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Characterization of fungal RTG2 genes in retrograde signaling of Saccharomyces cerevisiae.

Ünlü ES, Narayanan L, Gordon DM - FEMS Yeast Res. (2013)

Bottom Line: Changes in the functional status of mitochondria result in the transcriptional activation of a subset of nuclear-encoded genes in a process referred to as retrograde signaling.Our findings show that all four Rtg2p homologs are functional upon activation of retrograde signaling, although their degree of complementation varied.In addition, all Rtg2p homologs showed a marked reduction in Mks1p binding, which may contribute to their altered responses to retrograde signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Mississippi State University, P.O. Box GY, Starkville, MS 39762, USA.

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Aco1p and Cit2p protein profiles in rtg2Δ cells expressing Rtg2p homologs. 5 × 106 cells from an exponentially growing culture supplemented with glutamate were processed by alkaline lysis followed by TCA precipitation. The resulting whole cell extracts were separated by SDS-PAGE followed by immunoblot analysis using antibodies against Aco1p, the triple-hemagglutin epitope to detect Cit2p and Tom40p. Tom40p was included as a loading control. Aco1p and Cit2p protein levels in rtg2Δ cells expressing Rtg2p homologs from the native RTG2 promoter (a) and from the constitutive GPD promoter (b).
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fig02: Aco1p and Cit2p protein profiles in rtg2Δ cells expressing Rtg2p homologs. 5 × 106 cells from an exponentially growing culture supplemented with glutamate were processed by alkaline lysis followed by TCA precipitation. The resulting whole cell extracts were separated by SDS-PAGE followed by immunoblot analysis using antibodies against Aco1p, the triple-hemagglutin epitope to detect Cit2p and Tom40p. Tom40p was included as a loading control. Aco1p and Cit2p protein levels in rtg2Δ cells expressing Rtg2p homologs from the native RTG2 promoter (a) and from the constitutive GPD promoter (b).

Mentions: Activation of retrograde signaling results in the transcriptional upregulation of CIT2, the peroxisomal isoform of citrate synthase, and ACO1, an early TCA cycle enzyme. To determine the ability of RTG2 homologs to induce the transcriptional activation of these two genes, we first determined CIT2 and ACO1 transcript levels under basal conditions (+ glutamate) (Table 1). Quantitative real-time PCR analysis revealed a 10- to 14-fold reduction in CIT2 transcript levels when compared with cells expressing RTG2 from S. cerevisiae for the rtg2Δ mutant and for cells expressing the RTG2 homologs from A. gossypii and V. polyspora. A threefold reduction in the level of CIT2 transcript was found for cells expressing the C. glabrata RTG2 homolog, while little difference was detected for cells expressing RTG2 from K. lactis. Less variation was found for ACO1 transcript levels. All RTG2 homologs, except V. polyspora, had ACO1 transcript levels that were reduced by 1- to 4-fold compared with that of RTG2 from S. cerevisiae. A similar pattern was detected for Cit2p at the protein level. Cells expressing Rtg2p homologs from C. glabrata and K. lactis had levels of Cit2p and Aco1p similar to that of Rtg2p from S. cerevisiae. However, cells expressing A. gossypii and V. polyspora homologs showed Cit2p and Aco1p expression levels similar to that detected for rtg2Δ mutants (Fig. 2a). Increasing the expression of RTG2 homologs using the GPD promoter partially rescued Cit2p levels for A. gossypii and V. polyspora (Fig. 2b).


Characterization of fungal RTG2 genes in retrograde signaling of Saccharomyces cerevisiae.

Ünlü ES, Narayanan L, Gordon DM - FEMS Yeast Res. (2013)

Aco1p and Cit2p protein profiles in rtg2Δ cells expressing Rtg2p homologs. 5 × 106 cells from an exponentially growing culture supplemented with glutamate were processed by alkaline lysis followed by TCA precipitation. The resulting whole cell extracts were separated by SDS-PAGE followed by immunoblot analysis using antibodies against Aco1p, the triple-hemagglutin epitope to detect Cit2p and Tom40p. Tom40p was included as a loading control. Aco1p and Cit2p protein levels in rtg2Δ cells expressing Rtg2p homologs from the native RTG2 promoter (a) and from the constitutive GPD promoter (b).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814403&req=5

fig02: Aco1p and Cit2p protein profiles in rtg2Δ cells expressing Rtg2p homologs. 5 × 106 cells from an exponentially growing culture supplemented with glutamate were processed by alkaline lysis followed by TCA precipitation. The resulting whole cell extracts were separated by SDS-PAGE followed by immunoblot analysis using antibodies against Aco1p, the triple-hemagglutin epitope to detect Cit2p and Tom40p. Tom40p was included as a loading control. Aco1p and Cit2p protein levels in rtg2Δ cells expressing Rtg2p homologs from the native RTG2 promoter (a) and from the constitutive GPD promoter (b).
Mentions: Activation of retrograde signaling results in the transcriptional upregulation of CIT2, the peroxisomal isoform of citrate synthase, and ACO1, an early TCA cycle enzyme. To determine the ability of RTG2 homologs to induce the transcriptional activation of these two genes, we first determined CIT2 and ACO1 transcript levels under basal conditions (+ glutamate) (Table 1). Quantitative real-time PCR analysis revealed a 10- to 14-fold reduction in CIT2 transcript levels when compared with cells expressing RTG2 from S. cerevisiae for the rtg2Δ mutant and for cells expressing the RTG2 homologs from A. gossypii and V. polyspora. A threefold reduction in the level of CIT2 transcript was found for cells expressing the C. glabrata RTG2 homolog, while little difference was detected for cells expressing RTG2 from K. lactis. Less variation was found for ACO1 transcript levels. All RTG2 homologs, except V. polyspora, had ACO1 transcript levels that were reduced by 1- to 4-fold compared with that of RTG2 from S. cerevisiae. A similar pattern was detected for Cit2p at the protein level. Cells expressing Rtg2p homologs from C. glabrata and K. lactis had levels of Cit2p and Aco1p similar to that of Rtg2p from S. cerevisiae. However, cells expressing A. gossypii and V. polyspora homologs showed Cit2p and Aco1p expression levels similar to that detected for rtg2Δ mutants (Fig. 2a). Increasing the expression of RTG2 homologs using the GPD promoter partially rescued Cit2p levels for A. gossypii and V. polyspora (Fig. 2b).

Bottom Line: Changes in the functional status of mitochondria result in the transcriptional activation of a subset of nuclear-encoded genes in a process referred to as retrograde signaling.Our findings show that all four Rtg2p homologs are functional upon activation of retrograde signaling, although their degree of complementation varied.In addition, all Rtg2p homologs showed a marked reduction in Mks1p binding, which may contribute to their altered responses to retrograde signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Mississippi State University, P.O. Box GY, Starkville, MS 39762, USA.

Show MeSH
Related in: MedlinePlus