Fig. 3.
Reconstitution of eukaryotic MPPs and determination of the stability of Nif proteins toward MPPs in E. coli. (A) Schematic representation of plasmid construction. The red domes indicate ribosome binding sites. MPPβ and MPPα were arranged to form an operon under the control of Ptac/lacO and induced with IPTG for expression; Su9-GFP was cloned downstream of the PLtetO-1 promoter and induced with aTc (anhydrotetracycline) for expression (B) Functionality test of reconstituted MPPs from yeast and plants in E. coli. GFP expressed in E. coli was used as control (lane 1). For lanes 2–4, Su9-GFP was induced for expression with 400 ng/mL of aTc. “-” indicates no MPP expression modules were present (lanes 1 and 2); “+” indicates MPPs were expressed at basal level; “++” represents the expression of MPPs when induced with 200 μM of IPTG. Protein levels of the MPPs are shown in SI Appendix, Fig. S5. (C) Overview of NifD or NifD R98P variant expression plasmids. These were expressed in the operon-based nif gene cluster (29) together with the other components of the nitrogenase system from K. oxytoca under anaerobic conditions. (D) Comparison of the protein stability of NifD and its R98P variants in the intact nitrogenase system when expressed with reconstituted MPPs. The Nif expression module (expressing native NifD or its NifD R98P variant as in C) was cotransformed with either empty vector (“-”; lanes 1 and 4) or the MPP expression modules depicted in A (lanes 2, 3, 5, and 6; “+” indicates MPP expression at the basal level; “++” represents the expression of MPPs when induced with 200 μM [for Sc, O. sativa Os, and At MPPs] or 50 μM [for Nt MPP] IPTG). Red arrows refer to the NifDn fragment (defined in the Fig. 1 legend), which indicates in this instance that NifD was processed by the corresponding MPP. The NifDc fragment was not observed in many cases, perhaps because it is less stable in E. coli than in yeast. s.e., short-term exposure; l.e., long-term exposure.