The Secretory/Endomembrane System of Arabidopsis
The endomembrane system of all eukaryotes consists of those membrane-bound organelles that exchange lipid and cargo by vesicle trafficking. Despite the constant exchange of vesicles, each organelle/compartment has a relatively constant array of resident membrane and lumenal proteins. In the figure, resident proteins of the endoplasmic reticulum (ER) are red, those of the Golgi are orange, those of the vacuole are yellow, those of the endosomes (EE) are purple. Secreted proteins (green) end up either in the plasma membrane (PM) or are released into the extracellular matrix (ECM; equivalent to the “cell wall” or “apoplast”). Peroxisomal (Pex) proteins, some of which are first trafficked through the ER, are indicated in blue.

Vesicle Formation at the Donor Compartment
Vesicle formation is accomplished by a similar process at each compartment, though the individual protein machinery varies at the different compartments. The basic process is the same at each compartment: a coat-GTPase cooperates with a cargo-selective coat subunit to collect cargo molecules at the bud site, next a “cage” subunit is recruited from the cytosol to physically deform the lipid bilayer into a vesicle. Vesicle formation at the transitional ER (tER) is accomplished by the COP-II coat system (top panel at right). At the cis-Golgi, vesicle formation is accomplished by the COP-I coat system (second panel at right). Recovery of ER-resident proteins is accomplished by a cargo receptor called the KDEL-Receptor (KDEL-R, encoded by ERD2) which is a membrane protein that binds to ER-retrieval signals (KDEL-COOH) on the lumenal side, and recruits coat components on the cytosolic side. At the TGN, vacuolar cargo is selected by a cargo receptor (VS-R; see text) which recognizes vacuolar sorting signals on the lumenal side and recruits the clathrin adaptor complex (AP-1) on the cytosolic side (third panel at right). Clathrin coats (in this case using AP-2 adaptor) also operate at the PM to accomplish the first step of endocytosis (lower panel at right). In other organisms (i.e., mammals) there are many cargo receptors which serve to internalize extracellular cargo but little is known about such proteins in plants.

The Sub-Complexes of the Three Major Coat Systems.
Each coat system can be considered to represent three major sub-complexes: a coat-GTPase, a “cargo-selective” complex and a “cage” complex. In some cases, these individual subunits have a clear evolutionary relationship: The Sar and Arf GTPases share a common ancestor among the small GTPases, and the subunits of the four clathrin adaptors are clear examples of gene duplication and also share a common ancestor with the components of F-COP. The three cage subunits also share many common protein folds like the α-solenoid and WD-40 motifs, but this state may be convergent rather than ancestral (see Stag et al., 2007).

The Early Endomembrane System: ER and Golgi.
(A) The ER is typically observed as a reticulate pattern that ramifies throughout the cytosol, here visualized by confocal microscopy in a leaf epidermal cell expressing GFP-Calnexin (see: Irons et al., 2003). Early Golgi markers, such as GFP-ERD2 (the KDEL-receptor; see Boevink et al., 1998) show bright spots corresponding to the Golgi stacks (arrow), but also have a small percentage of protein that is also found in the ER (open arrow).

Endosomal Compartments in Plants
Endosomal compartments in plants have long been under examined due to a lack of suitable marker proteins, however, new marker proteins have recently been developed allowing observation of these compartments through both fluorescent and electron microscopy. (A)-(C) Partial co-localization of GFP-SKD1 and a fluorescent fusion protein of the endosomal Rab GTPase RHA1/RabF2a in Arabidopsis roots cells. Arrows indicate areas of colocalization. Bars = 10 m. (D)-(E) Immunogold localization of endosomal proteins in high-pressure frozen/freeze-substituted wild type Arabidopsis roots. Gold labeling shows localization of RHA1/RabF2a (D) and SKD1 (E) on multivesicular bodies or MVBs. Bars = 200 nm. (F)-(H) Tomographic models of prevacuolar compartments/MVBs during formation of protein storage vacuoles in the Arabidopsis embryo reconstructed from electron tomograms. The limiting membranes of MVBs have been made translucent in these tomographic models to visualize the internal vesicles (arrows) and electron dense aggregates of vacuolar storage proteins (stars). Images (A) to (E) are from Haas et al. (2007); images (F)-(H) are from Otegui et al. (2006). Bars= 100 nm

The Secretory Pathway, Endocytic Pathway and Retrograde/Recycling Pathways.
The traditional secretory pathways begins by translocation of proteins (yellow arrow) into the endoplasmic reticulum (ER), followed by transport in vesicles to the Golgi by bulk flow (default secretion, solid black line). From the Golgi, secretory proteins are transported to the Plasma Membrane (PM) or extra-cellular matrix (ECM, “cell wall”). Also in the Golgi, vacuolar proteins are re-directed to the vacuole (by way of the late endosome) due to specific sorting signals (vacuolar sorting, dashed brown line). Cargo that is endocytosed from the PM/ECM is transported through the endosomes by bulk flow (default endocytosis, solid blue line), first to the early endosomes, then to the late endosomes where endocytic cargo meets vacuolar cargo, and finally the cargo arrives in the vacuole. Many signal-mediated retrograde pathways operate to recycle specific cargo at most compartments (dashed green lines). Finally, several specialized sorting pathways serve to transport peroxisomal proteins from the ER to peroxisomes or to transport some glycosylated proteins from the Golgi to the plastid (dashed cyan lines; see text).

Docking and Fusion of a Vesicle at the Target Compartment.
Docking and fusion of a vesicle is also accomplished by a similar process at each trafficking step, again the individual protein machinery varies. Identification of the target compartment for a particular vesicle is mediated by a member of the Rab family of small GTPases which acts on both the vesicle and target membrane to recruit tethering and docking factors (TDF) and other effectors that serve to attach the vesicle to the target membrane. Once a vesicle is tethered, a member of the SM family of proteins mediates assembly of the target-(t)-SNAREs into a three SNARE-helix bundle that serves as a binding site for the vesicle-(v)-SNARE helix. Mutual twisting of the SNARE helices pulls the membranes into close proximity and drives fusion of the bilayers. (Only one set of SNAREs is shown, fusion is likely mediated by multiple SNARE complexes surrounding the fusion site.) Following fusion of the vesicle, lumenal cargo is delivered into the target compartment lumen and vesicle membrane proteins and lipids are now part of the target membrane. The final four-helix SNARE complex is resolved by the action of a complex of proteins (SNAP and NSF) that use ATP energy to unwind the helices of the individual SNARE proteins for subsequent rounds of fusion (t-SNAREs) or for recycling back to the donor compartment (v-SNARE).

Retromer and ESCRT Coat Machinery
The retromer coat is responsible for recycling of proteins (e.g., the vacuolar cargo receptor) from the endosomal compartments back to the Golgi. Coat formation is superficially similar to that of the other coat complexes (see Figure 3), with the complex of Vps26, Vps29 and Vps35 functioning to bind and collect cargo proteins and the sorting nexin/Vps5 subunit functioning as a cage, but the mechanisms appears to be distinct. Similarly, the ESCRT system has subunits that function in cargo recognition and some variation of a “cage”, but it is topologically reversed to create a vesicle inside the lumen of the endosomal compartments (though evidence of the protein coat having a role in membrane deformation as shown in the figure is controversial). The ESCRT-I, -II and –III sub-complexes act sequentially to identify cargo and form a “cage” that leads to the invagination in the endosomal membranes. A large ATPase (SKD1) then acts to release the ESCRT complexes back to the cytosol prior to enclosure of the lumenal vesicle. These internal vesicles are ultimately delivered to the vacuolar lumen where their contents are degraded.

The Plasma Membrane in Polarized Cells
In addition to simply forming the limiting membrane of the plant cell, the PM has recently been found to have multiple kinds of specialized sub-domains in cell that are polarized or that are responding to particular stresses or developmental signals. For example, in studies reported by Assaad et al (2004), an epidermal cell responds to infection by powdery mildew (Erysiphe cichoracearum; red) by establishment of a particular PM structure called papillae. Two PM-SNAREs, Qa-PEN1 (A) and Qa-SYP122 (B), visualized by confocal microscopy in cell expressing GFP- or CFP-fusions (respectively), become enriched in these domains and are essential for resisting the fungal infection. In a similar way, the Qa-SNARE KNOLLE is also important for establishment of the cell plate in dividing cells (C),here visualized in the root tips of plants expressing KNOLLE::CFP-KNOLLE. The best studied examples of polarization in plant cells has been shown by the auxin efflux (PINs) and influx (AUX1s) transporters. For example, in work reported in Kleine-Vehn et al. (2006) the authors performed whole-mount immunofluorescent microscopy to show how the protophloem cells of the root have independent apical and basal domains enriched in AUX1 and PIN1 (respectively (D-F)). Images (A) and (B) are from Assaad et al., 2004; images (D-F) are from Kleine-Vehn et al., 2006.

Vacuolar Compartments in Plants
In most mature vegetative cells, the majority of the cell volume is occupied by the large central lytic vacuole. This can be visualized by confocal microscopy of cells expressing 35S::GFP-δ-TIP (J. Zouhar, E. Avila, and N.V.R., unpublished) in either epidermal cells of the cotyledon (left) or hypocotyl cells (right).