Nitrogen Starvation and TorC1 Inhibition Differentially Affect Nuclear Localization of the Gln3 and Gat1 Transcription Factors Through the Rare Glutamine tRNACUG in Saccharomyces cerevisiae

摘要

A leucine, leucyl-tRNA synthetase–dependent pathway activates TorC1 kinase and its downstream stimulation of protein synthesis, a major nitrogen consumer. We previously demonstrated, however, that control of [Gln3][1], a transcription activator of catabolic genes whose products generate the nitrogenous precursors for protein synthesis, is not subject to leucine-dependent TorC1 activation. This led us to conclude that excess nitrogen-dependent down-regulation of [Gln3][1] occurs via a second mechanism that is independent of leucine-dependent TorC1 activation. A major site of [Gln3][1] and [Gat1][2] (another GATA-binding transcription activator) control occurs at their access to the nucleus. In excess nitrogen, [Gln3][1] and [Gat1][2] are sequestered in the cytoplasm in a Ure2-dependent manner. They become nuclear and activate transcription when nitrogen becomes limiting. Long-term nitrogen starvation and treatment of cells with the glutamine synthetase inhibitor methionine sulfoximine (Msx) also elicit nuclear [Gln3][1] localization. The sensitivity of [Gln3][1] localization to glutamine and inhibition of glutamine synthesis prompted us to investigate the effects of a glutamine tRNA mutation ( [sup70][3]-65 ) on nitrogen-responsive control of [Gln3][1] and [Gat1][2]. We found that nuclear [Gln3][1] localization elicited by short- and long-term nitrogen starvation; growth in a poor, derepressive medium; Msx or rapamycin treatment; or [ure2][4] Δ mutation is abolished in a [sup70][3]-65 mutant. However, nuclear [Gat1][2] localization, which also exhibits a glutamine tRNACUG requirement for its response to short-term nitrogen starvation or growth in proline medium or a [ure2][4] Δ mutation, does not require tRNACUG for its response to rapamycin. Also, in contrast with [Gln3][1], [Gat1][2] localization does not respond to long-term nitrogen starvation. These observations demonstrate the existence of a specific nitrogen-responsive component participating in the control of [Gln3][1] and [Gat1][2] localization and their downstream production of nitrogenous precursors. This component is highly sensitive to the function of the rare glutamine tRNACUG, which cannot be replaced by the predominant glutamine tRNACAA. Our observations also demonstrate distinct mechanistic differences between the responses of [Gln3][1] and [Gat1][2] to rapamycin inhibition of TorC1 and nitrogen starvation. [1]: http://www.yeastgenome.org/cgi-bin/locus.fpl?dbid=S000000842 [2]: http://www.yeastgenome.org/cgi-bin/locus.fpl?dbid=S000001873 [3]: http://www.yeastgenome.org/cgi-bin/locus.fpl?dbid=S000006690 [4]: http://www.yeastgenome.org/cgi-bin/locus.fpl?dbid=S000005173