FIGURE 8.

A, a truncated Gat1 peptide terminating at Gat1 Ser-233 (pRR1053) supports NCR-sensitive production of two major (IsoC and IsoD) and two minor (IsoE and IsoF) isoforms. Extracts of cells expressing wild type Gat1 (pKA62) were provided as controls for the production of IsoA and IsoB (lanes 1, 3, and 5) and a Gat1 M40L substitution (pRR979) producing only IsoB (lane 7). Note that lanes 6 and 7 in the upper panel were underloaded relative to lanes 1–5. The upper panel depicts a normal exposure of the Western blot in which the relative levels of the Gat1 isoforms produced under the various conditions can be directly compared. The lower panel is a longer exposure of that blot to visualize the minor bands that are not visible in the upper panel. There is a faint unidentified species between IsoA and IsoB in lanes 1, 3, and 5 of the lower panel. Culture conditions used in extract preparation appear as plus signs for proline (Pro), glutamine (Gln), or rapamycin-treated glutamine (Rap). Minus signs indicate the absence of that condition. Plasmid numbers and pertinent genotypes also appear above the Western blot images. B, chromosomal and plasmid-borne truncated GAT1-MYC¹³ (Gat1 Met-1 to Ser-233) genes support production of the same isoforms, exhibiting the same regulatory characteristics. Growth conditions are as in A. The plasmid (pRR1053) and strain (FV655) used in the experiment appear above the blot image. The blot depicted is an overexposure to permit evaluation of the minor IsoE and IsoF species. Shorter exposures yield the same results as observed in A. The gel concentration and/or running times of electrophoresis in this and subsequent blots containing IsoE and IsoF were increased from those in preceding figures to increase resolution.