Soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) mediate fusion between vesicular and target membranes. Usually, some of these transmembrane proteins reside on vesicular membranes in a single chain, while the others reside on target membranes in a three-protein complex
[39][40]. The assembly of SNAREs residing on both membranes to a stable four-helix bundle leads to membrane fusion. VAM3/SYP22 is a SNARE that localizes to both the MVB and the tonoplast. Since
vam3 mutant displayed depolarization of PIN1-GFP in leaves and vacuolar aggregation of PIN1-GFP was surrounded by mRFP-VAM3 labeled tonoplast under dark treatment, it is suggested that VAM3-mediated vacuolar trafficking is required for maintenance of PIN polarity
[41]. In addition, mutation in both SYP42 and SYP43 (
syp42 syp43), two SYP4 group members of TGN-localized SNARE
[40], led to defect of vacuolar accumulation of PIN2-GFP in the darkness
[42], also providing an indication of involvement of SYP4 in PIN vacuolar transport. Nonetheless, the regulatory effect of SNARE SYP4 is indirect and could be attributed to the influence on trafficking between the TGN and MVBs
[42].
7. The Retrograde Systems
The retromer is a conserved protein complex that regulates vesicular transport of transmembrane proteins from endosomes to the trans-Golgi network (TGN) and to the plasma membrane
[43]. It consists of a dimer of sorting nexins (SNXs), which contains PX domains binding to phosphatidylinositol 3-phosphate (PI3P) produced by VPS34 for membrane recruitment, and a trimer of VPS26, VPS29, and VPS35, which binds endocytic receptors for cargo selection
[44]. In Arabidopsis, the retromer protein VPS29, as well as the physical interactors VPS35 and VPS26, colocalize with SNX1 at the MVB and are required for MVB morphology
[16][45][46]. The complex controls endocytic recycling of specific cargos, such as PIN1 and PIN2, because loss of function of VPS29 just resulted in intracellular accumulation of PIN1-GFP and PIN2-GFP, but not AUX1-YFP and GFP-PIP2a, in SNX1-labled compartments
[46]. The improperly recycled PIN proteins in
vps29 mutant were then destinated for degradation
[16][46]. Similar alteration of PINs is also reported in
snx1 mutants
[16][47]. A posttranslational regulation is suggested in such context based on the finding that the decreased PIN2 protein levels in
snx1 mutants are independent of gene transcription
[16]. Recent work reveals that SNX1 forms a complex both with phosphatidylinositol 3-phosphate 5-kinase (FAB1) and its product phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P
2, which is required for the association of SNX1 with the endosome membrane) to facilitate maturation of SNX1 endosomes, and hence to impair PIN2 trafficking
[48].
8. The ESCRT Pathway
Sorting into the intralumenal vesicles of endosomes is a common step for membrane protein degradation in vacuoles. This process is controlled by the ESCRT (endosomal sorting complex required for transport), which is defined as a ubiquitin-dependent protein sorting pathway consisting of five distinct complexes, termed ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III, and vacuolar protein sorting4 (VPS4)
[49][50]. These complexes are sequentially required to interact with ubiquitinated membrane proteins and to drive their internalization into the intraluminal vesicles of MVBs. Therefore, the components of such complexes are involved in PIN degradation in vacuoles.
TOM (target of MYB) 1-likes (TOLs) have VHS (Vps27, Hrs, and STAM) and GAT (GGAs and TOM) domains similar to components of the ESCRT-0, which is essential for recruitment of the ESCRT-1 for initiating MVB-dependent cargo sorting. They function redundantly in recognizing ubiquitinated PINs and then regulating the endocytic sorting to vacuoles for protein degradation
[51]. Another ESCRT-0 subunit identified in plants is FYVE domain protein required for endosomal sorting1 (FREE1), which binds to PI3P, ubiquitin, and specifically interacts with VPS23A and VPS23B, subunits of the ESCRT-I complex, via PTAP-like tetrapeptide motifs
[52]. Mutation in FREE1 led to defects in MVB formation and mislocalization of vacuole-accumulated endocytosed PIN2 to the tonoplast
[52]. To some extent, the ESCRT-0 together with ubiquitin modification determines specificity of vacuolar targeting of membrane proteins, because multiple mutation of TOLs caused defect in forming WM compartments of PIN2-VENUS while not in that of FM4-64 labeled endomembrane
[51]. It is noteworthy that FREE1 does not merely regulate protein sorting to the vacuole but also autophagic degradation because of association between FREE1, SH3P2 (a unique plant autophagy regulator), and the PI3K complex I subunit ATG6
[53]. ELC is an Arabidopsis homolog of VPS23 displaying the binding activity to ubiquitin. It forms the ESCRT-I complex with homologs of VPS37 and VPS28
[54]. VPS28A and VPS28B are redundant components of the ESCRT-I complex involved in Arabidopsis embryonic development. In embryos of
vps28avps28b double mutant, PIN1-GFP displayed broadened expression profile, reduced polarity, and aberrant vacuole-like structures
[55]. Aberrant localization and polarity of PIN1-GFP and PIN2-GFP are also reported in the knockout mutant of VPS36, the ESCRT-II subunit that can bind ubiquitin and interact with the other ESCRT-II components VPS22 and VPS25
[56].
9. Cytoskeletons
Both microtubule and actin cytoskeletons that are responsible for intracellular vesicular trafficking have been implicated in regulating PIN targeting to the vacuole. Especially for filamentous actin (F-actin), auxin transporter inhibitors, such as 2,3,5-triiodobenzoic acid (TIBA) and 2-(1-pyrenoyl) benzoic acid (PBA), also serve as actin stabilizers and repress subcellular motility in plant cells
[57]. Nevertheless, the inhibition of PIN2 vacuolar targeting by latrunculin B (LatB)-induced actin depolymerization
[16] implies a different effect of F-actin that the stabilization may promote vesicular trafficking to the vacuole. This finding is supported by appearance of reduced PM abundance and increased intracellular aggregation of PIN2-GFP in
act7, the loss-of-function mutant of actin subunit ACT7 with excessive actin bundling
[58]. The contradiction is possibly due to inconsistent effects of various inhibitors on consequently F-actin dynamics or different roles of the actin cytoskeleton in subcellular trafficking in different scenarios. Comparing to the F-actin, few studies report the role of microtubules in PIN vacuolar sorting. Given that a microtubule-associated protein cytoplasmic linker associated protein1 (CLASP1) positively regulates microtubule depolymerization, the interaction with retromer component SNX1 indicates involvement of microtubules in PIN2 retrograde trafficking from endosomes
[59].
10. Plant Hormones
Plant hormones are critical regulators for PIN degradation. The most studied is auxin as shown in the experiment that treatment with either naphthylene-1-acetic acid (NAA), an artificially synthesized auxin, or 2,3,5-triiodobenzoic acid (TIBA), a polar auxin transport inhibitor, reduced PIN2 protein levels with unaffected transcription levels
[21][60]. The scenario of NAA-induced PIN2 degradation is further specified to PIN2 recovery decrease at the lower side of gravistimulated roots after the initial increase of PIN2 level. Meanwhile, at the upper side of the bending roots where PIN2 levels initially decrease, auxin depletion also promotes PIN2 vacuolar targeting for degradation
[22]. Both scenarios of PIN2 turnover regulation involve SCF
TIR1/AFB-based auxin signaling pathway and suggest optimal auxin levels for the stabilization of PIN2 proteins
[22][60]. Nonetheless, no auxin signaling components have been revealed with differential expression between the upper and lower part of root during gravistimulation, also indicating that the initial PIN2 degradation there might not be triggered by the lack of auxin. Ubiquitin modification, at least that of PIN2, might prompt new thinking for insight into regulatory mechanism of PIN degradation.
Cytokinin, frequently having crosstalk with auxin, acts as a PIN regulator at both transcriptional and post-translational levels. Take PIN1 for example, B-type ARR cytokinin response factors bind directly to the responsive elements, including PIN1 cytokinin response element (PCRE1), in PIN1 promoter to upregulate PIN1 expression in inflorescences
[61]. However, in terms of regulation of PIN degradation, cytokinin, during development of lateral root primordium, targets PIN1 to the vacuole for degradation through modulation of the endocytic trafficking. Interference of the cytokinin effect by latrunculin B-caused actin depolymerization, not by that of oryzalin-induced microtubule depolymerization, suggests the mediation of the actin cytoskeleton in the process
[62].
11. Environmental Stimuli
Gravity is the most mentioned trigger of PIN degradation, which has been discussed regarding PIN2 redistribution in root gravitropism. The perception of the gravity vector is tightly associated with statoliths, dense starch-filled organelles which settle to the vicinity of the plasma membrane of the columella of the root cap and the endodermal cells of aerial tissue under gravistimulation
[63]. Although the physical attachment of amyloplasts to the cytoskeleton and activation of calcium release from the endoplasmic reticulum by statoliths suggest the signal transduction during gravitropic response and reorient plant growth via auxin
[64], the detailed link of gravity perception to the establishment of auxin gradient remains unidentified to date. In addition, light negatively regulates PIN2 vacuolar targeting in HY5-dependent pathway, but the mechanism is still large in the shadow and 26S proteasome and COP9 signalosome, which directly modulates ubiquitin E3 ligase function for26 proteasome-mediated degradation, are involved in this process
[65].
12. Other Regulators
In addition, some other genes are reported to regulate PIN degradation. MODULATOR OF PIN2 (MOP2) and MOP3, which are identified by a genetic screen, control PIN stability in a nonredundant manner as mutation of either reduced PIN protein levels
[66], however, the underlying mechanism of these newly characterized PIN regulators is still unknown. During establishment of procambial cells in cotyledons, expression and localization are under the regulation of vasculature complexity and connectivity (VCC), a plant-specific transmembrane protein which expression is induced by auxin. As VCC mutated, depolarization and vacuolar accumulation of PIN1-GFP were enhanced
[67]. SSR1 is a mitochondrial protein that regulates the retrograde trafficking possibly through the influence on the retromer. Knock-out of SSR1 resulted in decrease of protein expression of canonical PINs in roots, especially for PIN2 that underwent rapid degradation
[68].
MAB1 encodes a mitochondrial E1ß subunit of the pyruvate dehydrogenase complex in the TCA cycle. The amounts of PIN1 and PIN2 at the PM were reduced in
mab1 in a transcriptional behavior, likely due to the reduction of PIN endocytic recycling and acceleration of PIN vacuolar sorting
[69]. Loss-of-function and dominant-negative (DN) mutations in ROP3 (Rho-related GTPase of plants 3) also cause reduced protein levels of PIN1-GFP and PIN3-GFP, which was inhibited by MG132 treatment.