The TAP and Myc-tags do not affect Gcn1 function

It was reasoned that if the antibodies against Gcn1 interact with a protein-binding domain of Gcn1, it may disrupt the interaction of Gcn1 with its binding partners. Therefore, the decision was made to use an epitope tagged version of Gcn1 to affinity purify Gcn1 containing protein complexes from cell extracts. Gcn1 tagged at the C-terminus with TAP or Myc epitopes were available in the Sattlegger lab.

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First it was investigated whether the tags affect the function of Gcn1. If tagging affects Gcn1 function, then Gcn2 activation would be impaired. Therefore, it is expected that strains with defective Gcn1 function will have a growth defect under amino acid starvation conditions.

To test this, plasmids carrying the Myc tagged GCN1 from a low copy (lc) or high copy (hc) plasmid, untagged GCN1 or vector alone (refer to Table 2.1) were introduced into the gcn1! strain. Wild type transformed with vector alone was used as a positive control. The resulting tranformants, strain containing chromosamally TAP tagged GCN1 and gcn1! strain were subjected to semi quantitative growth assays in the presence of amino acid starvation inducing drugs, 3-Amino Triazole (3-AT) or Sulfometuron methyl (SM). 3-AT causes starvation for histidine and it is a competitive inhibitor of the HIS3 gene product, imidazole glycerolphosphate dehydratase, an enzyme involved in histidine biosynthesis (112). SM causes starvation for the branched amino acids by inhibiting the enzyme involved in the biosynthesis of these amino acids (113).

Saturated cultures of the indicated strains (Figure 3.9) were grown in liquid minimal media and the cultures were tenfold serially diluted. 5 !l of each dilution and undiluted cultures were spotted on plates containing 3-AT or SM, and no SM as a control. After spotting the cultures, the plates were incubated at 30°C and the growth was monitored for 7 to 10 days.

As expected, the wild type grew well on both the control and starvation plates and the strain lacking Gcn1 was unable to grow on either SM or 3-AT containing plates, suggesting the successful induction of starvation by 3-AT and SM (Figure 3.9).

Similar to the wild type, strains expressing the TAP tagged GCN1 grew well on both the control and starvation plates. This result suggested that the TAP tagging has no effect on Gcn1 function, at least at the investigated SM concentration (Figure 3.9A).

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As shown, (Figure 3.9B), similarly to the wild type, strains carrying the Myc tagged (lc or hc) and untagged Gcn1 were able to grow on both the control and starvation plates. But at higher concentrations of 3-AT, strain that expressed

GCN1 from the lc plasmid displayed a mild growth defect.

Figure 3.9. Semi quantitative growth assay of TAP tagged Gcn1 (A) and Myc tagged Gcn1 (B). Indicated strains were grown in liquid minimal media with appropriate amino acids. Saturated yeast cultures were 10 fold serially diluted, four different dilutions and undiluted cultures were spotted on plates containing SM, 3-AT and no drug as a control. Growth was monitored every day for 7 to 10 days. The experiment was performed with two independent biological replicates.

Though both the lc and hc plasmids expressed Myc-tagged GCN1, the growth defect was observed only in the strain that expressed GCN1 from the lc plasmids. It is possible that GCN1 from the lc plasmids was not expressed to the level that was expressed from its chromosomal gene. Therefore, the expression level of Gcn1 in these strains was investigated.

According to the western blot result, GCN1 was expressed about 0.5 times more from the hc plasmid, and 0.5 times less from the lc plasmid, than endogenous GCN1 (Figure 3.10). It is surprising to see that the lc plasmid expressed less Gcn1 than the endogenous gene. Considering that lc plasmids reside as 2-3 copies in the cell (114), one would expect that it leads to higher

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Gcn1 levels than that in a wild type strain with chromosomal GCN1.

Figure 3.10. Expression levels of endogenous GCN1, and GCN1 from a low copy and high copy plasmid. A) 50 !g of cell extracts from the gcn1! (H2556) strain transformed with low copy (lc) or high copy (hc) and the wild type (H1511) strains (refer to Table 2.1 and Table 2.2) were resolved by SDS-PAGE and the proteins were subjected to immunoblotting using antibodies against Gcn1 or Pgk1. B) The Gcn1 and Pgk1 signals were quantified using the Multi Gauge V3.1 software (Fujifilm) and the Gcn1/Pgk1 signal ratio was calculated and normalized to that of the wild type. The experiment was performed with two independent biological replicates and the standard error is indicated.

The fact that the hc plasmid, which is supposed to be maintained with 100 copies per cell (114) expressed only 0.5 times more than the endogenous GCN1,

might indicate that the plasmid borne gene was not expressed efficiently, possibly because the promoter was truncated, or because the transcript was incomplete and prone to degradation. The low abundance of Gcn1 in the gcn1! strain harboring lc

GCN1 may explain why this strain showed slight 3-AT sensitivity.

Taken together, the results suggested that either Myc or TAP tagging does not have an effect on Gcn1 function.

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3.2.3 Formaldehyde cross-linking does not affect anti-Myc antibody mediated