Large scale purification of Gcn1 containing complexes, and identification of the

A large scale purification of Gcn1 containing complexes was necessary in order to have sufficient amount of samples for the subsequent identification of proteins in the Gcn1-containing complexes via Mass Spectrometry. Therefore, cells expressing Gcn1-Myc from a high copy plasmid, and the gcn1! strain containing empty vector, were grown to exponential phase. The cells were then subjected to formaldehyde (0.3%) cross-linking at room temperature for 10 minutes, cell

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extracts obtained and subjected to anti-Myc antibody mediated immunoprecipitation with the optimised conditions. Four independent immunoprecipitations were performed, the beads were pooled together at the end of the experiment before elution by boiling in protein loading dye. An aliquot of the eluate was analyzed by SDS-PAGE followed by Western blotting using antibodies against Myc, Gcn2, Gcn20 or Pgk1.

Figure 3.25. Gcn1 specific immunoprecipitation of Gcn1 binding proteins. The gcn1! (H2556) strain harbouring hc GCN1-Myc (refer to Table 2.1) was subjected to formaldehyde cross-linking as indicated in Figure 3.14, and the cell extract obtained from the cells were subjected to anti-Myc immunoprecipitation as indicated in Figure 3.21. Cell extract from a strain deleted for GCN1, GCN2 and GCN20 (""") was loaded along with the immunoprecipitate as a negative control.

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Gcn1, Gcn2 and Gcn20 were specifically immunoprecipitated in the Gcn1-Myc immunoprecipitate (Figure 3.25). A signal for Pgk1 was not detectable in the immunoprecipitates, suggesting that un-specifically bound proteins had been removed. As expected, compared to the non-cross linked samples, Gcn1, Gcn2 and Gcn20 were present in lower amounts in the 0.3% cross-linked samples. As expected, the amount of Gcn20 in the gcn1! sample was much less than in the

Gcn1-Myc input sample, as observed and suggested previously, this is because Gcn20 is less stable in the absence of Gcn1 (2).

As the immune complexes were eluted from the beads by boiling the beads in protein loading dye, these samples cannot be subjected to Mass Spectrometry (MS). Therefore it was decided to separate the samples via SDS- PAGE, stain the gel to visualize proteins and then subject the gel lanes to MS analysis. As the co-precipitated proteins were likely to be lowly abundant, and to prevent diluting the samples too much when resolving them in the gel, it was aimed to run the samples in SDS-PAGE just far enough that all proteins entered the gel. To first test how far the sample has to enter the gel to have all proteins entered the gel even of very large, 50 !g of protein from the Gcn1-Myc sample was denatured at 95°C and resolved by SDS-PAGE for 6, 4, 2 or 1 cm from the

slot followed by Western blotting with antibodies against Myc. The distance travelled by the samples was determined by measuring the distance between the dye front and slot.

A signal for Gcn1 was detected below the slot in samples that were run for 4 or 6 cm (Figure 3.26). In the samples ran for 2 and 1 cm the signal for Gcn1 was detected at the slot, raising the possibility that not all Gcn1 may fully entered the gel. These results indicated that Gcn1 can migrate below the slot if the samples are run for 4 or 6 cm below the well.

However, it was decided to run the samples on the gel for 5 cm, to be on the safe side that all Gcn1 would migrate below the slot and no Gcn1 will be lost in the subsequent staining procedure.

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Figure 3.26. Western blot of samples that were resolved by SDS-PAGE for 6, 4, 2 or 1 cm. 50 !g of cell extract from the Gcn1-Myc strain indicated in Figure 3.25 was resolved by SDS-PAGE for indicated distances from the slot followed by Western blotting with an antibodies against Myc.

The gel was then stained with colloidal Coomassie stain to visualize the bands (Figure 3.27). In each lane only two bands were visible. This suggests that the proteins co-precipitated by Gcn1-Myc were not abundant to visualize by Colloidal Coomassie staining. The two visible bands around 64 kDa and 50 kDa may represent heavy and light chains of the anti-Myc antibodies. The high amounts of immunoglobulin can easily mask the identification of low abundance peptides. Therefore, these visible bands were cut out of the gel and analyzed separately from the rest of the lanes, via MS. The MS analysis was done at the Centre for Protein Research, University of Otago.

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Figure 3.27. Colloidal Coomassie staining of SDS-PAGE. Immunoprecipitates from Figure 3.25 were resolved by SDS-PAGE for 5 cm and stained with colloidal Coomassie.

A summary of the work strategy employed for the identification of Gcn1 binding proteins is given in Figure 3.28.

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Figure 3.28. Work strategy of formaldehyde cross-linking followed by affinity purification and LC-MS-MS analysis. In order to create our in house Gcn1 interactome we have used an epitope tagged Gcn1 (Gcn1-Myc). The gcn1" cells carrying the hc plasmid borne Gcn1-Myc or vector alone were grown in minimal media to exponential phase and the cells were subjected to formaldehyde (0.3%) cross-linking in vivo at room temperature for 10 minutes. Extracts obtained from the cross-linked cells and untreated controls were subjected to anti-Myc antibody mediated co-immunoprecipitation in order to isolate proteins binding to the Gcn1-Myc. The co-precipitate was investigated for the presence of Gcn1 and known Gcn1 binding proteins (Gcn2 and Gcn20). After confirming that good amount of Gcn1 and the known Gcn1 binding proteins were detectable by Western blotting in the Gcn1-Myc co-precipitate, the samples were analyzed by MS. For MS analysis, the co-precipitates were subjected to SDS-PAGE electrophoresis and the gels were stained with colloidal Coomassie to visualize proteins. The stained gels were subjected to LC-MS-MS in order to identify proteins that were co-precipitated by the Gcn1-Myc.

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