Interaction assays of glutaredoxins with TGA2 in yeast

In order to map out the interaction domains of the GRX480 protein, DNA coding for different mutant versions of the wild type protein were generated using PCR specific primers and co-expressed in the yeast strain MAV203, with the transcription factor TGA2. The yeast MAV203 strain contained the ß-galactosidase gene reporter gene, downstream of a Gal4 promoter.

The glutaredoxin mutant versions included the double mutant and a version containing a deletion of the first 30 amino acids in the N-terminus (see Figure 6.1E). As a control, GRX370 was also co-expressed with TGA2 in the same yeast system. The glutaredoxins were expressed as fusion proteins to the yeast Gal4-DNA binding domain and a myc tag, while the transcription factor TGA2 was expressed as a fusion protein to the yeast Gal4-activation domain, and an HA tag. Control transformations were performed using plasmids lacking either TGA2 or glutaredoxin or both (Figure 6.3AI). After transformation, the yeasts were grown on selective medium lacking leucine and tryptophan. Individual colonies were analyzed for ß-galactosidase activity and protein expression respectively (Figure 6.3AII).

The double mutant version of GRX480 (GRX-DM) still interacted with TGA2. Of all the clones tested in 5 independent experiments (3-5 clones analyzed each time), they showed significant ONPG activity above the controls where there was no interaction occurring.

When compared with the wild type protein, only in about 32% of the clones was interaction with TGA2 less than in the wild type. In most cases, the strength of the interaction was within the range of the double mutant.

The deletion of the 30 N-terminal amino acids of GRX480 did not result in a loss in its ability to interact with TGA2. Interestingly, of the 17 clones tested, about 52% showed significantly stronger interaction than the double mutant protein, while in the rest, the interaction was within the range of the double mutant. In 3 independent experiments, they showed average ONPG activity higher than in the wild type.

(I)

TRP1 ADH-Promoter Gal4-BD Myc-tag GRX480

(B)

TRP1 ADH-Promoter Gal4-BD Myc-tag GRX480-DM

(C)

LEU ADH-Promoter Gal4-AD HA-tag

(F)

TRP1 ADH-Promoter Gal4-BD Myc-tag

(A)

TRP1 ADH-Promoter Gal4-BD Myc-tag GRX370

(E)

LEU ADH-Promoter Gal4-AD HA-tag TGA2

(G)

(D) TRP1 ADH-Promoter Gal4-BD Myc-tagGRX480-∆N30

Gal4 Promoter

Figure 6.3A: ONPG Assay for interaction between TGA2 and glutaredoxins.

I: Plasmid constructs used. Plasmids A (control lacking any glutaredoxin), B (with WT At1g28480 as GRX480), C (with double cysteine mutant of At1g28480 as GRX480-DM), D (with N-terminal deletion mutant of At1g28480 as GRX∆N30) and E (with GRX At5g40370 as GRX-370) contain the tryptophan selectable marker for yeast selection. The ADH promoter drives the expression of a fusion protein with the and N-terminal yeast Gal4-binding domain (Gal4BD) and myc tag. The plasmid constructs F (control lacking TGA2) and E (with TGA2) contain the Leucine selectable marker for yeast selection. The ADH promoter drives the expression of a fusion protein with the and N-terminal yeast Gal4-activating domain (Gal4AD) and HA tag. H, is the Gal4 promoter reporter construct present in the yeast MAV203 strain.

(II): ONPG Assay, showing average activity from clones measured. Yeast cells were co-transformed with the different GRX types or their control plasmid (GBD) together with TGA2 or the control plasmid (GAD) indicated as +/-. Individual clones showed similar behavior as above when the ONPG assay was repeated.

(I)

- GRX480 GDM GXdN30 GRX370 GRX + + + + + + + + + + + + + TGA2 1 1 2 3 1 2 3 1 2 3 1 2 3 clone TGA2

GRX

Loading ONPG Activity 140

120 100 80 60 40 20 0

(II)

Loading Myc-GBD

- + - + - + - + - + TGA2

- - GRX GRX GDM GDM GXdN30 GXdN30 GX370 GX370 GRX

mycGBD GRX

TGA2

Figure 6.3B: Expression of TGA and interacting glutaredoxin types in yeast.

The TGA2 was detected using antiserum raised against the C-terminal part of TGA2. GRX was detected using monoclonal antibody against the myc-tag.

(I): Protein extracts were prepared from individual clones from the experiment in Figure 6.3A(II) above which were used before for the ONPG assay. In the first lane, is the protein extract from a colony expressing TGA alone. Three colonies were analyzed in each case. Clone #3 containing WT At1g28480 (GRX480), and clone #2 containing the N-terminal deletion mutant (GXdN30) had two-fold stronger signals in the ONPG interaction assay.

(II): An independent experiment showing the expression of TGA2 and all the glutaredoxin types when co-transformed into yeast cells. In the control experiments, where one of the interacting proteins is absent, the TGA2 or GRX is also detected.

The wild type and N-terminal deletion mutant of At1g28480 showed more variation in their interaction with TGA2 (ONPG activity) than the GRX double mutant.

The question arose as to whether the variation of ONPG activity is proportional to the levels of expression of any of the interacting proteins. To answer this question, protein extracts from three individual clones with varying ONPG activity of the wild type, double mutant and N-terminal deletion mutant were separated on SDS PAGE and analyzed for their expression of GRX or TGA2 (Figure 6.3BI). Clone #3 of the wild type GRX480 and clone #2 of the N terminal deletion mutant which showed the strongest ONPG activity of up to 2 fold higher than the other two clones nevertheless expressed both GRX and TGA2 within the same range (Figure 6.3BI).

To investigate whether the redox active state of the wild type has any influence in its ability to interact with TGA2, yeast cells were subjected to oxidative stress in the presence of 1mM diamide for 5 hours. They nevertheless did not show a difference in their ONPG activity under these conditions (results not shown). It could very well be that an already oxidized state within the cells may have made no difference after diamide treatment, in terms of the activity of GRX480

.

There appears therefore to have been two populations of cells, with some showing interaction stronger than others, but it is not clear why they showed this behavior. Even though the wild type and N-terminal deletion mutant clones also had variable sizes on selective plates, it did not seem to correlate with their ONPG activity. The clones of all the other transformations had regular and comparable sizes.

The classical glutaredoxin GRX370 did not interact at all with the transcription factor TGA2. The ONPG activity was in the same range as all the negative controls (Figure 6.3AII).

All the glutaredoxin types tested in all the experiments and in controls were expressed in the yeast cells. The TGA2 was also expressed. This was the case in either the presence or absence of GRX or TGA (Figure 6.3BII).