Investigation into the formation of a protein-protein complex between ATP sulfurylase and APS reductase in onion (Allium cepa L.) : a thesis presented in partial fulfilment of the requirements for the degree of Masters in Science in Plant Biology at Massey University, Palmerston North, New Zealand
This thesis has attempted to obtain evidence, both in vitro and in vivo, of complex formation between chloroplastidic isoforms of APS-reductase (APSR; EC 126.96.36.199) and ATP-sulfiirylase (ATPS; EC 188.8.131.52) of Allium cepa (onion). Genes encoding APSR and ATPS from onion were expressed in E. coli, and the recombinant proteins were expressed and purified with glutathione Sepharose 4B chromatography and ion exchange chromatography. SDS-PAGE. separation and Coomassie blue staining revealed an ATPS recombinant protein with a molecular mass of 50 kDa, and full length and a truncated form of APSR, with molecular masses of 55 kDa and 45 kDa. respectively. Three different approaches were used to investigate complex formation in vitro. Using an ELlSA-based technique, an association of recombinant ATPS with recombinant APSR was shown at pH 7.4, with a proposed 1:1 stoichiometry. However, when these ELISA experiments were conducted at pH 9.4, no evidence for complex formation was observed, suggesting that the complex is dependent on the pH of the buffered solution used. The second method was the ligand binding assay where recombinant protein APSR was immobilised onto PVDF membrane and then incubated at 25°C with a solution containing ATPS protein. The detection of bound ATPS was achieved using anti- ATPS IgG, and it was possible to detect a putative ATPS-APSR complex at pH 7.4, although the complex was unable to be detected at pH 7.8 or pH 9.4. The third method used was the immunoprecipitation assay, where anti-ATPS IgG that was conjugated onto Sepharose resin was used to precipitate recombinant ATPS (and any proteins bound to ATPS). The proteins precipitated were identified firstly by their molecular mass and subsequent western analysis with either biotinylated ATPS IgG or APSR IgG antibodies. Using this technique, recombinant APSR was able to be precipitated from solution using recombinant ATPS, at pH 7.4. To investigate the role of glutathione as a possible co-factor in the mediation of the protein complex, the tripeptide was added to the buffer used (final concentration between 0-10 mM) in the ELISA experiments. These experiments showed that glutathione had no effect on the formation of the complex. However investigation of the role of glutathione using the immunoprecipitation assay (added at 5.0 mM) demonstrated that glutathione did result in an increase in the amount of the ATPS-APSR complex. The immunoprecipitation technique was also used in attempt to isolate the complex in vivo from onion chloroplast extracts. However the ATPS-APSR complex was unable to be detected using this technique. In this thesis, the term in vivo is used to refer to experiments with chloroplast extracts and so will include ATPS, APSR and other proteins that may also contribute to any ATPS-APSR complex. The term in vitro refers to the direct interaction between recombinant ATPS and APSR only. To determine if the ATPS-APSR complex had any effect on the control of ATPS activity, the specific activity of ATPS was measured in vitro using recombinant ATPS and APSR. Results from these experiments demonstrated that the complex formation did not alter the activity of ATPS. A second technique was used to detect the ATPS-APSR complex in vivo. An ATPS affinity column, made with anti-ATPS IgG antibodies conjugated to Sepharose, was firstly incubated with recombinant ATPS, and the chloroplast extract from onion was then passed through the column and any bound proteins were eluted with solutions with high or low pH. Using this technique, APSR was unable to be identified as a protein that associated with ATPS, but a number of other proteins, with molecular masses of ca. 48 kDa, 45 kDa, 40 kDa and 28 kDa, were identified as being putative protein partners to ATPS. Another approach to signal whether ATPS complex formation with other protein partners (including APSR) has occurred is to examine changes in the kinetic properties of the enzyme. To do this, the Km values of ATPS for inorganic phosphate were determined in two cultivars of onions, grown hydroponically with varying sulfur supply. Measurements that were taken prior to and during bulbing showed that for at least one cultivar (Texas Grano) with sufficient sulfur, an increase in the K
value from 3.8 nM (prior to bulbing), to 0.15 µM (during bulbing) was observed. It is possible that this change in K
value is due to the formation of a protein-protein complex.