Fig 4. Δlam2 cells, Δgtr1 cells and Δgtr2 cells lack the basal transcription of isp5⁺ and its transcriptional activation associated with nuclear Gaf1 localization induced by nitrogen depletion.

(A) mRNA levels of isp5⁺ were decreased in Δlam2 cells, Δgtr1 cells and Δgtr2 cells in a TORC1-dependent manner. Total RNA from the indicated cells as described in Fig 2B were subjected to quantitative RT-PCR for isp5⁺ similarly to Fig 3A. N = 3 for each group. Magnified parts of the graphs are shown in insets. ***P<0.001 for Turkey’s test following one-way ANOVA for the comparisons with the value of wild-type cells. ^###P<0.001 for Turkey’s test following one-way ANOVA compared with respective single knockout cells. (B) Nitrogen depletion-induced isp5⁺ transcriptional activation was abolished in Δlam2 cells and Δgtr2 cells. Wild-type cells (wt, HM123), Δlam2 cells (KP6607) and Δgtr2 cells (KP6608) harboring the Renilla luciferase reporter plasmid for isp5⁺ promoter (pKB8527) were grown to mid-log phase in EMM medium. The medium was replaced by EMM or nitrogen-depleted EMM (-NH₄Cl), and the cells were subjected to Renilla luciferase reporter assay. Peak values were normalized to that in wild-type cells in the control condition (EMM). N = 3 for each group. Magnified parts of the graphs are shown in insets. ***P<0.001, ns not significant for unpaired t-test for the planned comparisons with the same genotype in the control condition. ^###P<0.001 for Turkey’s test following one-way ANOVA compared with wild-type cells in the respective conditions (EMM or -NH₄Cl). (C) Nuclear localization of Gaf1 upon nitrogen depletion was impaired in Δlam2 cells, Δgtr2 cells and Δgtr1 cells. Wild-type cells (wt, KP6488), Δlam2 cells (KP6606), Δgtr2 cells (KP6610) and Δgtr1 cells (KP6612) expressing Gaf1-YFP under the native Gaf1 promoter were grown to mid-log phase in EMM medium. Fluorescent images of Gaf1-YFP were acquired before (0 min) or after nitrogen depletion (-NH₄Cl) for the indicated time. Scale bar, 10 μm.