Mitochondria-to-nuclear signaling is regulated by the subcellular localization of the transcription factors Rtg1p and Rtg3p - PubMed (original) (raw)

Mitochondria-to-nuclear signaling is regulated by the subcellular localization of the transcription factors Rtg1p and Rtg3p

T Sekito et al. Mol Biol Cell. 2000 Jun.

Free PMC article

Abstract

Cells modulate the expression of nuclear genes in response to changes in the functional state of mitochondria, an interorganelle communication pathway called retrograde regulation. In yeast, expression of the CIT2 gene shows a typical retrograde response in that its expression is dramatically increased in cells with dysfunctional mitochondria, such as in rho(o) petites. Three genes control this signaling pathway: RTG1 and RTG3, which encode basic helix-loop-helix leucine zipper transcription factors that bind as heterodimer to the CIT2 upstream activation site, and RTG2, which encodes a protein of unknown function. We show that in respiratory-competent (rho(+)) cells in which CIT2 expression is low, Rtg1p and Rtg3p exist as a complex largely in the cytoplasm, and in rho(o) petites in which CIT2 expression is high, they exist as a complex predominantly localized in the nucleus. Cytoplasmic Rtg3p is multiply phosphorylated and becomes partially dephosphorylated when localized in the nucleus. Rtg2p, which is cytoplasmic in both rho(+) and rho(o) cells, is required for the dephosphorylation and nuclear localization of Rtg3p. Interaction of Rtg3p with Rtg1p is required to retain Rtg3p in the cytoplasm of rho(+) cells; in the absence of such interaction, nuclear localization and dephosphorylation of Rtg3p is independent of Rtg2p. Our data show that Rtg1p acts as both a positive and negative regulator of the retrograde response and that Rtg2p acts to transduce mitochondrial signals affecting the phosphorylation state and subcellular localization of Rtg3p.

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Figures

Figure 1

Figure 1

Rtg1p and Rtg3p interact in both ρ+ and ρo petite cells. Whole-cell extracts were prepared from wild-type (WT) ρ+ and ρo cells and _rtg1_Δ, _rtg2_Δ, or _rtg3_Δ mutant derivatives of these strains. Extracts were adjusted to 3 mg/ml protein and incubated with 5 μl of antiserum raised against recombinant Rtg3p. The immunoprecipitates were then analyzed by Western blotting with antiserum raised against recombinant Rtg1p.

Figure 2

Figure 2

Subcellular localization of Rtg3p (A), Rtg1p (B), and Rtg2p (C) in wild-type (WT) and in various _rtg_Δ mutant derivatives of ρ+ and ρo cells. Constructs encoding C-terminal-tagged GFP derivatives of full-length Rtg1p, Rtg2p, and Rtg3p were transplaced into their respective chromosomal loci and expressed under the control of the each of the native promoters. Cells were grown in YNBR+cas medium. In wild-type ρ+ cells, Rtg3p-GFP (A, a) and Rtg1p-GFP (B, a) are largely cytoplasmic. In otherwise wild-type ρo cells, however, both Rtg3p-GFP (A, b) and Rtg1p-GFP (B, b) are concentrated in the nucleus. Rtg2p-GFP expressed from a single-copy gene transplaced into the RTG2 locus appears strictly cytoplasmic both in ρ+ and ρo cells (C, a and b). The effects of _rtg1_Δ, _rtg2_Δ, or _rtg3_Δ mutations on the subcellular localization of these GFP fusion proteins are shown. Localization of the GFP fusion proteins was determined by epifluorescence microscopy as described in MATERIALS AND METHODS.

Figure 3

Figure 3

Effects of the functional state of mitochondria and _rtg_Δ mutations on the phosphorylation state of Rtg3p and Rtg1p. Logarithmic phase cultures of wild-type (WT) ρ+ and ρo cells and _rtg_Δ mutant derivatives of these strains were grown in YPR medium, and cell-free extracts were prepared as described in MATERIALS AND METHODS. Aliquots of these extracts were analyzed by Western blotting with antisera raised against recombinant Rtg3p (A) or Rtg1p (B). In some cases the extracts were treated with 5 U of calf intestinal alkaline phosphatase (cip) before Western blot analysis, as indicated.

Figure 4

Figure 4

An _rtg1_Δ mutation is epistatic to an _rtg2_Δ mutation. (A) Subcellular localization of Rtg3p-GFP in ρ+ or ρo _rtg1_Δ _rtg2_Δ double-mutant cells was determined by epifluorescence microscopy. (B) Extracts were prepared from wild-type ρ+ and ρo cells and the indicated rtg mutant derivatives of these strains and analyzed by Western blotting with antiserum specific for Rtg3p.

Figure 5

Figure 5

Deletion mutants of Rtg3p-GFP. (A) Shown in order below that of wild-type (WT) Rtg3p-GFP are representations of mutants with deletion of the C or N terminus, the Zip, and loop-helix 2 (LH) domains of Rtg3p-GFP. The white bars separate the helix 1-loop-helix 2 domains. (B) A portion of the amino acid sequence in the basic region (b) of wild-type Rtg3p is shown and below it a representation of the deletion mutant lacking the basic region. A consensus bipartite NLS is indicated in bold.

Figure 6

Figure 6

Effects of domain deletions on various properties of Rtg3p-GFP. Constructs encoding the deletion mutants of Rtg3p-GFP indicated in Figure 5 were transplaced into the RTG3 locus of wild-type and various mutant derivatives of ρ+ cells. ρo derivatives of those strains were obtained by ethidium bromide mutagenesis. The various ρ+ and ρo strains and rtg mutant derivatives were analyzed in A for their ability to interact with Rtg1p by Western blotting with anti-Rtg1p antiserum of immunoprecipitates obtained by incubation of whole-cell extracts with anti-GFP antiserum, in B for their phosphorylation state by Western blot analysis using anti-Rtg3p antiserum, in C for their subcellular localization by epifluorescence microscopy, and in D and E for their ability to support CIT2 expression as determined by Northern blotting with a _CIT2_-specific probe as described in MATERIALS AND METHODS. RNA loads were normalized to the level of ACT1 mRNA using an _ACT1_-specific probe.

Figure 7

Figure 7

Rtg3p phosphorylation is subject to a feedback control. ρ+ wild-type (WT), _rtg3_Δ, and _rtg3_Δ280–298 deletion mutant allele transplaced into the chromosomal RTG3 locus, each transformed with a centromeric plasmid, pΔb-rtg3-GFP encoding the Rtg3pΔ280–298-GFP deletion mutant or with pRtg3-GFP encoding wild-type Rtg3p-GFP, were grown in YNBR+cas medium to midlogarithmic phase. Whole-cell extracts were prepared and analyzed by Western blotting using Rtg3p-specific antiserum. The positions of the plasmid-encoded GFP-tagged and chromosomally expressed Rtg3ps are indicated.

Figure 8

Figure 8

Model of the control of mitochondria-to-nuclear signaling. In cells with dysfunctional mitochondria, one or more signals, one of which is possibly the level of glutamate produced from the TCA cycle, are transmitted from mitochondria (bold, dashed arrow) via Rtg2p to a cytoplasmic complex between Rtg1p and a highly phosphorylated form of Rtg3p. This complex, which may include other factors not indicated, becomes transiently dissociated along with a dephosphorylation of Rtg3p. Rtg1p and Rtg3p then translocate to the nucleus and assemble for transcriptional activation at target gene R box sites, GTCAC. The phosphorylation state of cytoplasmic Rtg3p is sensitive to a feedback response, indicated by the light green arrow, in that the absence of Rtg1p–Rtg3p-dependent transcription in the nucleus activates further dephosphorylation and nuclear translocation of cytoplasmic Rtg3p. It is not known whether dephosphorylation of cytoplasmic Rtg3p is caused by inactivation of a kinase or activation of a phosphatase.

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