Glutamine tRNA CUG ‐Dependent, Nitrogen‐Responsive Nuclear Gln3 and Gat1 Localization

Abstract

A leucine-dependent pathway activates TorC1 kinase and its downstream stimulation of protein synthesis. However, Gln3 and Gat1, nitrogen-responsive transcription activators, don't respond to leucine-dependent TorC1 activation. This led us to conclude that excess nitrogen-dependent down regulation of Gln3 occurs via a second mechanism. A major site of Gln3/Gat1 control occurs at their access to the nucleus. In excess nitrogen, Gln3/Gat1 are sequestered in the cytoplasm in a Ure2-dependent manner. They become nuclear when nitrogen or glutamine is limiting. The sensitivity of Gln3 localization to glutamine and inhibition of glutamine synthesis by methionine sulfoximine (Msx) prompted us to investigate the effects of a glutamine tRNA mutation (sup70-65) on Gln3/Gat1 regulation. Contrary to prediction, we show nuclear Gln3 localization - elicited by short- and long-term nitrogen starvation, growth in proline, during Msx or rapamycin treatment or in a ure2Δ - is abolished by alteration of glutamine tRNACUG. In contrast, nuclear Gat1 localization, which also exhibits a glutamine tRNACUG requirement for its response to short-term nitrogen starvation, growth in proline medium or a ure2Δ, neither responds to Msx or long-term starvation nor requires tRNACUG for its response to rapamycin. Gln3/Gat1 requirements for the rare tRNACUG, cannot be replaced in growing cells by the predominant glutamine tRNACAA. These observations demonstrate the existence of a specific nitrogen-responsive component participating in the control of Gln3/Gat1 localization that is highly sensitive to the function of the rare glutamine tRNACUG. Our data also demonstrate distinct mechanistic differences between the regulation of Gln3 and Gat1. Supported by NIH grant GM-35642.