Fig. 1.
Analysis of the protease processing site within NifD. (A) Predicted protease processing region within NifD from K. oxytoca, colored in either blue or black at 10-residue intervals. (B and C) Two rounds of internal deletions within NifD. Intact NifD expressed in E. coli (NifDEc) was used as a positive control, and empty vector (EV) was used as a negative control. (B) 10-aa internal deletions within the predicted region to narrow down the processing region. Intact (unprocessed) NifD expressed in yeast exhibited a single band; see the lane marked NifD(Δ91–100 AA). Processed NifD exhibited three bands, including intact NifD, NifDc (C-terminal portion of processed NifD), and a much smaller fragment labeled NifDn (N-terminal portion of processed NifD). (C) 5-aa internal deletions within NifD region 91 to 100 aa and point mutations at specific amino acids. The R98K variant protects NifD from protease mediated degradation. (D) Conserved sequence around position R98 (indicated in red) among diverse NifD proteins (protein IDs provided in Dataset S1). WebLogo 3 was used to draw the sequence logo. (E) Testing the tolerance of NifDs and their corresponding R98K variants from A. vinelandii DJ (Av), R. capsulatus SB1003 (Rc), and Anabaena sp. PCC7120 (As) to protease-mediated degradation. Each NifD was labeled with a C-terminal 6×His tag. The Western blot assays shown in A, B, and E were conducted using extracts derived from purified yeast mitochondria as indicated above the lanes; mitochondrial targeting was achieved using the Su9 signal peptide.