Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 20 (24), 7209-19

RTG-dependent Mitochondria to Nucleus Signaling Is Negatively Regulated by the Seven WD-repeat Protein Lst8p

Affiliations

RTG-dependent Mitochondria to Nucleus Signaling Is Negatively Regulated by the Seven WD-repeat Protein Lst8p

Z Liu et al. EMBO J.

Abstract

In cells with reduced mitochondrial function, RTG1, 2 and 3 are required for expression of genes involved in glutamate synthesis. Glutamate negatively regulates RTG-dependent gene expression upstream of Rtg2p, which, in turn, acts upstream of the bHLH/Zip transcription factors, Rtg1p and Rtg3p. Here we report that some mutations [lst8-(2-5)] in LST8, an essential gene encoding a seven WD40-repeat protein required for targeting of amino acid permeases (AAPs) to the plasma membrane, bypass the requirement for Rtg2p and abolish glutamate repression of RTG-dependent gene expression. The lst8-1 mutation, however, which reduces plasma membrane expression of AAP, cannot bypass the Rtg2p requirement, but still suppresses glutamate repression of RTG target gene expression. We show that Lst8p negatively regulates RTG gene function, acting at two sites, one upstream of Rtg2p, affecting glutamate repression of RTG-dependent gene expression through Ssy1p, an AAP-like sensor of external amino acids, and the other between Rtg2p and Rtg1p-Rtg3p. These data, together with genome-wide transcription profiling, reveal pathways regulated by glutamate, and provide insight into the regulation of cellular responses to mitochondrial dysfunction.

Figures

None
Fig. 1. RTB2/LST8 is a negative regulator of CIT2 expression. (ARTB2 regulates CIT2 expression upstream of RTG3, and downstream of RTG2. Wild-type (PSY142), rtg2Δ (PSY142-rtg2), rtg2Δ lst8-5 (RBY426) and rtg2Δ rtb2-5 rtg3Δ (RBY427) cells were grown in YPD medium to mid-log phase and collected for β-galactosidase activity analysis to determine transcriptional activation of an integrated CIT2-lacZ reporter gene. β-galactosidase assays on whole-cell extracts were carried out in triplicate as described in Materials and methods. (BRTB2 is LST8. Wild-type PSY142 cells and rtg2Δ lst8-5 transformed with pRS416-LST8, lst8-5, rtg2Δ lst8-1 and lst8-1 derivatives were grown in YNBcasD medium plus uracil (omitted in cells transformed with pRS416-LST8) to mid-log phase, and CIT2-lacZ reporter gene expression was determined by β-galactosidase activities.
None
Fig. 2. Analysis of lst8 mutations. (A) Alignment of the seven WD repeats in Lst8p. Five or more conserved residues among seven WD repeats are highlighted in gray, and the consensus sequence formed is shown above the alignment; ϕ designates any hydrophobic residue and ψ designates any hydrophilic residue; T designates serine or threonine. Based on the alignment between Lst8p and the G protein β1 subunit (ter Haar et al., 1998), each WD repeat is likely to consist of four β-strands, indicated by arrows A, B, C and D. The last row gives a canonical numbering system for residues of a WD repeat. The red type indicates mutations identified in our screen: lst8-2, Gly146 to glutamate; lst8-3, Gly138 to aspartate; lst8-4 and lst8-6, Gly181 to aspartate; lst8-5, Gly171 to aspartate. The yellow type indicates the lst8-1 mutation identified as a synthetic lethal with sec13 (Roberg et al., 1997a). (B) Three-dimensional model of Lst8p. The model was created by Swiss-Model (Peitsch, 1996), an automated comparative protein modeling server, based on the homology between Lst8p and G protein β1 subunit. The image was prepared using programs Gl_render (http://www.hhmi.swmed.edu/external/Doc/G1_render.html), BOBSCRIPT (Esnouf, 1997), MOLSCRIPT (Kraulis, 1991) and Raster3D (Meritt, 1997). The positions of four β-strands A–D from WD repeat 6 are indicated. N and C designate the N- and C-terminal ends of Lst8p. Locations of the lst8-(2–5) mutations are indicated by the red dots, and the lst8-1 mutation by the yellow dot.
None
Fig. 3. The glutamate auxotrophy of an rtg2Δ mutation is rescued by the lst8-5 but not the lst8-1 mutation. Wild-type PSY142 cells, and rtg2Δ, lst8-5, rtg2Δ lst8-5, rtg2Δ rtb2-5 rtg3Δ, lst8-1 and rtg2Δ lst8-1 derivatives were streaked on YNBD medium supplemented or not with 0.02% glutamate as indicated, and with uracil, leucine and lysine, and incubated at 30°C for 2–3 days.
None
Fig. 4. The insensitivity of glutamate repression of CIT2-lacZ reporter gene expression in lst8-5 mutant cells is not the result of a defect in glutamate uptake. (ALst8-5 and lst8-1 mutants are largely insensitive to glutamate repression of CIT2-lacZ reporter gene expression. Wild-type (PSY142), lst8-1 and lst8-5 mutant strains with an integrated copy of CIT2-lacZ reporter gene were grown in YNB5%D medium with or without 0.2% glutamate. Whole-cell extracts were prepared and β-galactosidase activities were determined as described in Materials and methods. (B) Glutamate uptake in wild-type and lst8-5 mutant cells. Wild-type (PSY142) and lst8-5 mutant strains were grown in YNBD medium and glutamate uptake was assayed as described in Materials and methods. Glutamate was present at a final concentration of 0.01%. (C) Glutamate uptake into wild-type and gap1Δ dip5Δ mutant strains. Wild-type (S288C) and a gap1Δ dip5Δ derivative strain were grown in YNBD medium, and the glutamate uptake assay was carried out as described in (B). (DCIT2-lacZ reporter gene expression is still repressed by glutamate in gap1Δ dip5Δ mutant cells. Wild-type (S288C) and a gap1Δ dip5Δ mutant derivative strain were grown in YNB5%D medium with or without glutamate as indicated. Whole-cell extracts were prepared and β-galactosidase activities were determined as described in Materials and methods.
None
Fig. 5. Effects of shr3Δ, ssy1Δ and ptr3Δ mutations on glutamate repression, rtg2Δ suppression and glutamate auxotrophy. (ACIT2-lacZ expression. Transformants of wild-type (PLY126), shr3Δ (PLY151-ura3), ssy1Δ (HKY20), ptr3Δ (HKY31), rtg2Δ (PLY126-rtg2), shr3Δ rtg2Δ (PLY151-rtg2), ssy1Δ rtg2Δ (HKY20-rtg2) and ptr3Δ rtg2Δ (HKY31-rtg2) strains containing a CIT2-lacZ reporter gene on the centromere-based plasmid pCIT2-lacZ were grown to mid-log phase in the media indicated in the figure, and β-galactosidase activities were determined in extracts as described in Materials and methods. Strains rtg2Δ (PLY126-rtg2), shr3Δ rtg2Δ (PLY151-rtg2), ssy1Δ rtg2Δ (HKY20-rtg2) and ptr3Δ rtg2Δ (HKY31-rtg2) were not tested for CIT2-lacZ expression when grown in YNB5%D medium because they are glutamate auxotrophs. (B) The shr3Δ, ssy1Δ and ptr3Δ mutations cannot rescue glutamate auxotrophy of rtg2Δ cells. Cells were streaked on YNBD medium with or without 0.02% glutamate and incubated at 30°C for 2–3 days.
None
Fig. 6. The loss of glutamate repression of CIT2-lacZ reporter gene expression in ssy1Δ cells is uncoupled from glutamate uptake. (A) Glutamate uptake in ssy1Δ cells overexpressing Dip5 and Gap1. ssy1Δ (HKY20) cells were transformed with the 2µ control plasmid (empty vector), 2µ-TEF-DIP5 or 2µ-GAP1 plasmids, and the resultant transformants, including wild-type cells transformed with the 2µ control plasmid, were analyzed for glutamate uptake as described in Figure 4B. (B) Glutamate repression of CIT2-lacZ reporter gene expression in ssy1Δ cells overexpressing Dip5p and Gap1p. The various transformants described in (A) above were transformed with the pCIT2-lacZ reporter gene construct and the transformants were grown in YNBD medium with or without 0.2% glutamate. Whole-cell extracts were prepared and β-galactosidase activities were determined as described in Materials and methods.
None
Fig. 7. Effects of different amino acids on CIT2-lacZ reporter gene expression in wild-type and ssy1Δ cells. Wild-type (PLY126) and ssy1Δ (HKY20) strains transformed with a centromere-based plasmid pCIT2-lacZ were grown to mid-log phase in YNB5%D medium supplemented with 0.1% of the indicated amino acids. Whole-cell extracts were prepared and β-galactosidase activities were determined as described in Materials and methods.
None
Fig. 8. Model for dual regulation of RTG gene functions by Lst8p. Once on the cell surface, Ssy1p binds to glutamate or glutamine and sends an inhibitory signal to Rtg2p through Ptr3p. As revealed by the lst8-1 mutation, Lst8p might affect targeting or assembly of the Ssy1p–Ptr3p system, or its signal transduction function as suggested by Roberg et al. (1997a), and thus act upstream of Rtg2p. Lst8p is also proposed to function downstream of Rtg2p, as revealed by the lst8-(2–5) mutations. Intracellular glutamate is hypothesized to be sensed by Ptr3p (Klasson et al., 1999), or some additional component of the Ssy1p signaling pathway. Repressive signals generated by glutamate would lock Lst8p in its ‘RTG inhibition’ state so that less Lst8p would be available for its function in Ssy1p–Ptr3 signaling; this would have the effect of attenuating glutamate repression of RTG gene functions.

Similar articles

See all similar articles

Cited by 41 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback