Rapid Single-Step Affinity Purification of HA-Tagged Plant Mitochondria

Kuhnert, Franziska; Stefanski, Anja; Overbeck, Nina; Drews, Leonie; Reichert, Andreas S.; Weber, Andreas P.M.

doi:10.1104/pp.19.00732

Cited by 30 publications

(23 citation statements)

References 66 publications

(106 reference statements)

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“…To confirm that the proteins were indeed located in mitochondria of N. benthamiana, we performed a mitochondrial purification using an affinity-tag-based technique similar to those reported (22,23). We designed a fusion protein with a Twin-streptag consisting of mTurquoise fluorescent protein and metaxin, a mitochondrial outer membrane protein (Twin-strep::mTurquoise::metaxin).…”

Section: Design and Functional Validation Of Nifd Variants Resistant Tomentioning

confidence: 99%

Plant expression of NifD protein variants resistant to mitochondrial degradation

Allen

Gregg

Okada

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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To engineer Mo-dependent nitrogenase function in plants, expression of the structural proteins NifD and NifK will be an absolute requirement. Although mitochondria have been established as a suitable eukaryotic environment for biosynthesis of oxygen-sensitive enzymes such as NifH, expression of NifD in this organelle has proven difficult due to cryptic NifD degradation. Here, we describe a solution to this problem. Using molecular and proteomic methods, we found NifD degradation to be a consequence of mitochondrial endoprotease activity at a specific motif within NifD. Focusing on this functionally sensitive region, we designed NifD variants comprising between one and three amino acid substitutions and distinguished several that were resistant to degradation when expressed in both plant and yeast mitochondria. Nitrogenase activity assays of these resistant variants in Escherichia coli identified a subset that retained function, including a single amino acid variant (Y100Q). We found that other naturally occurring NifD proteins containing alternate amino acids at the Y100 position were also less susceptible to degradation. The Y100Q variant also enabled expression of a NifD(Y100Q)–linker–NifK translational polyprotein in plant mitochondria, confirmed by identification of the polyprotein in the soluble fraction of plant extracts. The NifD(Y100Q)–linker–NifK retained function in bacterial nitrogenase assays, demonstrating that this polyprotein permits expression of NifD and NifK in a defined stoichiometry supportive of activity. Our results exemplify how protein design can overcome impediments encountered when expressing synthetic proteins in novel environments. Specifically, these findings outline our progress toward the assembly of the catalytic unit of nitrogenase within mitochondria.

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Section: Design and Functional Validation Of Nifd Variants Resistant Tomentioning

confidence: 99%

Plant expression of NifD protein variants resistant to mitochondrial degradation

Allen

Gregg

Okada

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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“…Conversely, other small tagging systems are useful for identifying protein functions. Several peptide tags, such as the FLAG (Xiao et al, 2019;Cui et al, 2020;Miura et al, 2020;Uemura et al, 2020), HA (Shinozawa et al, 2019;Kuhnert et al, 2020), and c-MYC (Earley et al, 2006;Baudisch et al, 2018;Roshan et al, 2018) tags are used in plants because of their specificity. These tags may affect the solubility or insolubility of the expressed proteins and their activities, depending on the characteristics of the expressed proteins (Ki and Pack, 2020).…”

Section: Discussionmentioning

confidence: 99%

RAP Tag and PMab-2 Antibody: A Tagging System for Detecting and Purifying Proteins in Plant Cells

et al. 2020

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An affinity tag system requires both high affinity and specificity. The RAP tag epitope DMVNPGLEDRIE, derived from rat podoplanin (PDPN), is specifically recognized by PMab-2 monoclonal antibodies in rats. Here, we demonstrated that high levels of PMab-2 can be produced in Nicotiana benthamiana and plant-derived PMab-2 possesses similar activity to CHO-derived PMab-2, and the RAP tag presents a useful tagging system for detecting and purifying proteins from plant cells. The heavy chain of PMab-2 fused with KDEL, an endoplasmic reticulum retention sequence, and the light chain of the antibody were introduced into N. benthamiana by agroinfiltration. The expression of PMab-2 peaked 4 days after agroinfiltration, and approximately 0.3 mg/g fresh weight of the antibody was accumulated. After purification, the plant-derived PMab-2 successfully recognized rat PDPN expressed in CHO-K1 cells and exhibited almost the same binding activity as CHO-derived PMab-2. The RAP-tagged proteins expressed in plant cells were specifically recognized by PMab-2. These results indicate that PMab-2 can accumulate at high levels in N. benthamiana and is easily purified and that the RAP tagging system presents a useful tool for detecting and purifying proteins of interest in plant cells.

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“…beads) followed by washing steps, and deliver organelles with high purity that can be used directly for subsequent analysis. Several epitope-tagging protocols achieving organelle isolation via co-IP have been published, focusing exclusively on one type of organelle [36][37][38][39][40][41][42].…”

mentioning

confidence: 99%

“…Split-isopeptide Catcher-Tag systems could be used as specific, covalent alternatives to the non-covalent, affinity-based organelle isolation techniques, such as the biotin-streptavidin based isolation of nuclei or mitochondria published as INTACT [37] and IMTACT method [42], the Strep-Tactin based isolation of tagged mitochondria [40], the immunogenic chloroplast isolation via YFP [36], or epitope-tagging approaches for mitochondria isolation [38,39,41].…”

mentioning

confidence: 99%

“…Purification can also be achieved via epitope-tagging of desired organelles with the help of targeting fusion constructs, combined with the immobilization of specific antibodies on magnetic beads. Immunogenic isolation was described for mitochondria via HA-Tag [38,39,41], Strep-Tag [40] or for chloroplasts via YFP fused to a targeting sequence [36]. Similar purification methods described for mammalian or other systems are based on GFP [44] or on immobilized antibodies directed against organelle surface proteins e.g.…”

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confidence: 99%

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Tagging and catching: rapid isolation and efficient labeling of organelles using the covalent Spy-System in planta

et al. 2020

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Background: Up-to-now, several biochemical methods have been developed to allow specific organelle isolation from plant tissues. These procedures are often time consuming, require substantial amounts of plant material, have low yield or do not result in pure organelle fractions. Moreover, barely a protocol allows rapid and flexible isolation of different subcellular compartments. The recently published SpySystem enables the in vitro and in vivo covalent linkage between proteins and protein complexes. Here we describe the use of this system to tag and purify plant organelles. Results: We developed a simple and specific method to in vivo tag and visualize, as well as isolate organelles of interest from crude plant extracts. This was achieved by expressing the covalent split-isopeptide interaction system, consisting of SpyTag and SpyCatcher, in Nicotiana benthamiana leaves. The functionality of the SpySystem in planta, combined with downstream applications, was proven. Using organelle-specific membrane anchor sequences to program the sub-cellular localization of the SpyTag peptide, we could tag the outer envelope of chloroplasts and mitochondria. By co-expression of a cytosolic, soluble eGFP-SpyCatcher fusion protein, we could demonstrate intermolecular isopeptide formation in planta and proper organelle targeting of the SpyTag peptides to the respective organelles. For one-step organelle purification, recombinantly expressed SpyCatcher protein was immobilized on magnetic microbeads via covalent thiol-etherification. To isolate tagged organelles, crude plant filtrates were mixed with SpyCatcher-coated beads which allowed isolation of SpyTag-labelled chloroplasts and mitochondria. The isolated organelles were intact, showed high yield and hardly contaminants and can be subsequently used for further molecular or biochemical analysis. Conclusion: The SpySystem can be used to in planta label subcellular structures, which enables the one-step purification of organelles from crude plant extracts. The beauty of the system is that it works as a covalent toolbox. Labeling of different organelles with individual tags under control of cell-specific and/or inducible promoter sequences will allow the rapid organelle and cell-type specific purification. Simultaneous labeling of different organelles with specific Tag/Catcher combinations will enable simultaneous isolation of different organelles from one plant extract in future experiments.

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Rapid Single-Step Affinity Purification of HA-Tagged Plant Mitochondria

Cited by 30 publications

References 66 publications

Plant expression of NifD protein variants resistant to mitochondrial degradation

Plant expression of NifD protein variants resistant to mitochondrial degradation

RAP Tag and PMab-2 Antibody: A Tagging System for Detecting and Purifying Proteins in Plant Cells

Tagging and catching: rapid isolation and efficient labeling of organelles using the covalent Spy-System in planta

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