Bqt3 stabilizes Bqt4 in the INM. (A) Bqt4-dTM represents Bqt4 deleted for the TMH by removal of 19 amino acid residues from the C-terminal. Bqt4-dN represents Bqt4 deleted of the N-terminal 262 amino acid residues. (B) Two-hybrid interactions of Bqt4 and its deletion derivatives with Bqt3. AD and BD indicate the constructs based on the pGADT7 and pGBKT7 plasmids, respectively (see Materials and methods). (C) Localization of mCherry-Bqt3 in bqt4-full (CRLu61; h⁻ bqt3Δ::LEU2, aur1::mCherry-bqt3, bqt4Δ::ura4⁺ lys1::GFP-bqt4), bqt4-dTM (CRLu62; h⁻ bqt3Δ::LEU2, aur1::mCherry-bqt3, bqt4Δ::ura4⁺ lys1::GFP–bqt4-dTM), bqt4-dN (CRLu63; h⁻ bqt3Δ::LEU2, aur1::mCherry-bqt3, bqt4Δ::ura4⁺ lys1::GFP–bqt4-dN), and bqt4Δ (CRLu69; h⁻ bqt3Δ::LEU2, aur1::mCherry-bqt3, bqt4Δ::ura4⁺) was observed in mitotic cells. (D–F) Heterothallic h⁻ haploid cells expressing GFP-Bqt3 (D), -Bqt4 (E), and –Bqt4-dTM (F) were observed in the vegetative phase and after nitrogen starvation. Numbers on the top of each image indicate the time in hours after nitrogen starvation. (D) In bqt4Δ cells, levels of Bqt3 mRNA and protein remained unchanged. The strains examined are CRLt01 (h⁻ bqt3Δ::LEU2, aur1::GFP-bqt3, bqt4⁺) and CRLs87 (h⁻ bqt3Δ::LEU2, aur1::GFP-bqt3, bqt4Δ::ura4⁺). The graph shows the bqt3 mRNA level measured by real time PCR (mean of two experiments). (E) In bqt3Δ cells, Bqt4 protein was detected at low levels on the NE but disappeared after nitrogen starvation. Levels of Bqt4 protein measured by Western blotting were markedly decreased in bqt3Δ cells. The graph shows the bqt4 mRNA level, as measured by real-time PCR (mean of two experiments). The amount of bqt4 mRNA in bqt3Δ is comparable with that in bqt3⁺ in vegetative cells as well as after nitrogen starvation. The strains examined are CRLr06 (h⁻ bqt3⁺, bqt4Δ::ura4⁺, lys1::GFP-bqt4) and CRLp93 (h⁻ bqt3Δ::LEU2, bqt4Δ::ura4⁺, lys1::GFP-bqt4). (F) The Bqt4-dTM protein, which does not localize to the NE, escaped degradation in bqt3Δ cells. The strains examined are CRLr24 (h⁻ bqt3⁺, bqt4Δ::ura4⁺, lys1::GFP–bqt4-dTM) and CRLr25 (h⁻ bqt3Δ::LEU2, bqt4Δ::ura4⁺ lys1::GFP–bqt4-dTM). Bars, 10 µm.