Dynamics in PIN2 auxin carrier ubiquitylation in gravity-responding Arabidopsis roots

Plant tropisms are decisively influenced by dynamic adjustments in spatiotemporal distribution of the growth regulators auxin. Polar auxin transport requires activity of PIN-type auxin carrier proteins, with their distribution at the plasma membrane significantly contributing to the directionality of auxin flow. Control of PIN protein distribution involves regulation of their endocytosis and further sorting into the lytic vacuole for degradation and recently, protein ubiquitylation has been demonstrated to control degradative sorting of plasma membrane proteins in plants.1-6 Here we show dynamic adjustments in PIN2 ubiquitylation in gravity-stimulated roots, a response that coincides with establishment of a lateral PIN2 expression gradient. Our results imply that perception and transduction of gravity signals triggers differential ubiquitylation of PIN2, which might feed back on the coordination of auxin distribution in root meristems.     

Ubiquitin initiates sorting of Golgi and plasma membrane proteins into the vacuolar degradation pathway

ABSTRACT: BACKGROUND: In yeast and mammals, many plasma membrane (PM) proteins destined for degradation are tagged with ubiquitin. These ubiquitinated proteins are internalized into clathrin-coated vesicles and are transported to early endosomal compartments. There, ubiquitinated proteins are sorted by the endosomal sorting complex required for transport (ESCRT) machinery into the intraluminal vesicles of multivesicular endosomes. Degradation of these proteins occurs after endosomes fuse with lysosomes/lytic vacuoles to release their content into the lumen. In plants, some PM proteins, which cycle between the PM and endosomal compartments, have been found to be ubiquitinated, but it is unclear whether ubiquitin is sufficient to mediate internalization and thus acts as a primary sorting signal for the endocytic pathway. To test whether plants use ubiquitin as a signal for the degradation of membrane proteins, we have translationally fused ubiquitin to different fluorescent reporters for the plasma membrane and analyzed their transport. RESULTS: Ubiquitin-tagged PM reporters localized to endosomes and to the lumen of the lytic vacuole in tobacco mesophyll protoplasts and in tobacco epidermal cells. The internalization of these reporters was significantly reduced if clathrin-mediated endocytosis was inhibited by the coexpression of a mutant of the clathrin heavy chain, the clathrin hub. Surprisingly, a ubiquitin-tagged reporter for the Golgi was also transported into the lumen of the vacuole. Vacuolar delivery of the reporters was abolished upon inhibition of the ESCRT machinery, indicating that the vacuolar delivery of these reporters occurs via the endocytic transport route. CONCLUSIONS: Ubiquitin acts as a sorting signal at different compartments in the endomembrane system to target membrane proteins into the vacuolar degradation pathway: If displayed at the PM, ubiquitin triggers internalization of PM reporters into the endocytic transport route, but it also mediates vacuolar delivery if displayed at the Golgi. In both cases, ubiquitin-tagged proteins travel via early endosomes and multivesicular bodies to the lytic vacuole. This suggests that vacuolar degradation of ubiquitinated proteins is not restricted to PM proteins but might also facilitate the turnover of membrane proteins in the early secretory pathway.     

Carbon and nitrogen metabolism regulated by the ubiquitin-proteasome system

The ubiquitin-proteasome system (UPS) is a unique protein degradation mechanism conserved in the eukaryotic cell. In addition to the control of protein quality, UPS regulates diverse cellular signal transduction via the fine-tuning of target protein degradation. Protein ubiquitylation and subsequent degradation by the 26S proteasome are involved in almost all aspects of plant growth and development and response to biotic and abiotic stresses. Recent studies reveal that the UPS plays an essential role in adaptation to carbon and nitrogen availability in plants. Here we highlight ubiquitin ligase ATL31 and the homologue ATL6 target 14-3-3 proteins for ubiquitylation to be degraded, which control signaling for carbon and nitrogen metabolisms and C/N balance response. We also give an overview of the UPS function involved in carbon and nitrogen metabolisms.     

Palmitoylation cycles and regulation of protein function (Review)

The efficacy and success of many cellular processes is dependent on a tight orchestration of proteins trafficking to and from their site(s) of action in a time-controlled fashion. Recently, a dynamic cycle of palmitoylation/de-palmitoylation has been shown to regulate shuttling of several proteins, including the small GTPases H-Ras and N-Ras, and the GABA-synthesizing enzyme GAD65, between the Golgi compartment and either the plasma membrane or synaptic vesicle membranes. These proteins are peripheral membrane proteins that in the depalmitoylated state cycle rapidly on and off the cytosolic face of ER/Golgi membranes. Palmitoylation of one or more cysteines, by a Golgi localized palmitoyl transferase (PAT) results in trapping in Golgi membranes, and sorting to a vesicular pathway in route to the plasma membrane or synaptic vesicles. A depalmitoylation step by an acyl protein thioesterase (APT) releases the protein from membranes in the periphery of the cell resulting in retrograde trafficking back to Golgi membranes by a non-vesicular pathway. The proteins can then enter a new cycle of palmitoylation and depalmitoylation. This inter-compartmental trafficking is orders of magnitude faster than vesicular trafficking. Recent advances in identifying a large family of PATs, their protein substrates, and single PAT mutants with severe phenotypes, reveal their critical importance in development, synaptic transmission, and regulation of signaling cascades. The emerging knowledge of enzymes involved in adding and removing palmitate is that they provide an intricate regulatory network involved in timing of protein function and transport that responds to intracellular and extracellular signals.     

Phosphorylation-dependent Trafficking of Plasma Membrane Proteins in Animal and Plant Cells

In both unicellular and multicellular organisms, transmembrane (TM) proteins are sorted to and retained at specific membrane domains by endomembrane trafficking mechanisms that recognize sorting signals in the these proteins. The trafficking and distribution of plasma membrane (PM)-localized TM proteins (PM proteins), especially of those PM proteins that show an asymmetric distribution over the PM, has received much attention, as their proper PM localization is crucial for elementary signaling and transport processes, and defects in their localization often lead to severe disease symptoms or developmental defects. The subcellular localization of PM proteins is dynamically regulated by post-translational modifications, such as phosphorylation and ubiquitination. These modificaitons mostly occur on sorting signals that are located in the larger cytosolic domains of the cargo proteins. Here we review the effects of phosphorylation of PM proteins on their trafficking, and present the key examples from the animal field that have been subject to studies for already several decades, such as that of aquaporin 2 and the epidermal growth factor receptor. Our knowledge on cargo trafficking in plants is largely based on studies of the family of PIN FORMED (PIN) carriers that mediate the efflux of the plant hormone auxin. We will review what is known on the subcellular distribution and trafficking of PIN proteins, with a focus on how this is modulated by phosphorylation, and identify and discuss analogies and differences in trafficking with the well-studied animal examples.     

Sorting Out the Sorting Functions of Endosomes in Arabidopsis

In animals, sorting of membrane proteins following their internalization from the plasma membrane (PM) by endocytosis occurs through a series of different endosomal compartments. In plants, how and where these sorting events take place is still poorly understood and our current view of the endocytic pathway still largely relies on analogies made from the animal system. However, extensive differences seem to exist between animal and plant endosomal functions, as exemplified by the role of the trans-Golgi network (TGN) as an early endosomal compartment in plants or the functional diversification of conserved sorting complexes. By using the Arabidopsis root tip as a reference model, we and other have begun to shed light on the complexity of the plant endocytic pathways. Notably, we have recently characterized the functions of an endosomal compartment, the SNX1-endosomes, also referred to as the prevacuolar compartment (PVC) or multivesicular bodies (MVB), in the sorting of different cargo proteins, including two related auxin-efflux carriers, PIN1 and PIN2. We have shown that routing decisions take place at this endosomal level, such as the sorting of PIN2 toward the lytic vacuole for degradation or PIN1 toward the PM for recycling.Key Words: Arabidopsis, intracellular trafficking, endocytic recycling, endosomes, MVB, PVC, VPS29, SNX, PIN, cell polarity     

Focus Issue on Roots: Plant Nutrition: Root Transporters on the Move

Nutrient and water uptake from the soil is essential for plant growth and development. In the root, absorption and radial transport of nutrients and water toward the vascular tissues is achieved by a battery of specialized transporters and channels. Modulating the amount and the localization of these membrane transport proteins appears as a way to drive their activity and is essential to maintain nutrient homeostasis in plants. This control first involves the delivery of newly synthesized proteins to the plasma membrane by establishing check points along the secretory pathway, especially during the export from the endoplasmic reticulum. Plasma membrane-localized transport proteins are internalized through endocytosis followed by recycling to the cell surface or targeting to the vacuole for degradation, hence constituting another layer of control. These intricate mechanisms are often regulated by nutrient availability, stresses, and endogenous cues, allowing plants to rapidly adjust to their environment and adapt their development.Plants take up nutrients and water from the soil and transport them to the leaves to support photosynthesis and plant growth. However, most soils around the world do not provide optimal conditions for plant colonization. Consequently, plants have evolved sophisticated mechanisms to adjust to deficiency or excess of nutrients and water supply. Membrane transport proteins, including channels and transporters, play crucial roles in the uptake of nutrients and water from the soil and in their radial transport to the root vasculature. Newly synthesized membrane transport proteins have to be properly targeted to a defined compartment, usually the plasma membrane, to efficiently ensure their function. The trafficking of membrane transport proteins along the secretory pathway is tightly controlled and involves the recognition of exit signals by gatekeeper protein complexes. After reaching the plasma membrane, membrane transport proteins can be endocytosed and subsequently recycled to the cell surface or targeted to the vacuole for degradation. Because the subcellular localization of proteins directly influences their activity, modulating the localization of membrane transport proteins constitutes a powerful way to control nutrient and water uptake in plants. This review discusses the fundamental mechanisms at stake in membrane protein secretion and endocytosis, with a specific focus on membrane transport proteins, and how endogenous and exogenous cues affect their dynamics to integrate uptake of nutrients and water to plant growth conditions.     

Ubiquitylation of MHC class I by the K3 viral protein signals internalization and TSG101-dependent degradation

The Kaposi's sarcoma-associated herpes virus gene product K3 (KK3) subverts the MHC class I antigen presentation pathway by downregulating MHC class I from the plasma membrane. We now show that KK3 associates with MHC class I molecules and promotes ubiquitylation of class I after export from the endoplasmic reticulum. Ubiquitylation requires the KK3 N-terminal plant homeodomain and provides the signal for class I internalization at the plasma membrane. Once internalized, ubiquitylated MHC class I is targeted to the late endocytic pathway, where it is degraded. Depletion by small interfering RNA of TSG101, a ubiquitin enzyme 2 variant protein involved in late endosomal sorting, prevents class I degradation and preserves cell surface class I expression in KK3-expressing cells. These results suggest a mechanism by which the KK3-induced class I ubiquitylation provides a signal for both internalization and sorting to the late endosomal pathway for degradation. KK3 is the first viral gene product that subverts the trafficking of a host protein via the ubiquitin-dependent endosomal sorting machinery.     

Vacuolar degradation of two integral plasma membrane proteins, AtLRR84A and OsSCAMP1, is cargo ubiquitination-independent and prevacuolar compartment-mediated in plant cells

In plant cells, how integral plasma membrane (PM) proteins are degraded in a cargo ubiquitination-independent manner remains elusive. Here, we studied the degradative pathway of two plant PM proteins: AtLRR84A, a type I integral membrane protein belonging to the leucine-rich repeat receptor-like kinase protein family, and OsSCAMP1 (rice secretory carrier membrane protein 1), a tetraspan transmembrane protein located on the PM and trans-Golgi network (TGN) or early endosome (EE). Using wortmannin and ARA7(Q69L) mutant that could enlarge the multivesicular body (MVB) or prevacuolar compartment (PVC) as tools, we demonstrated that, when expressed as green fluorescent protein (GFP) fusions in tobacco BY-2 or Arabidopsis protoplasts, both AtLRR84A and OsSCAMP1 were degraded in the lytic vacuole via the internal vesicles of MVB/PVC in a cargo ubiquitination-independent manner. Such MVB/PVC-mediated vacuolar degradation of PM proteins was further supported by immunocytochemical electron microscopy (immunoEM) study showing the labeling of the fusions on the internal vesicles of the PVC/MVB. Thus, cargo ubiquitination-independent and PVC-mediated degradation of PM proteins in the vacuole is functionally operated in plant cells.     

BECN1/Beclin 1 sorts cell-surface APP/amyloid β precursor protein for lysosomal degradation

The regulation of plasma membrane (PM)-localized transmembrane protein/receptor trafficking has critical implications for cell signaling, metabolism and survival. In this study, we investigated the role of BECN1 (Beclin 1) in the degradative trafficking of PM-associated APP (amyloid β precursor protein), whose metabolism to amyloid-β, an essential event in Alzheimer disease, is dependent on divergent PM trafficking pathways. We report a novel interaction between PM-associated APP and BECN1 that recruits macroautophagy/endosomal regulatory proteins PIK3C3 and UVRAG. We found that BECN1 promotes surface APP internalization and sorting predominantly to endosomes and endolysosomes. BECN1 also promotes the targeting of a smaller fraction of internalized APP to LC3-positive phagophores, suggesting a role for BECN1-dependent PM macroautophagy in APP degradation. Furthermore, BECN1 facilitates lysosomal degradation of surface APP and reduces the secretion of APP metabolites (soluble ectodomains, sAPP). The association between APP and BECN1 is dependent on the evolutionarily conserved domain (ECD) of BECN1 (amino acids 267–337). Deletion of a BECN1 ECD subregion (amino acids 285–299) did not impair BECN1- PIK3C3 interaction, PtdIns3K function or macroautophagy, but was sufficient to impair the APP-BECN1 interaction and BECN1's effects on surface APP internalization and degradation, resulting in increased secretion of sAPPs. Interestingly, both the BECN1-APP association and BECN1-dependent APP endocytosis and degradative trafficking were negatively regulated by active AKT. Our results further implicate phosphorylation of the BECN1 Ser295 residue in the inhibition of APP degradation by AKT. Our studies reveal a novel function for BECN1 in the sorting of a plasma membrane protein for endolysosomal and macroautophagic degradation.     

PAT1a modulates intracellular transport and processing of amyloid precursor protein (APP), APLP1, and APLP2

Understanding the intracellular transport of the beta-amyloid precursor protein (APP) is a major key to elucidate the regulation of APP processing and thus beta-amyloid peptide generation in Alzheimer disease pathogenesis. APP and its two paralogues, APLP1 and APLP2 (APLPs), are processed in a very similar manner by the same protease activities. A putative candidate involved in APP transport is protein interacting with APP tail 1 (PAT1), which was reported to interact with the APP intracellular domain. We show that PAT1a, which is 99.0% identical to PAT1, binds to APP, APLP1, and APLP2 in vivo and describe their co-localization in trans-Golgi network vesicles or endosomes in primary neurons. We further demonstrate a direct interaction of PAT1a with the basolateral sorting signal of APP/APLPs. Moreover, we provide evidence for a direct role of PAT1a in APP/APLP transport as overexpression or RNA interference-mediated knockdown of PAT1a modulates APP/APLPs levels at the cell surface. Finally, we show that PAT1a promotes APP/APLPs processing, resulting in increased secretion of beta-amyloid peptide. Taken together, our data establish PAT1a as a functional link between APP/APLPs transport and their processing.     

Plant endosomal trafficking pathways

Endosomes regulate both the recycling and degradation of plasma membrane (PM) proteins, thereby modulating many cellular responses triggered at the cell surface. Endosomes also play a role in the biosynthetic pathway by taking proteins to the vacuole and recycling vacuolar cargo receptors. In plants, the trans-Golgi network (TGN) acts as an early/recycling endosome whereas prevacuolar compartments/multivesicular bodies (MVBs) take PM proteins to the vacuole for degradation. Recent studies have demonstrated that some of the molecular complexes that mediate endosomal trafficking, such as the retromer, the ADP-ribosylation factor (ARF) machinery, and the Endosomal Sorting Complexes Required for Transport (ESCRTs) have both conserved and specialized functions in plants. Whereas there is disagreement on the subcellular localization of the plant retromer, its function in recycling vacuolar sorting receptors (VSRs) and modulating the trafficking of PM proteins has been well established. Studies on Arabidopsis ESCRT components highlight the essential role of this complex in cytokinesis, plant development, and vacuolar organization. In addition, post-translational modifications of plant PM proteins, such as phosphorylation and ubiquitination, have been demonstrated to act as sorting signals for endosomal trafficking.     

Constant change: dynamic regulation of membrane transport by calcium signalling networks keeps plants in tune with their environment

Despite substantial variation and irregularities in their environment, plants must conform to spatiotemporal demands on the molecular composition of their cytosol. Cell membranes are the major interface between organisms and their environment and the basis for controlling the contents and intracellular organization of the cell. Membrane transport proteins (MTPs) govern the flow of molecules across membranes, and their activities are closely monitored and regulated by cell signalling networks. By continuously adjusting MTP activities, plants can mitigate the effects of environmental perturbations, but effective implementation of this strategy is reliant on precise coordination among transport systems that reside in distinct cell types and membranes. Here, we examine the role of calcium signalling in the coordination of membrane transport, with an emphasis on potassium transport. Potassium is an exceptionally abundant and mobile ion in plants, and plant potassium transport has been intensively studied for decades. Classic and recent studies have underscored the importance of calcium in plant environmental responses and membrane transport regulation. In reviewing recent advances in our understanding of the coding and decoding of calcium signals, we highlight established and emerging roles of calcium signalling in coordinating membrane transport among multiple subcellular locations and distinct transport systems in plants, drawing examples from the CBL‐CIPK signalling network. By synthesizing classical studies and recent findings, we aim to provide timely insights on the role of calcium signalling networks in the modulation of membrane transport and its importance in plant environmental responses.     

Protein transport in plant cells: in and out of the Golgi

In plant cells, the Golgi apparatus is the key organelle for polysaccharide and glycolipid synthesis, protein glycosylation and protein sorting towards various cellular compartments. Protein import from the endoplasmic reticulum (ER) is a highly dynamic process, and new data suggest that transport, at least of soluble proteins, occurs via bulk flow. In this Botanical Briefing, we review the latest data on ER/Golgi inter-relations and the models for transport between the two organelles. Whether vesicles are involved in this transport event or if direct ER-Golgi connections exist are questions that are open to discussion. Whereas the majority of proteins pass through the Golgi on their way to other cell destinations, either by vesicular shuttles or through maturation of cisternae from the cis- to the trans-face, a number of membrane proteins reside in the different Golgi cisternae. Experimental evidence suggests that the length of the transmembrane domain is of crucial importance for the retention of proteins within the Golgi. In non-dividing cells, protein transport out of the Golgi is either directed towards the plasma membrane/cell wall (secretion) or to the vacuolar system. The latter comprises the lytic vacuole and protein storage vacuoles. In general, transport to either of these from the Golgi depends on different sorting signals and receptors and is mediated by clathrin-coated and dense vesicles, respectively. Being at the heart of the secretory pathway, the Golgi (transiently) accommodates regulatory proteins of secretion (e.g. SNAREs and small GTPases), of which many have been cloned in plants over the last decade. In this context, we present a list of regulatory proteins, along with structural and processing proteins, that have been located to the Golgi and the 'trans-Golgi network' by microscopy.     

Binding of Plasma Membrane Lipids Recruits the Yeast Integral Membrane Protein Ist2 to the Cortical ER

Recruitment of cytosolic proteins to individual membranes is governed by a combination of protein–protein and protein–membrane interactions. Many proteins recognize phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] at the cytosolic surface of the plasma membrane (PM). Here, we show that a protein–lipid interaction can also serve as a dominant signal for the sorting of integral membrane proteins. Interaction with phosphatidly-inositolphosphates (PIPs) at the PM is involved in the targeting of the polytopic yeast protein Ist2 to PM-associated domains of the cortical endoplasmic reticulum (ER). Moreover, binding of PI(4,5)P₂ at the PM functions as a dominant mechanism that targets other integral membrane proteins to PM-associated domains of the cortical ER. This sorting to a subdomain of the ER abolishes proteasomal degradation and trafficking along the classical secretory (sec) pathway. In combination with the localization of IST2 mRNA to the bud tip and other redundant signals in Ist2, binding of PIPs leads to efficient accumulation of Ist2 at domains of the cortical ER from where the protein may reach the PM independently of the function of the sec-pathway.     

Endosomal functions in plants

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.     

The plant vacuolar sorting receptor AtELP is involved in transport of NH(2)-terminal propeptide-containing vacuolar proteins in Arabidopsis thaliana

Many soluble plant vacuolar proteins are sorted away from secreted proteins into small vesicles at the trans-Golgi network by transmembrane cargo receptors. Cleavable vacuolar sorting signals include the NH(2)-terminal propeptide (NTPP) present in sweet potato sporamin (Spo) and the COOH-terminal propeptide (CTPP) present in barley lectin (BL). These two proteins have been found to be transported by different mechanisms to the vacuole. We examined the ability of the vacuolar cargo receptor AtELP to interact with the sorting signals of heterologous and endogenous plant vacuolar proteins in mediating vacuolar transport in Arabidopsis thaliana. AtELP extracted from microsomes was found to interact with the NTPPs of barley aleurain and Spo, but not with the CTPPs of BL or tobacco chitinase, in a pH-dependent and sequence-specific manner. In addition, EM studies revealed the colocalization of AtELP with NTPP-Spo at the Golgi apparatus, but not with BL-CTPP in roots of transgenic Arabidopsis plants. Further, we found that AtELP interacts in a similar manner with the NTPP of the endogenous vacuolar protein AtALEU (Arabidopsis thaliana Aleu), a protein highly homologous to barley aleurain. We hypothesize that AtELP functions as a vacuolar sorting receptor involved in the targeting of NTPP-, but not CTPP-containing proteins in Arabidopsis.     

Multiple cytosolic and transmembrane determinants are required for the trafficking of SCAMP1 via an ER-Golgi-TGN-PM pathway

How polytopic plasma membrane (PM) proteins reach their destination in plant cells remains elusive. Using transgenic tobacco BY-2 cells, we previously showed that the rice secretory carrier membrane protein 1 (SCAMP1), an integral membrane protein with four transmembrane domains (TMDs), is localized to the PM and trans-Golgi network (TGN). Here, we study the transport pathway and sorting signals of SCAMP1 by following its transient expression in tobacco BY-2 protoplasts and show that SCAMP1 reaches the PM via an endoplasmic reticulum (ER)-Golgi-TGN-PM pathway. Loss-of-function and gain-of-function analysis of various green fluorescent protein (GFP) fusions with SCAMP1 mutations further demonstrates that: (i) the cytosolic N-terminus of SCAMP1 contains an ER export signal; (ii) the transmembrane domain 2 (TMD2) and TMD3 of SCAMP1 are essential for Golgi export; (iii) SCAMP1 TMD1 is essential for TGN-to-PM targeting; (iv) the predicted topology of SCAMP1 and its various mutants remain identical as demonstrated by protease protection assay. Therefore, both the cytosolic N-terminus and TMD sequences of SCAMP1 play integral roles in mediating its transport to the PM via an ER-Golgi-TGN pathway.