Plant expression of NifD protein variants resistant to mitochondrial degradation

Abstract

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.

Keywords: metabolic engineering; mitochondria; nitrogenase; protein engineering; synthetic biology.

Fig. 1.

A mitochondrial endoprotease degrades NifD. (A) Western blots (α-HA and α-NifK) of protein extracts after introduction of MTP::NifD::HA and MTP::NifK constructs into N. benthamiana and A. thaliana. Coinfiltration with MTP:NifK is indicated by + or −. A series of different promoters and MTPs were used to target each construct to mitochondria (SI Appendix, Fig. S1 and Tables S1 and S2 and Dataset S1). Open and filled triangles indicate unprocessed and processed forms of FAγ51::NifD::HA, respectively. An asterisk indicates the ∼48-kDa NifD secondary product. (B) Western blots (α-HA) of protein extracts for cytosolic (HA:NifD and HA::NifD::HA) or mitochondria (FAγ51::NifD::HA and FAγ51::HA::NifD::HA) targeted NifD constructs. A schematic of the constructs is shown (not to scale). Red dashed arrows indicate C- and N-terminal cleavage products found for FAγ51::HA::NifD::HA. Red shaded boxes represent an approximate area (not to scale) where cleavage was predicted to occur based on the sizes of the degradation products. MTP::NifD::HA products are designated as follows: open triangle, unprocessed; filled triangle, correctly processed; asterisk, secondary C-terminal ∼48-kDa cleavage product; and hash, secondary N-terminal cleavage product at ∼13 kDa. The band at ∼21 kDa is an unspecific background band.

Fig. 2.

Discovery of the NifD degradation region reveals a characteristic MPP site. (A) Western blots (α-HA) of protein extracts after introduction of MTP::NifD::HA genetic constructs coinfiltrated with MTP::NifK into N. benthamiana leaf cells. Constructs were based on FAγ51::NifD::HA (wild-type NifD), except wild-type amino acids were substituted in five amino acid alanine/glycine blocks (variants [var] 1 to 12). The initial region for broad coverage with alanine/glycine scanning is shown schematically at the top (not to scale), with the wild-type NifD sequence, and underneath the corresponding five amino-acid changes for each individual construct. MTP::NifD::HA bands are designated by open and filled triangles for unprocessed and correctly processed forms, respectively; the asterisk indicates the secondary cleavage product. (B) Comparison of amino acids 95 to 104 of NifD with a typical −2R MPP cleavage motif. The motif was visualized by using selected sequences from Δicp55 and wild-type A. thaliana, previously identified by Carrie et al. (27) and Huang et al. (26). For a complete set of mitochondrial cleavage sites, refer to SI Appendix, Fig. S4. Numbers at the top refer to the amino-acid position relative to the cleavage site. The sequences shown here contain a conserved Arg residue in position −2, characteristic of a −2R MPP cleavage site. NifD also contains an Arg in position −2 relative to the experimentally determined cleavage site. (C) A schematic of the MS identification of peptides from the degradation product. Eight peptides (red boxes; not to scale) were identified. The most N-terminal peptide is shown in detail in red, identifying the precise cleavage site shown by scissors.

Fig. 3.

(A) Location of the proposed secondary cleavage site RRN↓YYT shown in the crystal structure of the MoFe protein from K. pneumoniae (Protein Data Bank ID code1QGU). NifD is shown in blue, and NifK is shown in green. FeMoco is shown as spheres. The residues of the proposed cleavage site are represented by sticks. The cleavage site is in proximity to FeMoco and close to the interface of NifD and NifK. The numbering of NifD amino acids within the crystal structure was adjusted to correspond to the numbering of the full-length sequence, which contains two methionine residues at the start of the sequence. (B) ALVIS analysis of the amino acid (aa) distribution based on 1,476 putative NifD proteins around the proposed secondary cleavage site. The y axis shows the amino acid residue, and the x axis shows the position of the corresponding residues in K. oxytoca. The residues of the K. oxytoca NifD sequence “RAGRRNYYTG” are shown above in blue. Sequences that contain a Gln and Lys in position 100 are shown in purple and green, respectively. Amino acid residues that were present in variants are shown above (H99, K100, Q100, T101, A101, G101, and V102). SI Appendix, Table S3 lists the frequency distribution of the equivalent residues of K. oxytoca RRNYYT in detail.

Fig. 4.

Identification and functional analysis of cleavage-resistant NifD variants. (A) Western blot (α-HA) of protein extracts after introduction of MTP::NifD::HA variant constructs into N. benthamiana leaf cells coinfiltrated with MTP::NifK. Discrete amino acid changes compared to wild type are shown in bold/yellow, and variants found to be degradation resistant are identified by a red +. Open and filled triangles designate unprocessed and correctly processed forms of MTP::NifD, respectively; the asterisk indicates the secondary cleavage product. (B) ARAs of nitrogenase activity with variant NifD proteins in E. coli. A subset of the same modifications tested above were individually introduced into MIT v2.1, and ethylene was measured. Error bars represent the SEM from at least two biological replicates. A depiction of the MIT v2.1 assay is shown, where substitutions to NifD are incorporated into the nitrogenase cluster (for further details, see SI Appendix, Fig. S7). Negative controls contained no MIT v2.1 cluster, and positive controls included the cluster with the wild-type NifD sequence. Ppm, parts per million.

Fig. 5.

MTP::NifD(Y100Q)–linker–NifK polyprotein is degradation-resistant, soluble, correctly processed in plant mitochondria, and functional when expressed in E. coli. (A) Western blots (α-HA) of protein extracts after introduction of NifD genetic constructs into N. benthamiana leaf cells. The alaFAγ5::MTP is a modified version of FAγ51 that has been alanine-scanned to disrupt mitochondrial processing, thus producing a larger unprocessed protein. A schematic of each construct is described. Proteins were separated into soluble, insoluble, and total fractions. Insoluble and soluble fractions run slightly faster than total fractions due to different buffer conditions. (B) ARAs and ¹⁵N incorporation assays compare NifD–linker–NifK function to wild type in E. coli; also see SI Appendix, Fig. S7. For each modification, three biological replicates were measured in duplicate; error bars represent SEM.

Fig. 6.

The Y100Q mutation in NifD prevents secondary cleavage in yeast mitochondria. Western blot (α-HA) of protein extracts after introduction of NifD::HA genetic constructs into N. benthamiana leaf cells coinfiltrated with MTP::NifK or S. cerevisiae is shown. A schematic of the constructs is shown. The 6×His::NifD::HA construct produces a cytosolic targeted protein of a similar size to correctly processed MTP::NifD::HA. Open and filled triangles indicate unprocessed and processed forms of NifD, respectively. The asterisk indicates the secondary cleavage product. Coomassie-stained gel post transfer is shown underneath.

Fig. 7.

Comparison of the cleavage susceptibility of NifD proteins from other diazotrophs in plant mitochondria. Three NifD proteins tested contained the same RRNYY motif as K. oxytoca NifD, while the other three contained either glutamine or lysine in position 100. The upper Western blot (α-HA) shows the coexpression of NifD with each corresponding NifK protein. For comparison, the lower blot (α-HA) shows the expression of NifK only. The NifD and NifK proteins were targeted to mitochondria by using FAγ51. NifD proteins had a C-terminal HA tag, FAγ51::NifD::HA, and NifK proteins had an N-terminal HA tag, FAγ51::HA::NifK. The HA tag was added to NifK, as we were not able to use the NifK antibody for the detection of certain NifK proteins due to a reduced affinity. The bands for unprocessed and processed proteins are marked by an open and filled triangle, respectively. Triangles are black for NifD and white for NifK. The NifD degradation product is marked by an asterisk.