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Mitochondria act as cellular hubs of energy transformation and metabolite conversion in most eukaryotes. Plant mitochondrial electron transport chains are particularly flexible, featuring alternative components, such as ALTERNATIVE NAD(P)H DEHYDROGENASES and ALTERNATIVE OXIDASES (AOXs), that can bypass proton translocation steps. PLANT UNCOUPLING MITOCHONDRIAL PROTEINS (named PUMPs or plant UCPs) have been identified in plants as homologues of mammalian Uncoupling Proteins (UCPs), and their biochemical and physiological roles have been investigated in the context of mitochondrial energy metabolism. To dissect UCP function in Arabidopsis, the two most conserved (UCP1 and UCP2) have been targeted in recent work by combining mutant lines to circumvent potential functional redundancyin vivo. Such approaches rely on the assumption that both proteins reside in the inner mitochondrial membrane as a prerequisite for functional redundancy. Yet, contradicting results have been reported on UCP2 localization in plants. Here we provide evidence that, conversely to UCP1, which is an abundant inner mitochondrial membrane protein, UCP2 localizes to the Golgi rather than to mitochondria. Based on multiple lines of new and prior evidence, we summarize the consensus view that we have reached and provide an example of how open, critical exchange within the research community is able to constructively address ambiguities. Our observations and considerations provide direction to the ongoing discussion about the functions of UCP proteins. They further offer new perspectives for the study of Golgi membrane transport and subcellular targeting principles of membrane proteins. Since 20 to 30 % of genes in plant genomes are predicted to encode transmembrane proteins and the function of most of those proteins has not been experimentally investigated, we highlight the importance of using independent evidence for localization as a prerequisite for understanding physiological function of membrane proteins.

Insertion of membrane proteins into the lipid bilayer is a crucial step during their biosynthesis. Eukaryotic cells face many challenges in directing these proteins to their predestined target membrane. The hydrophobic signal peptide or transmembrane domain (TMD) of the nascent protein must be shielded from the aqueous cytosol and its target membrane identified followed by transport and insertion. Components that evolved to deal with each of these challenging steps range from chaperones to receptors, insertases, and sophisticated translocation complexes. One prominent translocation pathway for most proteins is the signal recognition particle (SRP)-dependent pathway which mediates co-translational translocation of proteins across or into the endoplasmic reticulum (ER) membrane. This textbook example of protein insertion is stretched to its limits when faced with secretory or membrane proteins that lack an amino-terminal signal sequence or TMD. Particularly, a large group of so-called tail-anchored (TA) proteins that harbor a single carboxy-terminal TMD require an alternative, post-translational insertion route into the ER membrane. In this review, we summarize the current research in TA protein insertion with a special focus on plants, address challenges, and highlight future research avenues., Update on different pathways and candidates that facilitate membrane insertion of tail-anchored proteins in eukaryotes with a special emphasis on recent insights in plant research.

Significance The GET pathway is required for the insertion of tail-anchored (TA) membrane proteins in the endoplasmic reticulum (ER) of yeast and mammals. Some orthologous genes had also been identified in higher plants with the exception of one of the two ER membrane receptors required for membrane insertion. Get2/CAML is required for the pathway’s cytosolic chaperone to dock and release its TA protein cargo. Here we report the identification of the elusive plant GET pathway receptor through an interaction screen in Arabidopsis. The candidate allows detection of further Get2/CAML orthologs in higher plants, revealing conservation and function of structural features across kingdoms. Additionally, our results demonstrate that these features, rather than sequence conservation, determine functionality of the candidate within the pathway., Type II tail-anchored (TA) membrane proteins are involved in diverse cellular processes, including protein translocation, vesicle trafficking, and apoptosis. They are characterized by a single C-terminal transmembrane domain that mediates posttranslational targeting and insertion into the endoplasmic reticulum (ER) via the Guided-Entry of TA proteins (GET) pathway. The GET system was originally described in mammals and yeast but was recently shown to be partially conserved in other eukaryotes, such as higher plants. A newly synthesized TA protein is shielded from the cytosol by a pretargeting complex and an ATPase that delivers the protein to the ER, where membrane receptors (Get1/WRB and Get2/CAML) facilitate insertion. In the model plant Arabidopsis thaliana, most components of the pathway were identified through in silico sequence comparison, however, a functional homolog of the coreceptor Get2/CAML remained elusive. We performed immunoprecipitation-mass spectrometry analysis to detect in vivo interactors of AtGET1 and identified a membrane protein of unknown function with low sequence homology but high structural homology to both yeast Get2 and mammalian CAML. The protein localizes to the ER membrane, coexpresses with AtGET1, and binds to Arabidopsis GET pathway components. While loss-of-function lines phenocopy the stunted root hair phenotype of other Atget lines, its heterologous expression together with the coreceptor AtGET1 rescues growth defects of Δget1get2 yeast. Ectopic expression of the cytosolic, positively charged N terminus is sufficient to block TA protein insertion in vitro. Our results collectively confirm that we have identified a plant-specific GET2 in Arabidopsis, and its sequence allows the analysis of cross-kingdom pathway conservation.

In flowering plants, the asymmetrical division of the zygote is the first hallmark of apical-basal polarity of the embryo and is controlled by a MAP kinase pathway that includes the MAPKKK YODA (YDA). In Arabidopsis, YDA is activated by the membrane-associated pseudokinase SHORT SUSPENSOR (SSP) through an unusual parent-of-origin effect: SSP transcripts accumulate specifically in sperm cells but are translationally silent. Only after fertilization is SSP protein transiently produced in the zygote, presumably from paternally inherited transcripts. SSP is a recently diverged, Brassicaceae-specific member of the BRASSINOSTEROID SIGNALING KINASE (BSK) family. BSK proteins typically play broadly overlapping roles as receptor-associated signaling partners in various receptor kinase pathways involved in growth and innate immunity. This raises two questions: How did a protein with generic function involved in signal relay acquire the property of a signal-like patterning cue, and how is the early patterning process activated in plants outside the Brassicaceae family, where SSP orthologs are absent? Here, we show that Arabidopsis BSK1 and BSK2, two close paralogs of SSP that are conserved in flowering plants, are involved in several YDA-dependent signaling events, including embryogenesis. However, the contribution of SSP to YDA activation in the early embryo does not overlap with the contributions of BSK1 and BSK2. The loss of an intramolecular regulatory interaction enables SSP to constitutively activate the YDA signaling pathway, and thus initiates apical-basal patterning as soon as SSP protein is translated after fertilization and without the necessity of invoking canonical receptor activation.

SUMMARYRoot hairs are tubular protrusions of the root epidermis that significantly enlarge the exploitable soil volume in the rhizosphere. Trichoblasts, the cell type responsible for root hair formation, switch from cell elongation to tip growth through polarization of the growth machinery to a pre-defined root hair initiation domain (RHID) at the plasma membrane. The emergence of this polar domain resembles the establishment of cell polarity in other eukaryotic systems [1–3]. Rho-type GTPases of plants (ROPs) are among the first molecular determinants of the RHID [4, 5] and later play a central role in polar growth [6]. Numerous studies have elucidated mechanisms that position the RHID in the cell [7–9] or regulate ROP activity [10–18]. The molecular players that target ROPs to the RHID and initiate outgrowth, however, have not been identified. We dissected the timing of the growth machinery assembly in polarizing hair cells and found that positioning of molecular players and outgrowth are temporally separate processes that are each controlled by specific ROP guanine nucleotide exchange factor (GEFs). A functional analysis of trichoblast-specific GEFs revealed GEF3 to be required for normal ROP polarization and thus efficient root hair emergence, while GEF4 predominantly regulates subsequent tip growth. Ectopic expression of GEF3 induced the formation of spatially confined, ROP-recruiting domains in other cell types, demonstrating the role of GEF3 to serve as a membrane landmark during cell polarization. Our findings suggest that morphogenetic programs in plants employ distinct regulatory modules for the alignment and activation of the cellular growth machinery.

The in vivo analysis of protein-protein interactions (PPIs) is a critical factor for gaining insights into cellular mechanisms and their biological functions. To that end, a constantly growing number of genetic tools has been established, some of which are using baker's yeast (Saccharomyces cerevisiae) as a model organism. Here, we provide a detailed protocol for the yeast mating-based split-ubiquitin system (mbSUS) to study binary interactions among or with full-length membrane proteins in their native subcellular environment. The system is based on the reassembly of two autonomously non-functional ubiquitin moieties attached to proteins of interest (POIs) into a native-like molecule followed by the release of a transcription factor. Upon its nuclear import, the activation of reporter gene expression gives a visual output via growth on interaction-selective media. Additionally, we apply a modification of the classical split-ubiquitin technique called CytoSUS that detects interactions of non-membrane/soluble proteins in their full-length form via translational fusion of an ER membrane anchor.

The in vivo analysis of protein-protein interactions (PPIs) is a critical factor for gaining insights into cellular mechanisms and their biological functions. To that end, a constantly growing number of genetic tools has been established, some of which are using baker's yeast (Saccharomyces cerevisiae) as a model organism. Here, we provide a detailed protocol for the yeast mating-based split-ubiquitin system (mbSUS) to study binary interactions among or with full-length membrane proteins in their native subcellular environment. The system is based on the reassembly of two autonomously non-functional ubiquitin moieties attached to proteins of interest (POIs) into a native-like molecule followed by the release of a transcription factor. Upon its nuclear import, the activation of reporter gene expression gives a visual output via growth on interaction-selective media. Additionally, we apply a modification of the classical split-ubiquitin technique called CytoSUS that detects interactions of non-membrane/soluble proteins in their full-length form via translational fusion of an ER membrane anchor.

Tail-anchored (TA) proteins are anchored to their corresponding membrane via a single transmembrane segment (TMS) at their C-terminus. In yeast, the targeting of TA proteins to the endoplasmic reticulum (ER) can be mediated by the guided entry of TA proteins (GET) pathway, whereas it is not yet clear how mitochondrial TA proteins are targeted to their destination. It is widely observed that some mitochondrial outer membrane (OM) proteins are mistargeted to the ER when overexpressed or when their targeting signal is masked. However, the mechanism of this erroneous sorting is currently unknown. In this study, we demonstrate the involvement of the GET machinery in mistargeting of non-optimal mitochondrial OM proteins to the ER. These findings suggest that the GET machinery can, in principle, recognize and guide mitochondrial and non-canonical TA proteins. Hence, under normal conditions, an active mitochondrial targeting pathway must exist that dominates the kinetic competition against other pathways.

Fluorescence-based protein-protein interaction techniques are vital tools for understanding in vivo cellular functions on a mechanistic level. However, only under the condition of highly efficient (co)transformation and accumulation can techniques such as Förster resonance energy transfer (FRET) realize their potential for providing highly accurate and quantitative interaction data. FRET as a fluorescence-based method unifies several advantages, such as measuring in an in vivo environment, real-time context, and the ability to include transient interactions as well as detecting the mere proximity of proteins. Here, we introduce a novel vector set that incorporates the benefit of the recombination-based 2in1 cloning system with the latest state-of-the-art fluorescent proteins for optimal coaccumulation and FRET output studies. We demonstrate its utility across a range of methods. Merging the 2in1 cloning system with new-generation FRET fluorophore pairs allows for enhanced detection, speeds up the preparation of clones, and enables colocalization studies and the identification of meaningful protein-protein interactions in vivo.

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins drive vesicle fusion in all eukaryotes and contribute to homeostasis, pathogen defense, cell expansion, and growth in plants. Two homologous SNAREs, SYP121 (=SYR1/PEN1) and SYP122, dominate secretory traffic to the Arabidopsis thaliana plasma membrane. Although these proteins overlap functionally, differences between SYP121 and SYP122 have surfaced, suggesting that they mark two discrete pathways for vesicular traffic. The SNAREs share primary cognate partners, which has made separating their respective control mechanisms difficult. Here, we show that the regulatory protein SEC11 (=KEULE) binds selectively with SYP121 to affect secretory traffic mediated by this SNARE. SEC11 rescued traffic block by dominant-negative (inhibitory) fragments of both SNAREs, but only in plants expressing the native SYP121. Traffic and its rescue were sensitive to mutations affecting SEC11 interaction with the N terminus of SYP121. Furthermore, the domain of SEC11 that bound the SYP121 N terminus was itself able to block secretory traffic in the wild type and syp122 but not in syp121 mutant Arabidopsis. Thus, SEC11 binds and selectively regulates secretory traffic mediated by SYP121 and is important for recycling of the SNARE and its cognate partners.

Protein-protein interactions (PPIs) play vital roles in all subcellular processes and a number of tools have been developed for their detection and analysis. Each method has its unique set of benefits and drawbacks that need to be considered prior to their application. In fact, researchers are spoilt for choice when it comes to deciding which method to use for the initial detection of a PPI, and which to corroborate the findings. With constant improvements in microscope development, the possibilities of techniques to study PPIs in vivo, and in real time, are continuously enhanced, and expanded. Here, we describe three common approaches, their recent improvements incorporating a 2in1-cloning approach, and their application in plant cell biology: ratiometric Bimolecular Fluorescence Complementation (rBiFC), FRET Acceptor Photobleaching (FRET-AB), and Fluorescent Lifetime Imaging (FRET-FLIM), using Nicotiana benthamiana leaves and Arabidopsis thaliana cell culture protoplasts as transient expression systems.

Protein-protein interactions (PPIs) play fundamental roles in all cellular processes. Especially membrane proteins facilitate a range of important biological functions in stimuli perception, signaling, and transport. Here we describe a detailed protocol for the yeast mating-based Split-Ubiquitin System (mbSUS) to study PPIs of ER membrane proteins in vivo. In contrast to the prominent Yeast Two-Hybrid, mbSUS enables analysis of full-length membrane proteins in their native cellular context. The system is based on the ubiquitin proteasome pathway leading to the release of an artificial transcription factor followed by activation of reporter genes to visualize PPIs. The mating-based approach is suitable for both small- and large-scale interaction studies. Additionally, we describe protocols to apply the recently established SUS Bridge assay (SUB) which is optimized for the detection of ternary protein interactions.

Protein-protein interactions (PPIs) play fundamental roles in all cellular processes. Especially membrane proteins facilitate a range of important biological functions in stimuli perception, signaling, and transport. Here we describe a detailed protocol for the yeast mating-based Split-Ubiquitin System (mbSUS) to study PPIs of ER membrane proteins in vivo. In contrast to the prominent Yeast Two-Hybrid, mbSUS enables analysis of full-length membrane proteins in their native cellular context. The system is based on the ubiquitin proteasome pathway leading to the release of an artificial transcription factor followed by activation of reporter genes to visualize PPIs. The mating-based approach is suitable for both small- and large-scale interaction studies. Additionally, we describe protocols to apply the recently established SUS Bridge assay (SUB) which is optimized for the detection of ternary protein interactions.

Protein-protein interactions (PPIs) play vital roles in all subcellular processes and a number of tools have been developed for their detection and analysis. Each method has its unique set of benefits and drawbacks that need to be considered prior to their application. In fact, researchers are spoilt for choice when it comes to deciding which method to use for the initial detection of a PPI, and which to corroborate the findings. With constant improvements in microscope development, the possibilities of techniques to study PPIs in vivo, and in real time, are continuously enhanced, and expanded. Here, we describe three common approaches, their recent improvements incorporating a 2in1-cloning approach, and their application in plant cell biology: ratiometric Bimolecular Fluorescence Complementation (rBiFC), FRET Acceptor Photobleaching (FRET-AB), and Fluorescent Lifetime Imaging (FRET-FLIM), using Nicotiana benthamiana leaves and Arabidopsis thaliana cell culture protoplasts as transient expression systems.

Next Section Abstract Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins are key players in cellular trafficking and coordinate vital cellular processes, such as cytokinesis, pathogen defense, and ion transport regulation. With few exceptions, SNAREs are tail-anchored (TA) proteins, bearing a C-terminal hydrophobic domain that is essential for their membrane integration. Recently, the Guided Entry of Tail-anchored proteins (GET) pathway was described in mammalian and yeast cells that serve as a blueprint of TA protein insertion [Schuldiner M, et al. (2008) Cell 134(4):634–645; Stefanovic S, Hegde RS (2007) Cell 128(6):1147–1159]. This pathway consists of six proteins, with the cytosolic ATPase GET3 chaperoning the newly synthesized TA protein posttranslationally from the ribosome to the endoplasmic reticulum (ER) membrane. Structural and biochemical insights confirmed the potential of pathway components to facilitate membrane insertion, but the physiological significance in multicellular organisms remains to be resolved. Our phylogenetic analysis of 37 GET3 orthologs from 18 different species revealed the presence of two different GET3 clades. We identified and analyzed GET pathway components in Arabidopsis thaliana and found reduced root hair elongation in Atget lines, possibly as a result of reduced SNARE biogenesis. Overexpression of AtGET3a in a receptor knockout (KO) results in severe growth defects, suggesting presence of alternative insertion pathways while highlighting an intricate involvement for the GET pathway in cellular homeostasis of plants.

Growth and development of plants is ultimately driven by light energy captured through photosynthesis. ATP acts as universal cellular energy cofactor fuelling all life processes, including gene expression, metabolism, and transport. Despite a mechanistic understanding of ATP biochemistry, ATP dynamics in the living plant have been largely elusive. Here we establish live MgATP2−assessment in plants using the fluorescent protein biosensor ATeam1.03-nD/nA. We generate Arabidopsis sensor lines and investigate the sensorin vitrounder conditions appropriate for the plant cytosol. We establish an assay for ATP fluxes in isolated mitochondria, and demonstrate that the sensor responds rapidly and reliably to MgATP2−changesin planta. A MgATP2−map of the Arabidopsis seedling highlights different MgATP2−concentrations between tissues and in individual cell types, such as root hairs. Progression of hypoxia reveals substantial plasticity of ATP homeostasis in seedlings, demonstrating that ATP dynamics can be monitored in the living plant.One-sentence SummarySensing of MgATP2−by fluorimetry and microscopy allows dissection of ATP fluxes of isolated organelles, and dynamics of cytosolic MgATP2−in vivo.Funding AgenciesThis work was supported by the Deutsche Forschungsgemeinschaft (DFG) through the Emmy-Noether programme (SCHW1719/1-1; M.S. and GR4251/1-1; C.G.), the Research Training Group GRK 2064 (M.S.; A.J.M.), the Priority Program SPP1710 (A.J.M.) and a grant (SCHW1719/5-1; M.S.) as part of the package PAK918. The Seed Fund grant CoSens from the Bioeconomy Science Center, NRW (A.J.M.; M.S.) is gratefully acknowledged. The scientific activities of the Bioeconomy Science Center were financially supported by the Ministry of Innovation, Science and Research within the framework of the NRW Strategieprojekt BioSC (No. 313/323-400-002 13). A.Co. received funding by the Ministero dell’Istruzione, dell’Università e della Ricerca through the FIRB 2010 programme (RBFR10S1LJ_001) and Piano di Sviluppo di Ateneo 2015 (Università degli Studi di Milano). M.Z. received funding by the Ministero dell’Istruzione, dell’Università e della Ricerca (Italy) through the PRIN 2010 programme (PRIN2010CSJX4F). S.W. and T.N. received travel support by the Deutscher Akademischer Austauschdienst (DAAD). V.D.C. was supported by the European Social Fund, Operational Programme 2007/2013, and an Erasmus+ Traineeship grant. M.D.F was supported by The Human Frontier Science Program (RPG0053/2012), and the Leverhulme Foundation (RPG-2015-437). I.M.M. was supported by a grant from the Danish Council for Independent Research - Natural Sciences. V.C.P. was supported by the Innovation and Technology Fund (Funding Support to Partner State Key Laboratories in Hong Kong) of the HKSAR.AbbreviationsAAC – ADP/ATP carrier; AK – adenylate kinase; cAT – carboxyatractyloside; CCCP – carbonyl cyanide m-chlorophenyl hydrazone; CFP – cyan fluorescent protein; CLSM – confocal laser scanning microscopy; ETC – electron transport chain; FRET – Förster Resonance Energy Transfer; LSFM – light sheet fluorescence microscopy.

Plant plasma membrane intrinsic proteins (PIPs) are aquaporins that facilitate the passive movement of water and small neutral solutes through biological membranes. Here, we report that post-Golgi trafficking of PIP2;7 in Arabidopsis thaliana involves specific interactions with two syntaxin proteins, namely, the Qc-SNARE SYP61 and the Qa-SNARE SYP121, that the proper delivery of PIP2;7 to the plasma membrane depends on the activity of the two SNAREs, and that the SNAREs colocalize and physically interact. These findings are indicative of an important role for SYP61 and SYP121, possibly forming a SNARE complex. Our data support a model in which direct interactions between specific SNARE proteins and PIP aquaporins modulate their post-Golgi trafficking and thus contribute to the fine-tuning of the water permeability of the plasma membrane.

The Arabidopsis thaliana Qa-SNARE SYP121 (=SYR1/PEN1) drives vesicle traffic at the plasma membrane of cells throughout the vegetative plant. It facilitates responses to drought, to the water stress hormone abscisic acid, and to pathogen attack, and it is essential for recovery from so-called programmed stomatal closure. How SYP121-mediated traffic is regulated is largely unknown, although it is thought to depend on formation of a fusion-competent SNARE core complex with the cognate partners VAMP721 and SNAP33. Like SYP121, the Arabidopsis Sec1/Munc18 protein SEC11 (=KEULE) is expressed throughout the vegetative plant. We find that SEC11 binds directly with SYP121 both in vitro and in vivo to affect secretory traffic. Binding occurs through two distinct modes, one requiring only SEC11 and SYP121 and the second dependent on assembly of a complex with VAMP721 and SNAP33. SEC11 competes dynamically for SYP121 binding with SNAP33 and VAMP721, and this competition is predicated by SEC11 association with the N terminus of SYP121. These and additional data are consistent with a model in which SYP121-mediated vesicle fusion is regulated by an unusual “handshaking” mechanism of concerted SEC11 debinding and rebinding. They also implicate one or more factors that alter or disrupt SEC11 association with the SYP121 N terminus as an early step initiating SNARE complex formation.

Identifying key players and their interactions is fundamental for understanding biochemical mechanisms at the molecular level. The ever-increasing number of alternative ways to detect protein-protein interactions (PPIs) speaks volumes about the creativity of scientists in hunting for the optimal technique. PPIs derived from single experiments or high-throughput screens enable the decoding of binary interactions, the building of large-scale interaction maps of single organisms, and the establishment of cross-species networks. This review provides a historical view of the development of PPI technology over the past three decades, particularly focusing on in vivo PPI techniques that are inexpensive to perform and/or easy to implement in a state-of-the-art molecular biology laboratory. Special emphasis is given to their feasibility and application for plant biology as well as recent improvements or additions to these established techniques. The biology behind each method and its advantages and disadvantages are discussed in detail, as are the design, execution, and evaluation of PPI analysis. We also aim to raise awareness about the technological considerations and the inherent flaws of these methods, which may have an impact on the biological interpretation of PPIs. Ultimately, we hope this review serves as a useful reference when choosing the most suitable PPI technique.

Background: Specific mutations in the chaperone protein CSPα cause adult-onset neuronal ceroid lipofuscinosis. Results: These mutants form SDS-resistant aggregates in a palmitoylation-dependent manner in cell lines and brain samples from mutation carriers. Conclusion: Palmitoylation induces disease-causing CSPα mutants to form SDS-resistant aggregates. Significance: Formation of SDS-resistant CSPα aggregates may underlie development of adult-onset neuronal ceroid lipofuscinosis., Recently, mutations in the DNAJC5 gene encoding cysteine-string protein α (CSPα) were identified to cause the neurodegenerative disorder adult-onset neuronal ceroid lipofuscinosis. The disease-causing mutations (L115R or ΔL116) occur within the cysteine-string domain, a region of the protein that is post-translationally modified by extensive palmitoylation. Here we demonstrate that L115R and ΔL116 mutant proteins are mistargeted in neuroendocrine cells and form SDS-resistant aggregates, concordant with the properties of other mutant proteins linked to neurodegenerative disorders. The mutant aggregates are membrane-associated and incorporate palmitate. Indeed, co-expression of palmitoyltransferase enzymes promoted the aggregation of the CSPα mutants, and chemical depalmitoylation solubilized the aggregates, demonstrating that aggregation is induced and maintained by palmitoylation. In agreement with these observations, SDS-resistant CSPα aggregates were present in brain samples from patients carrying the L115R mutation and were depleted by chemical depalmitoylation. In summary, this study identifies a novel interplay between genetic mutations and palmitoylation in driving aggregation of CSPα mutant proteins. We propose that this palmitoylation-induced aggregation of mutant CSPα proteins may underlie the development of adult-onset neuronal ceroid lipofuscinosis in affected families.

Plasma membrane intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water through the cell membrane. We previously showed that the traffic of the maize (Zea mays) PIP2;5 to the plasma membrane is dependent on the endoplasmic reticulum diacidic export motif. Here, we report that the post-Golgi traffic and water channel activity of PIP2;5 are regulated by the SNARE (for soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) SYP121, a plasma membrane resident syntaxin involved in vesicle traffic, signaling, and regulation of K(+) channels. We demonstrate that the expression of the dominant-negative SYP121-Sp2 fragment in maize mesophyll protoplasts or epidermal cells leads to a decrease in the delivery of PIP2;5 to the plasma membrane. Protoplast and oocyte swelling assays showed that PIP2;5 water channel activity is negatively affected by SYP121-Sp2. A combination of in vitro (copurification assays) and in vivo (bimolecular fluorescence complementation, Förster resonance energy transfer, and yeast split-ubiquitin) approaches allowed us to demonstrate that SYP121 and PIP2;5 physically interact. Together with previous data demonstrating the role of SYP121 in regulating K(+) channel trafficking and activity, these results suggest that SYP121 SNARE contributes to the regulation of the cell osmotic homeostasis.

The vesicle-trafficking protein SYP121 (SYR1/PEN1) was originally identified in association with ion channel control at the plasma membrane of stomatal guard cells, although stomata of the Arabidopsis syp121 loss-of-function mutant close normally in ABA and high Ca²⁺. We have now uncovered a set of stomatal phenotypes in the syp121 mutant that reduce CO₂ assimilation, slow vegetative growth and increase water use efficiency in the whole plant, conditional upon high light intensities and low relative humidity. Stomatal opening and the rise in stomatal transpiration of the mutant was delayed in the light and following Ca²⁺-evoked closure, consistent with a constitutive form of so-called programmed stomatal closure. Delayed reopening was observed in the syp121, but not in the syp122 mutant lacking the homologous gene product; the delay was rescued by complementation with wild-type SYP121 and was phenocopied in wild-type plants in the presence of the vesicle-trafficking inhibitor Brefeldin A. K⁺ channel current that normally mediates K⁺ uptake for stomatal opening was suppressed in the syp121 mutant and, following closure, its recovery was slowed compared to guard cells of wild-type plants. Evoked stomatal closure was accompanied by internalisation of GFP-tagged KAT1 K⁺ channels in both wild-type and syp121 mutant guard cells, but their subsequently recycling was slowed in the mutant. Our findings indicate that SYP121 facilitates stomatal reopening and they suggest that K⁺ channel traffic and recycling to the plasma membrane underpins the stress memory phenomenon of programmed closure in stomata. Additionally, they underline the significance of vesicle traffic for whole-plant water use and biomass production, tying SYP121 function to guard cell membrane transport and stomatal control.

Cytokinins are hormones that are involved in various processes of plant growth and development. The model of cytokinin signalling starts with hormone perception through membrane-localized histidine kinase receptors. Although the biochemical properties and functions of these receptors have been extensively studied, there is no solid proof of their subcellular localization. Here, cell biological and biochemical evidence for the localization of functional fluorophor-tagged fusions of Arabidopsis histidine kinase 3 (AHK3) and 4 (AHK4), members of the cytokinin receptor family, in the endoplasmic reticulum (ER) is provided. Furthermore, membrane-bound AHK3 interacts with AHK4 in vivo. The ER localization and putative function of cytokinin receptors from the ER have major impacts on the concept of cytokinin perception and signalling, and hormonal cross-talk in plants.

Throughout their development, plants balance cell surface area and volume with ion transport and turgor. This balance lies at the core of cellular homeostatic networks and is central to the capacity to withstand abiotic as well as biotic stress. Remarkably, very little is known of its mechanics, notably how membrane traffic is coupled with osmotic solute transport and its control. Here we outline recent developments in the understanding of so-called SNARE proteins that form part of the machinery for membrane vesicle traffic in all eukaryotes. We focus on SNAREs active at the plasma membrane and the evidence for specialisation in enhanced, homeostatic and stress-related traffic. Recent studies have placed a canonical SNARE complex associated with the plasma membrane in pathogen defense, and the discovery of the first SNARE as a binding partner with ion channels has demonstrated a fundamental link to inorganic osmotic solute uptake. Work localising the channel binding site has now identified a new and previously uncharacterised motif, yielding important clues to a plausible mechanism coupling traffic and transport. We examine the evidence that this physical interaction serves to balance enhanced osmotic solute uptake with membrane expansion through mutual control of the two processes. We calculate that even during rapid cell expansion only a minute fraction of SNAREs present at the membrane need be engaged in vesicle traffic at any one time, a number surprisingly close to the known density of ion channels at the plant plasma membrane. Finally, we suggest a framework of alternative models coupling transport and traffic, and approachable through direct, experimental testing.

To date the use of fluorescent reporter constructs in analysing membrane transport has been limited primarily to cell lines expressing stably either the tagged transporter protein(s) or markers to identify lineages of interest. Strategies for transient expression have yet to be exploited in transport analysis, despite their wide application in cellular imaging studies. Here we describe a Gateway-compatible, bicistronic vector, incorporating the constitutive Ubiqutin-10 gene promoter of Arabidopsis that gives prolonged expression after transient transformation and enables fluorescence marking of cells without a fusion construct. We show that Arabidopsis root epidermal cells are readily transformed by co-cultivation with Agrobacterium and are tractable for quantitative electrophysiological analysis. As a proof of principle, we transiently transformed Arabidopsis with the bicistronic vector carrying GFP as the fluorescent marker and, separately, the integral plasma membrane protein SYP121 essential for the inward K+ channel current. We demonstrate that transient expression of SYP121 in syp121 mutant plants is sufficient to rescue the K+ current in vivo. The combination of transient expression and use of the bicistronic vector promises significant advantages for studies of membrane transport and nutrient acquisition in roots

Eukaryotic cells expand using vesicle traffic to increase membrane surface area. Expansion in walled eukaryotes is driven by turgor pressure which depends fundamentally on the uptake and accumulation of inorganic ions. Thus, ion uptake and vesicle traffic must be controlled coordinately for growth. How this coordination is achieved is still poorly understood, yet is so elemental to life that resolving the underlying mechanisms will have profound implications for our understanding of cell proliferation, development, and pathogenesis, and will find applications in addressing the mineral and water use by plants in the face of global environmental change. Recent discoveries of interactions between trafficking and ion transport proteins now open the door to an entirely new approach to understanding this coordination. Some of the advances to date in identifying key protein partners in the model plant Arabidopsis and in yeast at membranes vital for cell volume and turgor control are outlined here. Additionally, new evidence is provided of a wider participation among Arabidopsis Kv-like K(+) channels in selective interaction with the vesicle-trafficking protein SYP121. These advances suggest some common paradigms that will help guide further exploration of the underlying connection between ion transport and membrane traffic and should transform our understanding of cellular homeostasis in eukaryotes.

Fluorescent tagging of proteins and confocal imaging techniques have become methods of choice in analysing the distributions and dynamic characteristics of proteins at the subcellular level. In common use are a number of strategies for transient expression that greatly reduce the preparation time in advance of imaging, but their applications are limited in success outside a few tractable species and tissues. We previously developed a simple method to transiently express fluorescently-tagged proteins in Arabidopsis root epidermis and root hairs. We describe here a set of Gateway-compatable vectors with fluorescent tags incorporating the ubiqutin-10 gene promoter (P(UBQ10) ) of Arabidopsis that gives prolonged expression of the fluorescently-tagged proteins, both in tobacco and Arabidopsis tissues, after transient transformation, and is equally useful in generating stably transformed lines. As a proof of principle, we carried out transformations with fluorescent markers for the integral plasma membrane protein SYP121, a member of the SNARE family of vesicle-trafficking proteins, and for DHAR1, a cytosolic protein that facilitates the scavenging of reactive oxygen species. We also carried out transformations with SYP121 and its interacting partner, the KC1 K(+) channel, to demonstrate the utility of the methods in bimolecular fluorescence complementation (BiFC). Transient transformations of Arabidopsis using Agrobacterium co-cultivation methods yielded expression in all epidermal cells, including root hairs and guard cells. Comparative studies showed that the P(UBQ10) promoter gives similar levels of expression to that driven by the native SYP121 promoter, faithfully reproducing the characteristics of protein distributions at the subcellular level. Unlike the 35S-driven construct, expression under the P(UBQ10) promoter remained elevated for periods in excess of 2 weeks after transient transformation. This toolbox of vectors and fluorescent tags promises significant advantages for the study of membrane dynamics and cellular development, as well as events associated with environmental stimuli in guard cells and nutrient acquisition in roots.

The SNARE (for soluble N-ethylmaleimide–sensitive factor protein attachment protein receptor) protein SYP121 (=SYR1/PEN1) of Arabidopsis thaliana facilitates vesicle traffic, delivering ion channels and other cargo to the plasma membrane, and contributing to plant cell expansion and defense. Recently, we reported that SYP121 also interacts directly with the K+ channel subunit KC1 and forms a tripartite complex with a second K+ channel subunit, AKT1, to control channel gating and K+ transport. Here, we report isolating a minimal sequence motif of SYP121 prerequisite for its interaction with KC1. We made use of yeast mating-based split-ubiquitin and in vivo bimolecular fluorescence complementation assays for protein–protein interaction and of expression and electrophysiological analysis. The results show that interaction of SYP121 with KC1 is associated with a novel FxRF motif uniquely situated within the first 12 residues of the SNARE sequence, that this motif is the minimal requirement for SNARE-dependent alterations in K+ channel gating when heterologously expressed, and that rescue of KC1-associated K+ current of the root epidermis in syp121 mutant Arabidopsis plants depends on expression of SNARE constructs incorporating this motif. These results establish the FxRF sequence as a previously unidentified motif required for SNARE–ion channel interactions and lead us to suggest a mechanistic framework for understanding the coordination of vesicle traffic with transmembrane ion transport.

SNARE (soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) proteins drive membrane fusion and contribute to membrane and protein targeting and delivery in all eukaryotic cells. SNAREs are essential to the mechanics of cell growth and development, and they facilitate a number of homeostatic and evoked responses in plants, from hormone signalling to pathogen defence. Additionally, there is now unambiguous evidence that SNAREs play roles in anchoring other membrane proteins and in facilitating ion channel gating through direct, physical interaction with channel proteins. What is the physiological significance of these additional features of plant SNAREs? We explore possible interpretations and suggest functions as scaffolds for effective signal transmission between proteins and, by analogy with a mechanical device invented by James Watt, as molecular governors to coordinate solute transport with cell expansion and growth.

The gaseous phytohormone ethylene regulates many developmental processes and responses to environmental conditions in higher plants. In Arabidopsis thaliana, ethylene perception and initiation of signaling are mediated by a family of five receptors which are related to prokaryotic two-component sensor histidine kinases. The transient expression of fluorescence-tagged receptors in tobacco (Nicotiana benthamiana) epidermal leaf cells demonstrated that all ethylene receptors are targeted to the ER endomembrane network and do not localize to the plasmalemma. In support of in planta overlay studies, the ethylene receptors form homomeric and heteromeric protein complexes at the ER in living plant cells, as shown by membrane recruitment assays. A comparable in vivo interaction pattern was found in the yeast mating-based split-ubiquitin system. The overlapping but distinct expression pattern of the ethylene receptor genes suggests a differential composition of the ethylene receptor complexes in different plant tissues. Our findings may have crucial functional implications on the ethylene receptor-mediated efficiency of hormone perception, induction of signaling, signal attenuation and output.

The A-type response regulator ARR4 is an element in the two-component signalling network of Arabidopsis. ARR4 interacts with the N-terminus of the red/far-red light photoreceptor phytochrome B (phyB) and functions as a modulator of photomorphogenesis. In concert with other A-type response regulators, ARR4 also participates in the modulation of the cytokinin response pathway. Here evidence is presented that ARR4 directly modulates the activity state of phyB in planta, not only under inductive but also under extended irradiation with red light. Mutation of the phosphorylatable aspartate to asparagine within the receiver domain creates a version of ARR4 that negatively affects photomorphogenesis. Additional evidence suggests that ARR4 activity is regulated by a phosphorelay mechanism that depends on the AHK family of cytokinin receptors. Accordingly, the ability of ARR4 to function on phyB is modified by exogenous application of cytokinin. These results implicate a cross-talk between cytokinin and light signalling mediated by ARR4. This cross-talk enables the plant to adjust light reponsiveness to endogenous requirements in growth and development.

Growth in plants depends on ion transport for osmotic solute uptake and secretory membrane trafficking to deliver material for wall remodelling and cell expansion. The coordination of these processes lies at the heart of the question, unresolved for more than a century, of how plants regulate cell volume and turgor. Here we report that the SNARE protein SYP121 (SYR1/PEN1), which mediates vesicle fusion at the Arabidopsis plasma membrane, binds the voltage sensor domains (VSDs) of K(+) channels to confer a voltage dependence on secretory traffic in parallel with K(+) uptake. VSD binding enhances secretion in vivo subject to voltage, and mutations affecting VSD conformation alter binding and secretion in parallel with channel gating, net K(+) concentration, osmotic content and growth. These results demonstrate a new and unexpected mechanism for secretory control, in which a subset of plant SNAREs commandeer K(+) channel VSDs to coordinate membrane trafficking with K(+) uptake for growth.

SNARE (soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) proteins drive vesicle traffic, delivering membrane and cargo to target sites within the cell and at its surface. They contribute to cell homeostasis, morphogenesis, and pathogen defense. A subset of SNAREs, including the Arabidopsis thaliana SNARE SYP121, are known also to coordinate solute uptake via physical interactions with K+ channels and to moderate their gating at the plasma membrane. Here, we identify a second subset of SNAREs that interact to control these K+ channels, but with opposing actions on gating. We show that VAMPs (vesicle-associated membrane proteins), which target vesicles to the plasma membrane, also interact with and suppress the activities of the inward-rectifying K+ channels KAT1 and KC1. Interactions were evident in yeast split-ubiquitin assays, they were recovered in vivo by ratiometric bimolecular fluorescence complementation, and they were sensitive to mutation of a single residue, Tyr-57, within the longin domain of VAMP721. Interaction was also recovered on exchange of the residue at this site in the homolog VAMP723, which normally localizes to the endoplasmic reticulum and otherwise did not interact. Functional analysis showed reduced channel activity and alterations in voltage sensitivity that are best explained by a physical interaction with the channel gates. These actions complement those of SYP121, a cognate SNARE partner of VAMP721, and lead us to propose that the channel interactions reflect a “hand-off” in channel control between the two SNARE proteins that is woven together with vesicle fusion.

Plants and animals employ innate immune systems to cope with microbial infection. Pattern-triggered immunity relies on the recognition of microbe-derived patterns by pattern recognition receptors (PRRs). Necrosis and ethylene-inducing peptide 1-like proteins (NLPs) constitute plant immunogenic patterns that are unique, as these proteins are produced by multiple prokaryotic (bacterial) and eukaryotic (fungal, oomycete) species. Here we show that the leucine-rich repeat receptor protein (LRR-RP) RLP23 binds in vivo to a conserved 20-amino-acid fragment found in most NLPs (nlp20), thereby mediating immune activation in Arabidopsis thaliana. RLP23 forms a constitutive, ligand-independent complex with the LRR receptor kinase (LRR-RK) SOBIR1 (Suppressor of Brassinosteroid insensitive 1 (BRI1)-associated kinase (BAK1)-interacting receptor kinase 1), and recruits a second LRR-RK, BAK1, into a tripartite complex upon ligand binding. Stable, ectopic expression of RLP23 in potato (Solanum tuberosum) confers nlp20 pattern recognition and enhanced immunity to destructive oomycete and fungal plant pathogens, such as Phytophthora infestans and Sclerotinia sclerotiorum. PRRs that recognize widespread microbial patterns might be particularly suited for engineering immunity in crop plants.

The molecular machinery that catalyzes S-acylation reactions at the Golgi comprises zDHHC enzymes with major differences in substrate affinity and S-acylation activity. It is proposed that the coexistence of these two groups of enzymes in cells is important to allow the Golgi S-acylation machinery to modify a wide and diverse set of substrates., S-acylation, the attachment of fatty acids onto cysteine residues, regulates protein trafficking and function and is mediated by a family of zDHHC enzymes. The S-acylation of peripheral membrane proteins has been proposed to occur at the Golgi, catalyzed by an S-acylation machinery that displays little substrate specificity. To advance understanding of how S-acylation of peripheral membrane proteins is handled by Golgi zDHHC enzymes, we investigated interactions between a subset of four Golgi zDHHC enzymes and two S-acylated proteins—synaptosomal-associated protein 25 (SNAP25) and cysteine-string protein (CSP). Our results uncover major differences in substrate recognition and S-acylation by these zDHHC enzymes. The ankyrin-repeat domains of zDHHC17 and zDHHC13 mediated strong and selective interactions with SNAP25/CSP, whereas binding of zDHHC3 and zDHHC7 to these proteins was barely detectable. Despite this, zDHHC3/zDHHC7 could S-acylate SNAP25/CSP more efficiently than zDHHC17, whereas zDHHC13 lacked S-acylation activity toward these proteins. Overall the results of this study support a model in which dynamic intracellular localization of peripheral membrane proteins is achieved by highly selective recruitment by a subset of zDHHC enzymes at the Golgi, combined with highly efficient S-acylation by other Golgi zDHHC enzymes.

Dynamic networks of protein-protein interactions regulate numerous cellular processes and determine the ability to respond appropriately to environmental stimuli. However, the investigation of protein complex formation in living plant cells by methods such as fluorescence resonance energy transfer has remained experimentally difficult, time consuming and requires sophisticated technical equipment. Here, we report the implementation of a bimolecular fluorescence complementation (BiFC) technique for visualization of protein-protein interactions in plant cells. This approach relies on the formation of a fluorescent complex by two non-fluorescent fragments of the yellow fluorescent protein brought together by association of interacting proteins fused to these fragments (Hu et al., 2002). To enable BiFC analyses in plant cells, we generated different complementary sets of expression vectors, which enable protein interaction studies in transiently or stably transformed cells. These vectors were used to investigate and visualize homodimerization of the basic leucine zipper (bZIP) transcription factor bZIP63 and the zinc finger protein lesion simulating disease 1 (LSD1) from Arabidopsis as well as the dimer formation of the tobacco 14-3-3 protein T14-3c. The interaction analyses of these model proteins established the feasibility of BiFC analyses for efficient visualization of structurally distinct proteins in different cellular compartments. Our investigations revealed a remarkable signal fluorescence intensity of interacting protein complexes as well as a high reproducibility and technical simplicity of the method in different plant systems. Consequently, the BiFC approach should significantly facilitate the visualization of the subcellular sites of protein interactions under conditions that closely reflect the normal physiological environment.

Over the past decade, confocal microscopy and the ever-expanding toolchest of fluorescent protein (xFP) markers and technologies have become routine methods for the biological laboratory. A common use of xFP fluorophores is in localizing proteins and the subcellular structures with which they associate, including analyzing their distribution and dynamics and the interactions of proteins in vivo. Additionally, a number of so-called optical highlighters have proven especially useful in analyzing the kinetics of these processes in pulse-chase studies of protein relocation(s) following an experimental challenge. Here we focus on exemplary methods in transformation and live-cell imaging in plant cells, with the expectation that researchers will find these and the accompanying resources useful as a starting point in developing their own expertise.

Protein-protein interactions are vital to all living cells. Many different in vivo and in vitro approaches are published which enable detection and determination of interactions between two proteins. However, most of these techniques are not designed to test for multi-protein interactions although oligomeric protein complexes are probably more likely to be the status quo in vivo than simple binary interactions. This chapter describes how a well-established system for detection of protein-protein interactions can be exploited for assaying and screening of different kinds of oligomeric interactions. The split-ubiquitin bridge assay (SUB) is based on the split-ubiquitin system and enables detection of trimeric protein complexes. SUB assays can be used to check several putative interaction couples and to screen for novel interaction partners in different ways.

Over the past decade, confocal microscopy and the ever-expanding toolchest of fluorescent protein (xFP) markers and technologies have become routine methods for the biological laboratory. A common use of xFP fluorophores is in localizing proteins and the subcellular structures with which they associate, including analyzing their distribution and dynamics and the interactions of proteins in vivo. Additionally, a number of so-called optical highlighters have proven especially useful in analyzing the kinetics of these processes in pulse-chase studies of protein relocation(s) following an experimental challenge. Here we focus on exemplary methods in transformation and live-cell imaging in plant cells, with the expectation that researchers will find these and the accompanying resources useful as a starting point in developing their own expertise.

Protein-protein interactions are vital to all living cells. Many different in vivo and in vitro approaches are published which enable detection and determination of interactions between two proteins. However, most of these techniques are not designed to test for multi-protein interactions although oligomeric protein complexes are probably more likely to be the status quo in vivo than simple binary interactions. This chapter describes how a well-established system for detection of protein-protein interactions can be exploited for assaying and screening of different kinds of oligomeric interactions. The split-ubiquitin bridge assay (SUB) is based on the split-ubiquitin system and enables detection of trimeric protein complexes. SUB assays can be used to check several putative interaction couples and to screen for novel interaction partners in different ways.

Calcineurin B-like proteins (CBLs) function as membrane-anchored Ca2+ sensors that, when activated, recruit a specific set of Ser-Thr kinases, calcineurin B-like protein-interacting protein kinases (CIPKs), to their sites of action (for review, see [Luan et al., 2009][1]; [Kudla et al., 2010][2]).

Fluorescent tagging of proteins and confocal imaging techniques have become methods of choice in analysing the distributions and dynamic characteristics of proteins at the subcellular level. In common use are a number of strategies for transient expression that greatly reduce the preparation time in advance of imaging, but their applications are limited in success outside a few tractable species and tissues. We previously developed a simple method to transiently express fluorescently-tagged proteins in Arabidopsis root epidermis and root hairs. We describe here a set of Gateway-compatable vectors with fluorescent tags incorporating the ubiqutin-10 gene promoter (P(UBQ10) ) of Arabidopsis that gives prolonged expression of the fluorescently-tagged proteins, both in tobacco and Arabidopsis tissues, after transient transformation, and is equally useful in generating stably transformed lines. As a proof of principle, we carried out transformations with fluorescent markers for the integral plasma membrane protein SYP121, a member of the SNARE family of vesicle-trafficking proteins, and for DHAR1, a cytosolic protein that facilitates the scavenging of reactive oxygen species. We also carried out transformations with SYP121 and its interacting partner, the KC1 K(+) channel, to demonstrate the utility of the methods in bimolecular fluorescence complementation (BiFC). Transient transformations of Arabidopsis using Agrobacterium co-cultivation methods yielded expression in all epidermal cells, including root hairs and guard cells. Comparative studies showed that the P(UBQ10) promoter gives similar levels of expression to that driven by the native SYP121 promoter, faithfully reproducing the characteristics of protein distributions at the subcellular level. Unlike the 35S-driven construct, expression under the P(UBQ10) promoter remained elevated for periods in excess of 2 weeks after transient transformation. This toolbox of vectors and fluorescent tags promises significant advantages for the study of membrane dynamics and cellular development, as well as events associated with environmental stimuli in guard cells and nutrient acquisition in roots.

A few membrane vesicle trafficking (SNARE) proteins in plants are associated with signaling and transmembrane ion transport, including control of plasma membrane ion channels. Vesicle traffic contributes to the population of ion channels at the plasma membrane. Nonetheless, it is unclear whether these SNAREs also interact directly to affect channel gating and, if so, what functional impact this might have on the plant. Here, we report that the Arabidopsis thaliana SNARE SYP121 binds to KC1, a regulatory K+ channel subunit that assembles with different inward-rectifying K+ channels to affect their activities. We demonstrate that SYP121 interacts preferentially with KC1 over other Kv-like K+ channel subunits and that KC1 interacts specifically with SYP121 but not with its closest structural and functional homolog SYP122 nor with another related SNARE SYP111. SYP121 promoted gating of the inward-rectifying K+ channel AKT1 but only when heterologously coexpressed with KC1. Mutation in any one of the three genes, SYP121, KC1, and AKT1, selectively suppressed the inward-rectifying K+ current in Arabidopsis root epidermal protoplasts as well as K+ acquisition and growth in seedlings when channel-mediated K+ uptake was limiting. That SYP121 should be important for gating of a K+ channel and its role in inorganic mineral nutrition demonstrates an unexpected role for SNARE–ion channel interactions, apparently divorced from signaling and vesicle traffic. Instead, it suggests a role in regulating K+ uptake coordinately with membrane expansion for cell growth.

Dynamic and reversible protein-protein interactions have a pivotal function in all living cells. For instance, protein-protein interactions are involved in the assembly and regulation of multimeric enzymes and transcription factors, various signal response pathways, intracellular sorting and movement of proteins and membrane vesicles, cell-to-cell protein transport, and many others. Here we provide a detailed protocol for the mating-based split-ubiquitin system (mbSUS), which is a sensitive and user-friendly alternative to the classical yeast two-hybrid system in particular. mbSUS relies on the ubiquitin-degradation pathway as a sensor for protein-protein interactions. Thus, mbSUS is predominantly suitable for the determination of full-length proteins localized in the cytoplasm and in or at membrane compartments, without the need for their truncation and nuclear mislocation. In addition, we present a set of Gateway compatible mbSUS vectors that allow the rapid generation of constructs for fast and efficient interaction studies. An additional vector is introduced that allows the extension of mbSUS for the analysis of oligomeric protein complex formation and competition assays in vivo. In summary, mbSUS provides an additional versatile tool for protein-protein interaction studies, which is complementary to in planta assays such as BiFC and FRET.

Dynamic and reversible protein-protein interactions have a pivotal function in all living cells. For instance, protein-protein interactions are involved in the assembly and regulation of multimeric enzymes and transcription factors, various signal response pathways, intracellular sorting and movement of proteins and membrane vesicles, cell-to-cell protein transport, and many others. Here we provide a detailed protocol for the mating-based split-ubiquitin system (mbSUS), which is a sensitive and user-friendly alternative to the classical yeast two-hybrid system in particular. mbSUS relies on the ubiquitin-degradation pathway as a sensor for protein-protein interactions. Thus, mbSUS is predominantly suitable for the determination of full-length proteins localized in the cytoplasm and in or at membrane compartments, without the need for their truncation and nuclear mislocation. In addition, we present a set of Gateway compatible mbSUS vectors that allow the rapid generation of constructs for fast and efficient interaction studies. An additional vector is introduced that allows the extension of mbSUS for the analysis of oligomeric protein complex formation and competition assays in vivo. In summary, mbSUS provides an additional versatile tool for protein-protein interaction studies, which is complementary to in planta assays such as BiFC and FRET.