switches that transduce signals by exchanging between the GTP- and GDP-bound conditions. Guanine nucleotide exchange factors (GEFs), GDP dissociation inhibitors (GDIs), and GTPase-activating proteins are regulators of small GTP-binding proteins.

GEFs activate small GTPases, which in turn interact with specific effectors to stimulate downstream pathways. GAPs trigger the intrinsic GTPase activity, thereby accelerating the inactivation of the GTPases' regulatory activity. GEFs convert the GDP-bound inactive form of the GTPases to the GTP-bound active form by stimulating the dissociation of GDP from the GDP-bound form. In the "active" state, the GTP-bound GTPases interact with various downstream effector proteins that execute diverse cellular functions. GTPases are inactivated through either the intrinsic capability of the GTPase to hydrolyze GTP to GDP + Pi or an interaction with another protein group, the GTPase-activating proteins (GAPs). These proteins

#### **Figure 1.**

*The membrane trafficking pathways are grouped into three major categories: (i) the biosynthetic secretory pathway, (ii) the endocytic pathway, and (iii) the vacuolar transport pathway (modified from Inada and Ueda [2]).*

**25**

**Figure 2.**

*donor membrane [2, 4].*

*Endomembrane Trafficking in Plants*

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

proteins within the endomembrane system.

**2. Membrane trafficking pathways**

**2.1 The biosynthetic secretory pathway**

*2.1.1.1 ER-to-Golgi protein transport*

*2.1.1 Anterograde transport (forward trafficking)*

sorted from the Golgi into the chloroplast [13].

catalyze the hydrolytic activity of GTPases, which then return to the inactive state GDP-bound state [6]. The improvement of fluorescent protein-labeled GTPases and cargo molecules has enhanced the assignment of subcellular locations for these

*The general machinery of membrane trafficking. Each trafficking pathway is mediated by the following steps: (i) budding of the transport vesicle from the donor membrane, which is mediated by ARF/SAR1 GTPase (and coat proteins in many cases); (ii) transport and targeting of the transport vesicle; (iii) tethering of the vesicle by tethering proteins under the regulation of RAB GTPase and fusion of the transport vesicle to the target membrane mediated by SNARE proteins; and (iv) recycling of the transport machinery components to the* 

The traffic between organelles is bi-directional. (i) Starting at the ER and leading toward the destination organelles (the forward trafficking) is called anterograde

The conventional trafficking pathway starts at the ER; protein synthesis and modification occurs and undergoes further modification [10]. Proteins leave the ER via COPII carriers to reach the Golgi. ER and Golgi compartments are closely associated with each other to ease the movement of cargo between them [11]. This ER-to-Golgi transport is termed "anterograde transport" and is mediated by COPII proteins, which are highly conserved in eukaryotes [12]. From the ER, synthesized proteins are exported to the cis-Golgi and are transported via Golgi stacks where protein modifications occur. Modified proteins are sorted into the extracellular space or storage and lytic organelles from the Golgi. In plants, proteins can also be

transport, and (ii) the reverse pathway is called retrograde transport.

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

#### **Figure 2.**

*Electrodialysis*

membrane (**Figure 2**).

of small GTP-binding proteins.

and/or the extracellular space. (ii) *The endocytic pathway* functions in the recycling of PM-localized and extracellular factors between the PM and the endosomal compartments. (iii) *The vacuolar transport pathway* drives the transportation of

Each trafficking pathway is mediated by the following steps: (i) budding of the transport vesicle from the donor membrane, which is mediated by ARF/SAR1 GTPase (and coat proteins in many cases); (ii) transport and targeting of the transport vesicle; (iii) tethering of the vesicle by tethering proteins under the regulation of RAB GTPase and fusion of the transport vesicle to the target membrane mediated by soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNARE); and (iv) recycling of the transport machinery components to the donor

In eukaryotic cells, small GTP-binding proteins involve the largest family of signaling proteins. The activation of small GTP-binding proteins (GTPases) is essential for vesicle formation from a donor membrane. Four main subfamilies have been identified in plants: (i) ADP-ribosylation factor (*ARF*)/secretion-associated RAS superfamily (*SAR*), (ii) *RAB*, (iii) Rho-like proteins in plants (*ROP*), and (iv) *RAN* [5–7]. Over the evolution of eukaryotic organisms, the conservation of GTPases explains their significance in cellular signaling processes [7–9]. Small GTPases serve as molecular switches that transduce signals by exchanging between the GTP- and GDP-bound conditions. Guanine nucleotide exchange factors (GEFs), GDP dissociation inhibitors (GDIs), and GTPase-activating proteins are regulators

GEFs activate small GTPases, which in turn interact with specific effectors to stimulate downstream pathways. GAPs trigger the intrinsic GTPase activity, thereby accelerating the inactivation of the GTPases' regulatory activity. GEFs convert the GDP-bound inactive form of the GTPases to the GTP-bound active form by stimulating the dissociation of GDP from the GDP-bound form. In the "active" state, the GTP-bound GTPases interact with various downstream effector proteins that execute diverse cellular functions. GTPases are inactivated through either the intrinsic capability of the GTPase to hydrolyze GTP to GDP + Pi or an interaction with another protein group, the GTPase-activating proteins (GAPs). These proteins

*The membrane trafficking pathways are grouped into three major categories: (i) the biosynthetic secretory pathway, (ii) the endocytic pathway, and (iii) the vacuolar transport pathway (modified from Inada and* 

newly synthesized protein to the vacuole (**Figure 1**) [3].

**24**

**Figure 1.**

*Ueda [2]).*

*The general machinery of membrane trafficking. Each trafficking pathway is mediated by the following steps: (i) budding of the transport vesicle from the donor membrane, which is mediated by ARF/SAR1 GTPase (and coat proteins in many cases); (ii) transport and targeting of the transport vesicle; (iii) tethering of the vesicle by tethering proteins under the regulation of RAB GTPase and fusion of the transport vesicle to the target membrane mediated by SNARE proteins; and (iv) recycling of the transport machinery components to the donor membrane [2, 4].*

catalyze the hydrolytic activity of GTPases, which then return to the inactive state GDP-bound state [6]. The improvement of fluorescent protein-labeled GTPases and cargo molecules has enhanced the assignment of subcellular locations for these proteins within the endomembrane system.

The traffic between organelles is bi-directional. (i) Starting at the ER and leading toward the destination organelles (the forward trafficking) is called anterograde transport, and (ii) the reverse pathway is called retrograde transport.

## **2. Membrane trafficking pathways**

#### **2.1 The biosynthetic secretory pathway**

#### *2.1.1 Anterograde transport (forward trafficking)*

## *2.1.1.1 ER-to-Golgi protein transport*

The conventional trafficking pathway starts at the ER; protein synthesis and modification occurs and undergoes further modification [10]. Proteins leave the ER via COPII carriers to reach the Golgi. ER and Golgi compartments are closely associated with each other to ease the movement of cargo between them [11]. This ER-to-Golgi transport is termed "anterograde transport" and is mediated by COPII proteins, which are highly conserved in eukaryotes [12]. From the ER, synthesized proteins are exported to the cis-Golgi and are transported via Golgi stacks where protein modifications occur. Modified proteins are sorted into the extracellular space or storage and lytic organelles from the Golgi. In plants, proteins can also be sorted from the Golgi into the chloroplast [13].

The accumulation of secretory cargo, deformation of the membrane, and formation of transport vesicles are mediated by COPII. In mammalian cells and in most of the plant cell types, the ER and Golgi are in close proximity, and the COPII cycles on and off the ER with a fast turnover rate [14, 15]. COPII coat proteins are mostly distributed in the cytosol and concentrate at ERES that appear in association with motile Golgi stacks in plant cells. These proteins also accumulate in punctate structures that are not associated with the Golgi (**Figure 2B**).

The recruitment of COPII coat proteins involves SAR1 GTPase and its GDP/ GTP exchange factor, SEC12 [16, 17]. The *Arabidopsis* genome encodes five genes for SAR1, seven genes SEC23, three genes for SEC24, two genes SEC13, and two SEC31 isoforms [18].

The assembly of COPII occurs at distinct sites on ribosome-free transitional ER (tER) or ER exit sites (ERESs) [19]. The cytosolic GTPase SAR1 is activated by the ER membrane-associated GEF SEC12, and then SAR1 associates with the ER lipid bilayer membrane, and after the COPII coat composed of the SEC23-24 and SEC13-31 heterodimer complexes is recruited [20–22]. The cargo recruitment complex involving SEC23-24 and SAR1 sorts transport and ER resident proteins [23, 24]. The COPII coat includes four proteins, assembled as an internal receptor/cargo-binding dimer of SEC23 and SEC24 and an outer cage dimer of SEC31 and SEC13. SEC16 is important for ER protein export by recognizing the COPII assembly region at the ERES [25]. The cargo selection is achieved by the SEC23/SEC24-SAR1 complex (pre-budding complex) [26]. This complex recruits SEC13-SEC31, which offer the outer layer of the coat and manage membrane deformation to constitute COPII vesicles. The SEC16 and SED4 are the other additional proteins for the COPII assembly. SEC16 comprises COPII coat component domains and has an important role as a scaffold for coat assembly [27]. SEC16 is a key organizer of ERESs in yeast and mammalian cells [28, 29]. The two encoded from *Arabidopsis* SEC16 genes resemble the human small isoform, and it was shown that they are important for ER export and tER organization in HeLa cells [29].

COPII is also involved in the physical deformation of the ER membrane that drives the COPII carrier formation [30]. SAR1-mediated GTP hydrolysis leads to COPII carrier un-coating and follows the exposure of the carrier membrane to fusion with the Golgi membrane [31].

In the GTP-bound conformation, SAR1 protein binds directly to the lipid bilayer which it does by an N-terminal amphipathic alpha-helix [32]. In the GDP-bound conformation, SAR1 binds membranes with lower affinity [25, 31].

The SED4 is responsible for the rate of ER-to-Golgi transport as an integral membrane protein at the ER membrane [32]. The deletion of SED4 causes to reduce the transportation rate of ER-to-Golgi in *S. cerevisiae* wild-type cells [33]. The SED4 and SEC12 have close homology with the cytoplasmic domain, but no GEF activity has been reported in *S. cerevisiae* [34].

It has been reported that SAR1 reduces the mechanical rigidity of the lipid bilayer membrane to which it binds in yeast [35]. Because of the ability of SAR1, it was suggested that membrane-bound SAR1-GTP decreases the energetic cost for the other COPII coat proteins (Sec13, Sec31, etc.) to generate curvature [35].

In *Arabidopsis*, three SAR1 homologs have been identified (AtSARA1a, AtSARA1b, and AtSARA1c). AtSARA1A and AtSARA1B have a 93% amino acid sequence identity [36]. The AtSARA1a expression level correlated with the secretion activity level from ER membranes. The AtSARA1a mRNA upregulation has been reported to cause the blockage of ER transport to the cis-Golgi compartment [37]. The COPII protein-encoding genes are ubiquitously expressed except SAR1 (At1g09180) and a SEC31 (At1g18830) isoform by microarray analyses [18]. Tissue

**27**

**Figure 3.**

*route (modified from Ebine et al. [46]).*

*Endomembrane Trafficking in Plants*

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

specificity was observed for the SEC31 isoform At1g18830, while all other genes appear to be ubiquitously expressed in all tissues and developmental stages [18]. SAR1 accumulation was observed to concentrate predominantly in crude ER fractions of *Pisum sativum* L. seedlings [38]. The COPII protein coat recruitment by SAR1p has been intensively studied [39]. In human development and disease, the SEC24 and SAR1 isoforms have specificity for the trafficking of selective cargo [40, 41]. In plant cells, specific amino acid sequences in the primary proteins affect the selective export of membrane cargo [42]. Diacidic sequence induces the accumulation of SEC24A to ERESs. The interaction occurs between the K channel KAT1, which contains the

Different export signals in SEC24 proteins might lead to selective accumulation of cargo in COPII carriers in mammalian and plant cells. It was suggested that more efficient intracellular trafficking is likely achieved by cargo specialization of COPII isoforms in multicellular organisms [45]. **Figure 3** shows the basic diagram of the

The COPII machinery has significant importance for ER-to-Golgi transport in the early secretory pathway in plants [48]. The retention of secretory cargo molecules or membrane proteins that cycle between the ER and Golgi apparatus leads to blockage of the ER export [14, 43, 48]. COPII machinery is involved in biotic and abiotic stress responses in plants. It has been shown that functional SEC24A is essential for systemic turnip mosaic virus movement by interaction in a signal specific manner with the N-terminal domain 6-kDa viral protein 6 K [49]. In high-temperature conditions, overexpression of SEC31A has been reported in the IRE1 mutant which

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.

*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* 

specific amino acid sequence in the cytosolic tail, and SEC24A [43, 44].

leads to improvement of the male sterility phenotype in *Arabidopsis* [50].

retrograde and anterograde transport in plant cells [47].

*2.1.2 Retrograde transport (reverse trafficking)*

*2.1.2.1 Retrograde transport and COPI*

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

*Electrodialysis*

isoforms [18].

HeLa cells [29].

fusion with the Golgi membrane [31].

has been reported in *S. cerevisiae* [34].

The accumulation of secretory cargo, deformation of the membrane, and formation of transport vesicles are mediated by COPII. In mammalian cells and in most of the plant cell types, the ER and Golgi are in close proximity, and the COPII cycles on and off the ER with a fast turnover rate [14, 15]. COPII coat proteins are mostly distributed in the cytosol and concentrate at ERES that appear in association with motile Golgi stacks in plant cells. These proteins also accumulate in punctate

The recruitment of COPII coat proteins involves SAR1 GTPase and its GDP/ GTP exchange factor, SEC12 [16, 17]. The *Arabidopsis* genome encodes five genes for SAR1, seven genes SEC23, three genes for SEC24, two genes SEC13, and two SEC31

The assembly of COPII occurs at distinct sites on ribosome-free transitional ER (tER) or ER exit sites (ERESs) [19]. The cytosolic GTPase SAR1 is activated by the ER membrane-associated GEF SEC12, and then SAR1 associates with the ER lipid bilayer membrane, and after the COPII coat composed of the SEC23-24 and SEC13-31 heterodimer complexes is recruited [20–22]. The cargo recruitment complex involving SEC23-24 and SAR1 sorts transport and ER resident proteins [23, 24]. The COPII coat includes four proteins, assembled as an internal receptor/cargo-binding dimer of SEC23 and SEC24 and an outer cage dimer of SEC31 and SEC13. SEC16 is important for ER protein export by recognizing the COPII assembly region at the ERES [25]. The cargo selection is achieved by the SEC23/SEC24-SAR1 complex (pre-budding complex) [26]. This complex recruits SEC13-SEC31, which offer the outer layer of the coat and manage membrane deformation to constitute COPII vesicles. The SEC16 and SED4 are the other additional proteins for the COPII assembly. SEC16 comprises COPII coat component domains and has an important role as a scaffold for coat assembly [27]. SEC16 is a key organizer of ERESs in yeast and mammalian cells [28, 29]. The two encoded from *Arabidopsis* SEC16 genes resemble the human small isoform, and it was shown that they are important for ER export and tER organization in

COPII is also involved in the physical deformation of the ER membrane that drives the COPII carrier formation [30]. SAR1-mediated GTP hydrolysis leads to COPII carrier un-coating and follows the exposure of the carrier membrane to

In the GTP-bound conformation, SAR1 protein binds directly to the lipid bilayer which it does by an N-terminal amphipathic alpha-helix [32]. In the GDP-bound

The SED4 is responsible for the rate of ER-to-Golgi transport as an integral membrane protein at the ER membrane [32]. The deletion of SED4 causes to reduce the transportation rate of ER-to-Golgi in *S. cerevisiae* wild-type cells [33]. The SED4 and SEC12 have close homology with the cytoplasmic domain, but no GEF activity

It has been reported that SAR1 reduces the mechanical rigidity of the lipid bilayer membrane to which it binds in yeast [35]. Because of the ability of SAR1, it was suggested that membrane-bound SAR1-GTP decreases the energetic cost for the other COPII coat proteins (Sec13, Sec31, etc.) to generate curvature [35]. In *Arabidopsis*, three SAR1 homologs have been identified (AtSARA1a, AtSARA1b, and AtSARA1c). AtSARA1A and AtSARA1B have a 93% amino acid sequence identity [36]. The AtSARA1a expression level correlated with the secretion activity level from ER membranes. The AtSARA1a mRNA upregulation has been reported to cause the blockage of ER transport to the cis-Golgi compartment [37]. The COPII protein-encoding genes are ubiquitously expressed except SAR1 (At1g09180) and a SEC31 (At1g18830) isoform by microarray analyses [18]. Tissue

conformation, SAR1 binds membranes with lower affinity [25, 31].

structures that are not associated with the Golgi (**Figure 2B**).

**26**

specificity was observed for the SEC31 isoform At1g18830, while all other genes appear to be ubiquitously expressed in all tissues and developmental stages [18].

SAR1 accumulation was observed to concentrate predominantly in crude ER fractions of *Pisum sativum* L. seedlings [38]. The COPII protein coat recruitment by SAR1p has been intensively studied [39]. In human development and disease, the SEC24 and SAR1 isoforms have specificity for the trafficking of selective cargo [40, 41]. In plant cells, specific amino acid sequences in the primary proteins affect the selective export of membrane cargo [42]. Diacidic sequence induces the accumulation of SEC24A to ERESs. The interaction occurs between the K channel KAT1, which contains the specific amino acid sequence in the cytosolic tail, and SEC24A [43, 44].

Different export signals in SEC24 proteins might lead to selective accumulation of cargo in COPII carriers in mammalian and plant cells. It was suggested that more efficient intracellular trafficking is likely achieved by cargo specialization of COPII isoforms in multicellular organisms [45]. **Figure 3** shows the basic diagram of the retrograde and anterograde transport in plant cells [47].

The COPII machinery has significant importance for ER-to-Golgi transport in the early secretory pathway in plants [48]. The retention of secretory cargo molecules or membrane proteins that cycle between the ER and Golgi apparatus leads to blockage of the ER export [14, 43, 48]. COPII machinery is involved in biotic and abiotic stress responses in plants. It has been shown that functional SEC24A is essential for systemic turnip mosaic virus movement by interaction in a signal specific manner with the N-terminal domain 6-kDa viral protein 6 K [49]. In high-temperature conditions, overexpression of SEC31A has been reported in the IRE1 mutant which leads to improvement of the male sterility phenotype in *Arabidopsis* [50].

#### *2.1.2 Retrograde transport (reverse trafficking)*
