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.
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.
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.
1. Protein expression
2. Preparation of cell-free extracts
3. Detecting protein degradation by immunoblotting
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 (≥90±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.
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
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). Furthermore, in any given time-course experiment, all samples should be 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 authors have nothing to disclose.
The work leading to this publication has received funding from the Marie Curie COFUND programme “U-Mobility”, 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.
Protein assay kit | Bio-Rad | 500-0001 | |
Proteinase inhibitor cocktail | Sigma-Aldrich | S8820 | |
Mini-Protean system | Bio-Rad | 165-8000 | |
Semi-dry western blotting SD electrotransfer system | Bio-Rad | 170-3940 | |
Affinity Purified Rabbit Anti-Ha | icllab | RHGT-45A-Z | |
Goat anti-Rabbit IgG Peroxidase Conjugate | Thermo Scientific | 31460 | |
BioTrace, NT nitrocellulose transfer membrane | Pall Corportation | 27377-000 | |
Immobilon western chemiluminescent HRP substrate | EMD Millipore | WBKL S0 050 | |
MG132 | EMD Millipore | 474790-1MG | |
Lactacystin | Sigma-Aldrich | L6785 | |
Thermo Scientific Pierce Fast Western Blot Kit, ECL Substrate | Pierce | 35055 |