The work leading to this publication has received funding from the Marie Curie COFUND programme &#8220;U-Mobility&#8221;, cofinanced by the University of Malaga and the European Union 7th Framework Programme (FP7/2007-2013) under GA No. 246550. The work in our laboratory is supported by grants from NIH, USDA/NIFA, NSF, BARD, and BSF to V.C.

1. Protein expression
<ol>
	<li>Choice of expression system 
	Select the system, i.e., vectors and vector delivery method, best suited for expression of the protein of interest in the specific model organism/cell. Note that our assay requires expression of the tested proteins in easily detectible amounts, which is best achieved by transient transformation of large numbers of cells. In plants, for example, this is best accomplished using binary plasmids as expression vectors and Agrobacterium as delivery system.</li>
	<li>Construction of binary expression vectors 
	Clone the coding sequence(s) of the protein(s) of interest into an expression vector either alone or in translational fusion to an epitope tag. Use vectors suitable for the chosen expression system and standard molecular biology procedures for gene cloning. For Agrobacterium-mediated gene delivery, employ binary vectors and introduce them, also using standard protocols, into an Agrobacterium strain, such as EHA105, for subsequent inoculation of plant tissues.</li>
	<li>Choice of plant species 
	Select the plant species in which the protein(s) of interest will be expressed. Note that, while any plant species susceptible to Agrobacterium-mediated genetic transformation can be used, our plant species of choice is Nicotiana benthamiana, which is easily grown, highly susceptible to Agrobacterium, and has large leaves which are easily inoculated.</li>
	<li>Plant growth 
	Grow one plant for 4 to 6 weeks in a pot with Pro-Mix BX in a pot (20 cm x 20 cm x 20 cm) under environmentally-controlled conditions (e.g., growth chamber) of long day (i.e., 16 h of 130-150 &#181;E m-2 s-1 light at 23&#176;C and 8 h dark at 20&#176;C) and 40-65% relative humidity. 
	1.5.2 Fertilize occasionally with commercially available products per manufacturer&#8217;s instructions. Once the plant is grown, select leaves with the size of 50 mm x 70 mm or larger (these length measurements do not include the petiole) for Agrobacterium inoculation.</li>
	<li>Inoculation with Agrobacterium 
	Grow Agrobacterium strain harboring the binary construct expressing the tested protein(s) from step 1.3 overnight at 28&#176;C in YEP medium (1% peptone, 1% yeast extract, and 0.5% NaCl) supplemented with appropriate antibiotics (e.g., 100 mg/L streptomycin and 10 mg/L rifampicin for pPZP-RSC2-based vectors24-25). 
	1.6.2 Centrifuge the cells, resuspended to OD600 =0.5 in infiltration buffer [10 mM MgCl2, 10 mM Mes (pH 5.6), 100 &#181;M acetosyringone], and incubate for 2 h at room temperature. Infiltrate the culture into the abaxial side of a leaf, using a 1-mL needleless syringe; note that the leaves are inoculated in situ, while is attached to the plant. 
	1.6.3 After infiltration, grow the plant for 72 h under the light regime of 16 h 130-150 &#181;E m-2 s-1 light at 23&#176;C/8 h dark at 20&#176;C before harvesting.</li>
	<li>Application of proteasomal inhibitors 
	To support the notion that the tested protein(s) is degraded via a proteasomal pathway, use specific proteasomal inhibitors MG132 and lactacystin28-29, which should reduce or even block degradation. Four hours before harvesting (see Step 2.1), infiltrate the Agrobacterium-inoculated leaf areas with 10 &#956;M MG132 (EMD Millipore) or 10 &#956;M lactacystin (Sigma-Aldrich) or mock-treat the leaf with the respective solvents, i.e., 0.1% DMSO or distilled water.</li>
</ol>
2. Preparation of cell-free extracts
<ol>
	<li>Leaf harvesting 
	Harvest the inoculated areas of the leaf, usually ca. 200-400 mg of fresh weight, and grind them into fine powder in liquid nitrogen. Alternatively, bead-beat the tissues, using any bead-beater (e.g., Biospec) or a dental amalgamator (e.g., TPC Advanced Technologies) directly in the degradation buffer (see Step 2.2).</li>
	<li>Protein extraction 
	Prepare total protein extract by placing the ground tissue into 500 &#956;L of degradation buffer [50mM Tris-HCL (pH 7.5), 100 mM NaCl, 10 mM MgCl2, 5 mM DTT, 5 mM adenosine 5&#180;-triphosphate, and 1x protease inhibitor cocktail (Sigma-Aldrich)]. Note that the protease inhibitor cocktail mainly affects serine, cysteine, aspartic, and metalloproteases and does not interfere with the 26S protease. Clarify the extract by two sequential centrifugations at 12,000 x g for 5 min.</li>
	<li>Protein degradation reaction 
	Transfer equal volumes of extracts, usually 20 &#956;L, to microfuge tubes and incubate them at room temperature for increasing periods of time. Typically, sample time zero, and 5, 10, 15, 20, and 30 min time points. Stop reactions by boiling in SDS gel-sample buffer, and analyze them by western blotting.</li>
</ol>
3. Detecting protein degradation by immunoblotting
<ol>
	<li>Gel electrophoresis 
	3.1.1 Resolve the protein samples by SDS-polyacrylamide gel electrophoresis and electrotransfer the resolved proteins to a nitrocellulose membrane according to the standard protocol30. 
	3.1.2 Determine protein concentration using the Bradford method (Bio-Rad), and load 50-80 &#181;g of total protein per lane. Make sure that all samples are loaded equally. For loading controls, compare intensities of a ubiquitous protein species, such as putative RuBisCo large chains which migrate as a major band with a relative electrophoretic mobility of around 50 kDa; they can be detected on Coomassie blue-stained gels or on Ponceau S- or fluorescent SYPRO Ruby-stained nitrocellulose membranes.</li>
	<li>Blocking 
	Block the membrane with 5% skim milk in TBST (10 mM Tris-HCl, 140 mM NaCl, 0.05% Tween 20, pH 7.4) for 1 h at room temperature.</li>
	<li>Primary antibody 
	Dilute primary anti-epitope antibody in 1% skim milk in TBST to the concentration recommended by the manufacturer and incubate with the blocked membrane for 1 h at room temperature or for overnight at 4&#186;C with gentle agitation.</li>
	<li>Rinsing 
	Rinse the membrane is rinsed in 20 ml TBST for 15 min once, and twice for 5 min at room temperature with gentle agitation.</li>
	<li>Secondary antibody 
	Dilute secondary antibody (e.g., anti-rabbit IgG) conjugated with horse radish peroxidase (HRP) in 1% skim milk in TBST as recommended by the manufacturer and incubate with the membrane for 1 h at room temperature with gentle agitation.</li>
	<li>Detection 
	Rinse the membrane again as described in Step 3.4. After the final rinse in TBST, visualize the proteins of interest with an HRP chemiluminescent substrate, most commonly using an ECL kit. &#160;</li>
</ol>

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No conflicts of interest declared.

This assay relies on the expression of the tested proteins in plant tissues; thus, the potential proteasomal degradation process obviously occurs already within the living tissues. We assay protein destabilization, however, only in the extracts, with the time zero sample serving as the initial reference point. Hence, we define it as a cell-free assay.
One important aspect for the success of this assay is the correct choice of the expression vector from which the tested protein(s) will be produced. Unless specific antibodies against the tested protein are available, it should be epitope-tagged for detection by western blotting. The tagged protein should be expressed from an Agrobacterium binary vector. Whereas many such vectors are available26, we suggest using vectors derived from our modular pSAT plasmid system24, which allows one-step insertion of expression cassettes into the pPZP-RCS2 binary plasmids24-25. Another advantage of the pSAT system is that it allows expression of multiple genes from the same vector25, which is especially useful for experiments to assay several proteasomal substrates or to study the effects of interactors of the tested substrate; for example coexpression of the Agrobacterium VirD5 protein that interacts with the proteasomal substrate VirF results in VirF protection from proteasomal degradation22. Importantly, the expanding pSAT series of vector already includes vectors for epitope tagging27. Regardless of the chosen cloning strategy, the sequence encoding the epitope-tagged protein of interest should be introduced in a binary vector for subsequent expression in plant tissues. Because this assay uses transient expression, the binary vector does not have to contain selection markers for stable transformation, although their presence is not detrimental to the assay efficiency.

Although coexpression of several proteins of interest is best achieved using multigene expression vectors, it can also be done by combining equivalent volumes of liquid cultures of two or three Agrobacterium strains, each of which carries a separate binary construct.

Another critical point is that, in many cases, the proteasomal degradation assay involves expression not only of the proteasomal substrate, but also of a component of the SCF pathway. For example, the experimental goal might be to test whether an F-box protein recognizes and targets for degradation a specific substrate. In this case, this epitope-tagged substrate should be coexpressed with the untagged F-box protein.

For more detailed analysis of the data, the extent of protein degradation can be easily quantified by measuring the relative intensity of the western signals using the latest ImageJ software (NIH)31. When performing such quantification, it is important not to over-develop the western blots to avoid signal saturation. Data quantification also requires that all samples are loaded on the SDS-polyacrylamide gel equally in respect to their protein content as protein degradation in this assay is detected by reduction in the intensity of the appropriate protein band. This is achieved by determination of the protein content of each cell extract and by subsequent verification of equal loading of each lane (see step 3.1).&#160;Furthermore, in any given time-course experiment, all samples should be&#160;derived from the same batch of extract helps to ensure equal loading.

The cell-free assay for proteasomal degradation described here uses plants as an experimental system. However, the same experimental design could be used for any other organism. The assay also can be extended by further focusing on the SCF pathway. For example, the SCF-dependent mechanism of protein degradation can be demonstrated using a dominant-negative form of the SCF component CULLIN1 (CUL1DN). Specifically, a mutant of Arabidopsis CUL1 with deleted amino acid residues between positions 1 and 420 has been shown to interact with the Skp1/ASK1 component of the SCF complex, but not with RBX1, which represents the catalytic core of SCF22. Inhibition or reduction of substrate degradation following coexpression of CUL1DN indicates involvement of the SCF pathway.

Our assay utilizes proteins of interest expressed in plant tissues, in their natural environment. One alternative to this approach is to purify recombinant proteins, add them to plant extracts, and follow their degradation by western analysis32. We do not recommend this tactic as methodology of choice because recombinant proteins do not always faithfully reflect the native biological properties; however, if expression in planta is not feasible, the use of recombinant proteins certainly represents a valuable alternative.

The ubiquitin/26S proteasome pathway is emerging as a widespread mechanism for control of diverse biological reactions, including transcriptional regulation, cell-cycle progression and signal transduction, receptor down-regulation or endocytosis, among others processes1-4. In this pathway, the target protein is tagged by ubiquitin residues which are first attached via a thiolester bond to ubiquitin-activating enzyme E1 and then translocated to a cysteine amino acid residue of ubiquitin-conjugation enzyme E2; finally, E2 interacts with ubiquitin ligase E3, resulting in polyubiquitination of the protein substrate. Ultimately, the polyubiquitinated proteins are recognized and degraded by the 26S proteasome. In this mechanism, the E3 enzyme specifies the substrate and acts as the key regulatory component of the ubiquitin/26S proteasome system (UPS). E3 ligases can act independently, such as RING domain ligases, or as part of a multisubunit SCF (Skp1/Cullin/F-box protein) complex, such as F-box domain ligases. SCF-mediated proteasomal degradation pathways are involved in regulation of transcription, cell cycle, signal transduction5-10 and many other major cellular functions.
Besides these critical roles in regulation of cellular processes, UPS takes the central stage in many plant-pathogen interactions. For example, increasing evidence suggests that several plant pathogens, including Agrobacterium tumefaciens, rely on the host UPS for to facilitate the infection process11. Agrobacterium elicits neoplastic growths on plants, which represent its natural hosts, and it can also transform a wide range of other eukaryotes, from fungi1,2 to human cells12,13. During its infection, Agrobacterium exports a DNA element (T-DNA) and several virulence (Vir) proteins into the host cell12-13. One of these proteins is VirF, the first F-box protein found to be encoded by a prokaryotic genome14. As part of the SCF ubiquitin ligase complex, VirF, and its functional host homolog VBF15, facilitate Agrobacterium infection via the UPS-mediated protein degradation, which presumably facilitates uncoating of the invading bacterial T-DNA from its accompanying bacterial and host proteins, VirE2 and VIP1, respectively16,17. Interestingly, many F-box proteins, including VirF, are intrinsically unstable due to their own proteolysis, which is mediated by autoubiquitination activity18,19 or by other E3 ligases for which F-box proteins may serve as substrates20-23.

When studying biochemical activities of F-box proteins, other ubiquitin ligases, and/or their substrates, it would be very useful to employ a simple and reliable assay for proteasomal degradation. Here we describe one such protocol for analyzing protein stability in a cell-free system. In this assay, the stability of the UPS substrate is analyzed in the presence or absence of one of the essential components of the proteasomal degradation pathway, such as an F-box protein, in a cell-free system. Generally, we express the tested protein(s) in plant tissues, prepare cell-free extracts from these tissues and monitor the amounts of the protein(s) of interest by western blotting. The UPS-dependent mechanism of protein degradation is demonstrated by inclusion of specific proteasomal inhibitors and/or using coexpression of dominant-negative form of an SCF component, Cullin. Whereas we illustrate this assay using proteasomal degradation of the Arabidopsis VIP117 protein by the F-box protein VBF15, it may be employed to investigate the stability of any other proteasomal substrates.

<table><tbody><tr><td>Protein assay kit</td><td>Bio-Rad</td><td>500-0001</td><td></td></tr><tr><td>Proteinase inhibitor cocktail&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S8820</td><td></td></tr><tr><td>Mini-Protean system</td><td>Bio-Rad</td><td>165-8000</td><td></td></tr><tr><td>Semi-dry western blotting SD electrotransfer system</td><td>Bio-Rad</td><td>170-3940</td><td></td></tr><tr><td>Affinity Purified Rabbit Anti-Ha</td><td>ICL Lab</td><td>RHGT-45A-Z</td><td></td></tr><tr><td>Goat anti-Rabbit IgG Peroxidase Conjugate</td><td>Thermo Scientific</td><td>31460</td><td></td></tr><tr><td>BioTrace, NT nitrocellulose transfer membrane</td><td>Pall Corporation</td><td>27377-000</td><td></td></tr><tr><td>Immobilon western chemiluminescent HRP substrate</td><td>EMD Millipore</td><td>WBKL S0 050</td><td></td></tr><tr><td>MG132</td><td>EMD Millipore</td><td>474790-1MG</td><td></td></tr><tr><td>Lactacystin</td><td>Sigma-Aldrich</td><td>L6785</td><td></td></tr><tr><td>Thermo Scientific Pierce Fast Western Blot Kit, ECL Substrate</td><td>Pierce</td><td>35055</td><td></td></tr></tbody></table>

assaying proteasomal degradation in a cell-free system in plants

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions.
The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.

Figure 1, adapted from Zaltsman et al.17, illustrates representative experiments for detection of proteasomal degradation in a cell-free system. Specifically, we demonstrate destabilization of a plant defense-related protein VIP1 by the VBF F-box protein via the SCFVBF pathway in N. benthamiana. Arabidopsis VBF and HA-tagged VIP1 (HA-VIP1) proteins were transiently coexpressed, and HA-VIP1 content protein within extracts of the expressing leaves was analyzed by western blotting. This analysis demonstrated that VIP1 amounts were reduced to a significant degree when coexpressed with VBF, but remained relatively stable in the absence of VBF coexpression (Figure 1A). Note that a slight reduction in the VIP1 content without VBF may be due to the low level of the endogenous tobacco VBF activity. The proteasomal degradation mechanism of VIP1 destabilization by VBF was inferred from its inhibition by MG132 (Figure 1A). Quantification of the results in Figure 1A demonstrated almost complete (&#8805;90&#177;5%) VIP1 by VBF, which was blocked by MG132 (Figure 1B). Note that slightly higher levels of VIP1 in the presence of MG132 most likely are explained by inhibition of the endogenous VBF activity17.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51293/51293fig1highres.jpg" src="/files/ftp_upload/51293/51293fig1.jpg" /> 
Figure 1. VBF promotes proteasomal degradation of VIP1. HA-VIP1 was expressed alone or coexpressed with VBF in N. benthamiana leaves. The resulting protein extracts were incubated for the indicated time periods and analyzed by western blotting using anti-HA antibodies. The putative RuBisCo large chain was used as loading control (A). Quantified western blot signal was expressed as percent of the signal obtained in the absence of VBF at the start of the incubation period. The data represent average values of three independent experiments with indicated standard deviations (B). This figure was adapted from Zaltsman et al.17

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Assaying Proteasomal Degradation in a Cell-free System in Plants

Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular &#8220;tags&#8221; for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular ...

assaying-proteasomal-degradation-in-a-cell-free-system-in-plants

Nanyang Technological University

Research

JoVE Journal

Biology

14.4K Views.  Stony Brook University, State University of New York. Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular “tags” for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.

Video: Assaying Proteasomal Degradation in a Cell-free System in Plants

mRNA Interactome Capture from Plant Protoplasts

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional gene regulation (PTGR) of messenger RNAs (mRNAs). In the past few years, a number of mRNA-bound proteomes from yeast and mammalian cell lines have been successfully isolated through the use of a novel method called &#34;mRNA interactome capture,&#34; which allows for the identification of mRNA-binding proteins (mRBPs) directly from a physiological environment. The method is composed of in vivo ultraviolet (UV) crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo(dT) beads, and the subsequent identification of the crosslinked proteins by mass spectrometry (MS). Very recently, by applying the same method, several plant mRNA-bound proteomes have been reported simultaneously from different Arabidopsis tissue sources: etiolated seedlings, leaf tissue, leaf mesophyll protoplasts, and cultured root cells. Here, we present the optimized mRNA interactome capture method for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type that serves as a versatile tool for experiments that include various cellular assays. The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the mRNA-bound proteome obtained from a medium-scale experiment (107 cells), RBPs noted to have RNA-binding capacity were found to be overrepresented, and many novel RBPs were identified. The experiment can be scaled up (109 cells), and the optimized method can be applied to other plant cell types and species to broadly isolate, catalog, and compare mRNA-bound proteomes in plants.

Here, we present an interactome capture protocol applied to Arabidopsis thaliana leaf mesophyll protoplasts. This method critically relies on in vivo UV crosslinking and allows for the isolation and identification of plant mRNA-binding proteins from a physiological environment.

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional ...

Biochemistry

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based phosphoproteomics has transformed the way of studying plant signal transduction. However, requirement of lots of starting materials and prolonged MS measurement time to achieve the depth of coverage has been the limiting factor for the high throughput study of global phosphoproteomic changes in plants. To improve the sensitivity and throughput of plant phosphoproteomics, we have developed a stop and go extraction (stage) tip based phosphoproteomics approach coupled with Tandem Mass Tag (TMT) labeling for the rapid and comprehensive analysis of plant phosphorylation perturbation in response to osmotic stress. Leveraging the simplicity and high throughput of stage tip technique, the whole procedure takes approximately one hour using two tips to finish phosphopeptide enrichment, fractionation, and sample cleaning steps, suggesting an easy-to-use and high efficiency of the approach. This approach not only provides an in-depth plant phosphoproteomics analysis (&#62; 11,000 phosphopeptide identification) but also demonstrates the superior separation efficiency (&#60; 5% overlap) between adjacent fractions. Further, multiplexing has been achieved using TMT labeling to quantify the phosphoproteomic changes of wild-type and snrk2 decuple mutant plants. This approach has successfully been used to reveal the phosphorylation events of Raf-like kinases in response to osmotic stress, which sheds light on the understanding of early osmotic signaling in land plants.

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Presented here is a phosphoproteomic approach, namely stop and go extraction tip based phosphoproteomic, which provides high-throughput and deep coverage of Arabidopsis phosphoproteome. This approach delineates the overview of osmotic stress signaling in Arabidopsis.

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based ...

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...

Developmental Biology

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. Maintenance of proteasome activity was implicated in many key cellular processes, like cell's stress response 3, cell cycle regulation and cellular differentiation 4 or in immune system response 5. The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases 4, 6. Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging 7, 8, 9, 10. Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)&#x2013;dgn fusion protein (GFP&#x2013;dgn, unstable) and a variant carrying a frameshift mutation (GFP&#x2013;dgnFS, stable 11) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP&#x2013;dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry.

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

A method to monitor ubiquitin-proteasome activity in living cells is described. A degron-destabilized GFP- (GFP-dgn) and a stable GFP-dgnFS fusion protein are generated and transduced into the cell using a lentiviral expression vector. This technique allows to generate a stable GFP-dgn/GFP-dgnFS expressing cell line in which ubiquitin-proteasome activity can be easily assessed using epifluorescence or flow cytometry.

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. ...

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels.
Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation.
This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways.

Here we describe a robust biological assay for quantifying the relative rate of proteolysis by the ubiquitin-proteasome system. The assay readout is yeast growth rate in liquid culture, which is dependent on the cellular levels of a reporter protein comprising a degradation signal fused to an essential metabolic marker.

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic ...

An Easy Method for Plant Polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...

Assays for the Degradation of Misfolded Proteins in Cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...

Protein Aggregate Formation Assay: A Method to Detect and Quantify Protein Aggregation in Cultured Cells upon Induction by Proteasome Inhibitor

Source: Lee, H. et al. <a href="https://www.jove.com/v/56425">Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells.</a> J. Vis. Exp. (2017)
In this video, we demonstrate a cell-based protein aggregation assay using proteasome inhibitors, which block proteasome activity, preventing misfolded, mutant proteins, fused to a fluorescent label, from undergoing ubiquitin-dependent proteasomal degradation, leading to their accumulation within the cell cytoplasm. The protein aggregates are then visualized and quantified by fluorescence microscopy.

Source: Lee, H. et al. Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells. J. Vis. Exp. (2017)
...

Assaying Proteasomal Degradation in a Cell-free System in Plants

Summary

Explore More Videos

Chapters in this video

mRNA Interactome Capture from Plant Protoplasts

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

An Easy Method for Plant Polysome Profiling

Assays for the Degradation of Misfolded Proteins in Cells

Protein Aggregate Formation Assay: A Method to Detect and Quantify Protein Aggregation in Cultured Cells upon Induction by Proteasome Inhibitor