Plant Plasma Membrane Proteins Research Articles

Plant endosomes as protein sorting hubs

Endocytosis, secretion, and endosomal trafficking are key cellular processes that control the composition of the plasma membrane. Through the coordination of these trafficking pathways, cells can adjust the composition, localization, and turnover of proteins and lipids in response to developmental or environmental cues. Upon being incorporated into vesicles and internalized through endocytosis, plant plasma membrane proteins are delivered to the trans-Golgi network (TGN). At the TGN, plasma membrane proteins are recycled back to the plasma membrane or transferred to multivesicular endosomes (MVEs), where they are further sorted into intralumenal vesicles for degradation in the vacuole. Both types of plant endosomes, TGN and MVEs, act as sorting organelles for multiple endocytic, recycling, and secretory pathways. Molecular assemblies such as retromer, ESCRT (endosomal sorting complex required for transport) machinery, small GTPases, adaptor proteins, and SNAREs associate with specific domains of endosomal membranes to mediate different sorting and membrane-budding events. In this review, we discuss the mechanisms underlying the recognition and sorting of proteins at endosomes, membrane remodeling and budding, and their implications for cellular trafficking and physiological responses in plants.

Endosomally Localized RGLG-Type E3 RING-Finger Ligases Modulate Sorting of Ubiquitylation-Mimic PIN2.

Intracellular sorting and the abundance of sessile plant plasma membrane proteins are imperative for sensing and responding to environmental inputs. A key determinant for inducing adjustments in protein localization and hence functionality is their reversible covalent modification by the small protein modifier ubiquitin, which is for example responsible for guiding proteins from the plasma membrane to endosomal compartments. This mode of membrane protein sorting control requires the catalytic activity of E3 ubiquitin ligases, amongst which members of the RING DOMAIN LIGASE (RGLG) family have been implicated in the formation of lysine 63-linked polyubiquitin chains, serving as a prime signal for endocytic vacuolar cargo sorting. Nevertheless, except from some indirect implications for such RGLG activity, no further evidence for their role in plasma membrane protein sorting has been provided so far. Here, by employing RGLG1 reporter proteins combined with assessment of plasma membrane protein localization in a rglg1 rglg2 loss-of-function mutant, we demonstrate a role for RGLGs in cargo trafficking between plasma membrane and endosomal compartments. Specifically, our findings unveil a requirement for RGLG1 association with endosomal sorting compartments for fundamental aspects of plant morphogenesis, underlining a vital importance for ubiquitylation-controlled intracellular sorting processes.

Correlative Light-Environmental Scanning Electron Microscopy of Plasma Membrane Efflux Carriers of Plant Hormone Auxin.

Fluorescence light microscopy provided convincing evidence for the domain organization of plant plasma membrane (PM) proteins. Both peripheral and integral PM proteins show an inhomogeneous distribution within the PM. However, the size of PM nanodomains and protein clusters is too small to accurately determine their dimensions and nano-organization using routine confocal fluorescence microscopy and super-resolution methods. To overcome this limitation, we have developed a novel correlative light electron microscopy method (CLEM) using total internal reflection fluorescence microscopy (TIRFM) and advanced environmental scanning electron microscopy (A-ESEM). Using this technique, we determined the number of auxin efflux carriers from the PINFORMED (PIN) family (NtPIN3b-GFP) within PM nanodomains of tobacco cell PM ghosts. Protoplasts were attached to coverslips and immunostained with anti-GFP primary antibody and secondary antibody conjugated to fluorochrome and gold nanoparticles. After imaging the nanodomains within the PM with TIRFM, the samples were imaged with A-ESEM without further processing, and quantification of the average number of molecules within the nanodomain was performed. Without requiring any post-fixation and coating procedures, this method allows to study details of the organization of auxin carriers and other plant PM proteins.

Cross-talk between clathrin-dependent post-Golgi trafficking and clathrin-mediated endocytosis in Arabidopsis root cells.

Coupling of post-Golgi and endocytic membrane transport ensures that the flow of materials to/from the plasma membrane (PM) is properly balanced. The mechanisms underlying the coordinated trafficking of PM proteins in plants, however, are not well understood. In plant cells, clathrin and its adaptor protein complexes, AP-2 and the TPLATE complex (TPC) at the PM, and AP-1 at the trans-Golgi network/early endosome (TGN/EE), function in clathrin-mediated endocytosis (CME) and post-Golgi trafficking. Here, we utilized mutants with defects in clathrin-dependent post-Golgi trafficking and CME, in combination with other cytological and pharmacological approaches, to further investigate the machinery behind the coordination of protein delivery and recycling to/from the TGN/EE and PM in Arabidopsis (Arabidopsis thaliana) root cells. In mutants with defective AP-2-/TPC-dependent CME, we determined that clathrin and AP-1 recruitment to the TGN/EE as well as exocytosis are significantly impaired. Likewise, defects in AP-1-dependent post-Golgi trafficking and pharmacological inhibition of exocytosis resulted in the reduced association of clathrin and AP-2/TPC subunits with the PM and a reduction in the internalization of cargoes via CME. Together, these results suggest that post-Golgi trafficking and CME are coupled via modulation of clathrin and adaptor protein complex recruitment to the TGN/EE and PM.

A cell surface-exposed protein complex with an essential virulence function in Ustilago maydis

Plant pathogenic fungi colonizing living plant tissue secrete a cocktail of effector proteins to suppress plant immunity and reprogramme host cells. Although many of these effectors function inside host cells, delivery systems used by pathogenic bacteria to translocate effectors into host cells have not been detected in fungi. Here, we show that five unrelated effectors and two membrane proteins from Ustilago maydis, a biotrophic fungus causing smut disease in corn, form a stable protein complex. All seven genes appear co-regulated and are only expressed during colonization. Single mutants arrest in the epidermal layer, fail to suppress host defence responses and fail to induce non-host resistance, two reactions that likely depend on translocated effectors. The complex is anchored in the fungal membrane, protrudes into host cells and likely contacts channel-forming plant plasma membrane proteins. Constitutive expression of all seven complex members resulted in a surface-exposed form in cultured U. maydis cells. As orthologues of the complex-forming proteins are conserved in smut fungi, the complex may become an interesting fungicide target.

Selective Enrichment Coupled with Proteomics to Identify S-Acylated Plasma Membrane Proteins in Arabidopsis.

Protein S-acylation, predominately in the form of palmitoylation, is a reversible lipid post-translational modification on cysteines that plays important roles in protein localization, trafficking, activity, and complex assembly. The functions and regulatory mechanisms of S-acylation have been extensively studied in mammals owing to remarkable development of high-resolution proteomics and the discovery of the S-acylation-related enzymes. However, the advancement of S-acylation studies in plants lags behind that in mammals, mainly due to the lack of knowledge about proteins responsible for this process, such as protein acyltransferases and their substrates. In this article, a set of systematic protocols to study global S-acylation in Arabidopsis seedlings is described. The procedures are presented in detail, including preparation of Arabidopsis seedlings, enrichment of plasma membrane (PM) proteins, ensuing enrichment of S-acylated proteins/peptides based on the acyl-biotin exchange method, and large-scale identification of S-acylated proteins/peptides via mass spectrometry. This approach enables researchers to study S-acylation of PM proteins in plants in a systematic and straightforward way. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of Arabidopsis seedling materials Basic Protocol 2: Isolation and enrichment of plasma membrane proteins Support Protocol 1: Determination of protein concentration using BCA assay Basic Protocol 3: Enrichment of S-acylated proteins by acyl-biotin exchange method Support Protocol 2: Protein precipitation by methanol/chloroform method Basic Protocol 4: Trypsin digestion and proteomic analysis Alternate Protocol: Pre-resin digestion and peptide-level enrichment.

The cell wall regulates dynamics and size of plasma-membrane nanodomains in Arabidopsis

Plant plasma-membrane (PM) proteins are involved in several vital processes, such as detection of pathogens, solute transport, and cellular signaling. For these proteins to function effectively there needs to be structure within the PM allowing, for example, proteins in the same signaling cascade to be spatially organized. Here we demonstrate that several proteins with divergent functions are located in clusters of differing size in the membrane using subdiffraction-limited Airyscan confocal microscopy. Single particle tracking reveals that these proteins move at different rates within the membrane. Actin and microtubule cytoskeletons appear to significantly regulate the mobility of one of these proteins (the pathogen receptor FLS2) and we further demonstrate that the cell wall is critical for the regulation of cluster size by quantifying single particle dynamics of proteins with key roles in morphogenesis (PIN3) and pathogen perception (FLS2). We propose a model in which the cell wall and cytoskeleton are pivotal for regulation of protein cluster size and dynamics, thereby contributing to the formation and functionality of membrane nanodomains.

Structural basis for plant plasma membrane protein dynamics and organization into functional nanodomains.

Plasma Membrane is the primary structure for adjusting to ever changing conditions. PM sub-compartmentalization in domains is thought to orchestrate signaling. Yet, mechanisms governing membrane organization are mostly uncharacterized. The plant-specific REMORINs are proteins regulating hormonal crosstalk and host invasion. REMs are the best-characterized nanodomain markers via an uncharacterized moiety called REMORIN C-terminal Anchor. By coupling biophysical methods, super-resolution microscopy and physiology, we decipher an original mechanism regulating the dynamic and organization of nanodomains. We showed that targeting of REMORIN is independent of the COP-II-dependent secretory pathway and mediated by PI4P and sterol. REM-CA is an unconventional lipid-binding motif that confers nanodomain organization. Analyses of REM-CA mutants by single particle tracking demonstrate that mobility and supramolecular organization are critical for immunity. This study provides a unique mechanistic insight into how the tight control of spatial segregation is critical in the definition of PM domain necessary to support biological function.

Single Event Resolution of Plant Plasma Membrane Protein Endocytosis by TIRF Microscopy.

Endocytosis is a key process in the internalization of extracellular materials and plasma membrane proteins, such as receptors and transporters, thereby controlling many aspects of cell signaling and cellular homeostasis. Endocytosis in plants has an essential role not only for basic cellular functions but also for growth and development, nutrient delivery, toxin avoidance, and pathogen defense. The precise mechanisms of endocytosis in plants remain quite elusive. The lack of direct visualization and examination of single events of endocytosis has greatly hampered our ability to precisely monitor the cell surface lifetime and the recruitment profile of proteins driving endocytosis or endocytosed cargos in plants. Here, we discuss the necessity to systematically implement total internal reflection fluorescence microcopy (TIRF) in the Plant Cell Biology community and present reliable protocols for high spatial and temporal imaging of endocytosis in plants using clathrin-mediated endocytosis as a test case, since it represents the major route for internalization of cell-surface proteins in plants. We developed a robust method to directly visualize cell surface proteins using TIRF microscopy combined to a high throughput, automated and unbiased analysis pipeline to determine the temporal recruitment profile of proteins to single sites of endocytosis, using the departure of clathrin as a physiological reference for scission. Using this ‘departure assay’, we assessed the recruitment of two different AP-2 subunits, alpha and mu, to the sites of endocytosis and found that AP2A1 was recruited in concert with clathrin, while AP2M was not. This validated approach therefore offers a powerful solution to better characterize the plant endocytic machinery and the dynamics of one’s favorite cargo protein.

Rice Plasma Membrane Proteomics Reveals Magnaporthe oryzae Promotes Susceptibility by Sequential Activation of Host Hormone Signaling Pathways.

Plant plasma membrane (PM) plays important roles in immune response. Here, we utilized quantitative mass spectrometry to explore rice PM protein composition and dynamic changes during Magnaporthe oryzae infection. We report, thus far, the largest rice PM proteome dataset with 3,906 identified proteins, among which 484 proteins were differentially expressed after M. oryzae infection. One third of the identified proteins are predicted to have at least one transmembrane domain. Half of the identified proteins are predicted to have binding functions and over one third of the proteins have enzyme-related functions. In addition, Gene Ontology analyses revealed that abscisic acid (ABA) and cytokinin (CK) signaling were sequentially activated after M. oryzae infection in rice. We found that the activation of ABA signaling and the suppression of rice immune response occurred at the early infection stage, while the activation of CK signaling, the upregulation of sugar transporter genes expression, and the nutrient efflux of infected rice cells occurred at later infection stage. Thus, we further propose that M. oryzae activates ABA signaling to repress rice immune signaling for initial invasion and redirects nutrient efflux of infected cells for massive growth at the later infection stage.

Enquiry into the Topology of Plasma Membrane-Localized PIN Auxin Transport Components

Auxin directs plant ontogenesis via differential accumulation within tissues depending largely on the activity of PIN proteins that mediate auxin efflux from cells and its directional cell-to-cell transport. Regardless of the developmental importance of PINs, the structure of these transporters is poorly characterized. Here, we present experimental data concerning protein topology of plasma membrane-localized PINs. Utilizing approaches based on pH-dependent quenching of fluorescent reporters combined with immunolocalization techniques, we mapped the membrane topology of PINs and further cross-validated our results using available topology modeling software. We delineated the topology of PIN1 with two transmembrane (TM) bundles of five α-helices linked by a large intracellular loop and a C-terminus positioned outside the cytoplasm. Using constraints derived from our experimental data, we also provide an updated position of helical regions generating a verisimilitude model of PIN1. Since the canonical long PINs show a high degree of conservation in TM domains and auxin transport capacity has been demonstrated for Arabidopsis representatives of this group, this empirically enhanced topological model of PIN1 will be an important starting point for further studies on PIN structure–function relationships. In addition, we have established protocols that can be used to probe the topology of other plasma membrane proteins in plants.

Determination of Dynamics of Plant Plasma Membrane Proteins with Fluorescence Recovery and Raster Image Correlation Spectroscopy.

A number of fluorescence microscopy techniques are described to study dynamics of fluorescently labeled proteins, lipids, nucleic acids, and whole organelles. However, for studies of plant plasma membrane (PM) proteins, the number of these techniques is still limited because of the high complexity of processes that determine the dynamics of PM proteins and the existence of cell wall. Here, we report on the usage of raster image correlation spectroscopy (RICS) for studies of integral PM proteins in suspension-cultured tobacco cells and show its potential in comparison with the more widely used fluorescence recovery after photobleaching method. For RICS, a set of microscopy images is obtained by single-photon confocal laser scanning microscopy (CLSM). Fluorescence fluctuations are subsequently correlated between individual pixels and the information on protein mobility are extracted using a model that considers processes generating the fluctuations such as diffusion and chemical binding reactions. As we show here using an example of two integral PM transporters of the plant hormone auxin, RICS uncovered their distinct short-distance lateral mobility within the PM that is dependent on cytoskeleton and sterol composition of the PM. RICS, which is routinely accessible on modern CLSM instruments, thus represents a valuable approach for studies of dynamics of PM proteins in plants.

The dynamics of plant plasma membrane proteins: PINs and beyond.

Plants are permanently situated in a fixed location and thus are well adapted to sense and respond to environmental stimuli and developmental cues. At the cellular level, several of these responses require delicate adjustments that affect the activity and steady-state levels of plasma membrane proteins. These adjustments involve both vesicular transport to the plasma membrane and protein internalization via endocytic sorting. A substantial part of our current knowledge of plant plasma membrane protein sorting is based on studies of PIN-FORMED (PIN) auxin transport proteins, which are found at distinct plasma membrane domains and have been implicated in directional efflux of the plant hormone auxin. Here, we discuss the mechanisms involved in establishing such polar protein distributions, focusing on PINs and other key plant plasma membrane proteins, and we highlight the pathways that allow for dynamic adjustments in protein distribution and turnover, which together constitute a versatile framework that underlies the remarkable capabilities of plants to adjust growth and development in their ever-changing environment.

Analysis of global ubiquitylation and ubiquitin-binding domains involved in endosomal trafficking.

Ubiquitylation is a reversible posttranslational modification that regulates various cellular pathways. Ubiquitylation of a plasma membrane protein was shown to serve as a signal for endocytosis of plasma membrane proteins in yeast and mammals as well as in plants. As more and more plant plasma membrane proteins are reported to be regulated through their ubiquitylation status, methods to analyze ubiquitylation and ubiquitin binding would be useful for the characterization of proteins involved in endocytosis of ubiquitylated cargo proteins.

Retromer Subunits VPS35A and VPS29 Mediate Prevacuolar Compartment (PVC) Function in Arabidopsis

Intracellular protein routing is mediated by vesicular transport which is tightly regulated in eukaryotes. The protein and lipid homeostasis depends on coordinated delivery of de novo synthesized or recycled cargoes to the plasma membrane by exocytosis and their subsequent removal by rerouting them for recycling or degradation. Here, we report the characterization of protein affected trafficking 3 (pat3) mutant that we identified by an epifluorescence-based forward genetic screen for mutants defective in subcellular distribution of Arabidopsis auxin transporter PIN1-GFP. While pat3 displays largely normal plant morphology and development in nutrient-rich conditions, it shows strong ectopic intracellular accumulations of different plasma membrane cargoes in structures that resemble prevacuolar compartments (PVC) with an aberrant morphology. Genetic mapping revealed that pat3 is defective in vacuolar protein sorting 35A (VPS35A), a putative subunit of the retromer complex that mediates retrograde trafficking between the PVC and trans-Golgi network. Similarly, a mutant defective in another retromer subunit, vps29, shows comparable subcellular defects in PVC morphology and protein accumulation. Thus, our data provide evidence that the retromer components VPS35A and VPS29 are essential for normal PVC morphology and normal trafficking of plasma membrane proteins in plants. In addition, we show that, out of the three VPS35 retromer subunits present in Arabidopsis thaliana genome, the VPS35 homolog A plays a prevailing role in trafficking to the lytic vacuole, presenting another level of complexity in the retromer-dependent vacuolar sorting.

Plasma Membrane Protein Ubiquitylation and Degradation as Determinants of Positional Growth in Plants

Being sessile organisms, plants evolved an unparalleled plasticity in their post-embryonic development, allowing them to adapt and fine-tune their vital parameters to an ever-changing environment. Crosstalk between plants and their environment requires tight regulation of information exchange at the plasma membrane (PM). Plasma membrane proteins mediate such communication, by sensing variations in nutrient availability, external cues as well as by controlled solute transport across the membrane border. Localization and steady-state levels are essential for PM protein function and ongoing research identified cis- and trans-acting determinants, involved in control of plant PM protein localization and turnover. In this overview, we summarize recent progress in our understanding of plant PM protein sorting and degradation via ubiquitylation, a post-translational and reversible modification of proteins. We highlight characterized components of the machinery involved in sorting of ubiquitylated PM proteins and discuss consequences of protein ubiquitylation on fate of selected PM proteins. Specifically, we focus on the role of ubiquitylation and PM protein degradation in the regulation of polar auxin transport (PAT). We combine this regulatory circuit with further aspects of PM protein sorting control, to address the interplay of events that might control PAT and polarized growth in higher plants.

Plasma membrane protein ubiquitylation and degradation as determinants of positional growth in plants

Being sessile organisms, plants evolved an unparalleled plasticity in their post-embryonic development, allowing them to adapt and fine-tune their vital parameters to an ever-changing environment. Crosstalk between plants and their environment requires tight regulation of information exchange at the plasma membrane (PM). Plasma membrane proteins mediate such communication, by sensing variations in nutrient availability, external cues as well as by controlled solute transport across the membrane border. Localization and steady-state levels are essential for PM protein function and ongoing research identified cis- and trans-acting determinants, involved in control of plant PM protein localization and turnover. In this overview, we summarize recent progress in our understanding of plant PM protein sorting and degradation via ubiquitylation, a post-translational and reversible modification of proteins. We highlight characterized components of the machinery involved in sorting of ubiquitylated PM proteins and discuss consequences of protein ubiquitylation on fate of selected PM proteins. Specifically, we focus on the role of ubiquitylation and PM protein degradation in the regulation of polar auxin transport (PAT). We combine this regulatory circuit with further aspects of PM protein sorting control, to address the interplay of events that might control PAT and polarized growth in higher plants. Figure 1Open in figure viewerPowerPoint Christian Luschnig (Corresponding author)

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.

Phosphoproteomic analysis reveals major default phosphorylation sites outside long intrinsically disordered regions of Arabidopsis plasma membrane proteins

BackgroundGenome-wide statistics established that long intrinsically disordered regions (over 30 residues) are predicted in a large part of proteins in all eukaryotes, with a higher ratio in trans-membrane proteins. At functional level, such unstructured and flexible regions were suggested for years to favour phosphorylation events. In plants, despite increasing evidence of the regulation of transport and signalling processes by phosphorylation events, only few data are available without specific information regarding plasma membrane proteins, especially at proteome scale.ResultsUsing a dedicated phosphoproteomic workflow, 75 novel and unambiguous phosphorylation sites were identified in Arabidopsis plasma membrane. Bioinformatics analysis showed that this new dataset concerned mostly integral proteins involved in key functions of the plasma membrane (such as transport and signal transduction, including protein phosphorylation). It thus expanded by 15% the directory of phosphosites previously characterized in signalling and transport proteins. Unexpectedly, 66% of phosphorylation sites were predicted to be located outside long intrinsically disordered regions. This result was further corroborated by analysis of publicly available data for the plasma membrane.ConclusionsThe new phosphoproteomics data presented here, with published datasets and functional annotation, suggest a previously unexpected topology of phosphorylation in the plant plasma membrane proteins. The significance of these new insights into the so far overlooked properties of the plant plasma membrane phosphoproteome and the long disordered regions is discussed.

Fluorescence recovery after photobleaching reveals high cycling dynamics of plasma membrane aquaporins in Arabidopsis roots under salt stress

The constitutive cycling of plant plasma membrane (PM) proteins is an essential component of their function and regulation under resting or stress conditions. Transgenic Arabidopsis plants that express GFP fusions with AtPIP1;2 and AtPIP2;1, two prototypic PM aquaporins, were used to develop a fluorescence recovery after photobleaching (FRAP) approach. This technique was used to discriminate between PM and endosomal pools of the aquaporin constructs, and to estimate their cycling between intracellular compartments and the cell surface. The membrane trafficking inhibitors tyrphostin A23, naphthalene-1-acetic acid and brefeldin A blocked the latter process. By contrast, a salt treatment (100 mm NaCl for 30 min) markedly enhanced the cycling of the aquaporin constructs and modified their pharmacological inhibition profile. Two distinct models for PM aquaporin cycling in resting or salt-stressed root cells are discussed.