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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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Related in: MedlinePlus

Complementation of rtg2Δ glutamate auxotrophy by Rtg2p homologs. 10-fold serial dilutions of cells expressing the indicated RTG2 genes were spotted to -Trp synthetic dropout media with and without glutamate. Growth was scored after 3 days of growth at 30 °C. Complementation profiles for Rtg2p homologs expressed from the native RTG2 promoter are shown in (a); complementation profiles for Rtg2p homologs expressed from the constitutive GPD promoter are shown in (b).
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fig01: Complementation of rtg2Δ glutamate auxotrophy by Rtg2p homologs. 10-fold serial dilutions of cells expressing the indicated RTG2 genes were spotted to -Trp synthetic dropout media with and without glutamate. Growth was scored after 3 days of growth at 30 °C. Complementation profiles for Rtg2p homologs expressed from the native RTG2 promoter are shown in (a); complementation profiles for Rtg2p homologs expressed from the constitutive GPD promoter are shown in (b).

Mentions: In cells defective for electron transport activity, the retrograde signaling pathway has been linked to cellular glutamate homeostasis through its control of the expression of the first three enzymes of the Krebs cycle (Liu & Butow, 1999). This RTG regulatory pathway functions to increase α-ketoglutarate production ensuring a sufficient supply of glutamate for various biosynthetic reactions. Like cells deleted for cit1 cit2, or aco1, rtg2 deletion mutants are strict glutamate auxotrophs (Gangloff et al., 1990; Kim et al., 1986; Liao & Butow, 1993). Therefore, to determine whether the putative fungal RTG2 homologs encode functional Rtg2p counterparts, we first tested their ability to complement the glutamate auxotrophy of rtg2Δ. When expressed from the endogenous RTG2 promoter, all Rtg2p homologs complemented growth on media lacking glutamate except for RTG2 from A. gossypii (Fig. 1a). Given that all homologs were expressed from the S. cerevisiae RTG2 promoter rather than their endogenous promoters, differences in promoter recognition is not likely a source of this noncomplementation. To determine whether enhanced expression improves complementation, we retested glutamate auxotrophy using RTG2 homologs expressed from the constitutive GPD promoter (Fig. 1b). Under these conditions, all Rtg2p homologs complemented the growth of rtg2Δ, suggesting that all homologs are functional for glutamate auxotrophy in S. cerevisiae. Western blot analysis confirmed the expression level of Rtg2p from A. gossypii was reduced relative to the levels of other Rtg2p homologs (Fig. S1). Quantitative PCR and protein half-life studies suggest that neither a reduction in mRNA levels nor increased protein turnover are likely explanations for decreased A. gossypii Rtg2p levels (data not shown). The potential of a translational mechanism of regulation influencing the expression of A. gossypii Rtg2p in the S. cerevisiae remains to be determined.


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

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

Complementation of rtg2Δ glutamate auxotrophy by Rtg2p homologs. 10-fold serial dilutions of cells expressing the indicated RTG2 genes were spotted to -Trp synthetic dropout media with and without glutamate. Growth was scored after 3 days of growth at 30 °C. Complementation profiles for Rtg2p homologs expressed from the native RTG2 promoter are shown in (a); complementation profiles for Rtg2p homologs expressed from the constitutive GPD promoter are shown in (b).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig01: Complementation of rtg2Δ glutamate auxotrophy by Rtg2p homologs. 10-fold serial dilutions of cells expressing the indicated RTG2 genes were spotted to -Trp synthetic dropout media with and without glutamate. Growth was scored after 3 days of growth at 30 °C. Complementation profiles for Rtg2p homologs expressed from the native RTG2 promoter are shown in (a); complementation profiles for Rtg2p homologs expressed from the constitutive GPD promoter are shown in (b).
Mentions: In cells defective for electron transport activity, the retrograde signaling pathway has been linked to cellular glutamate homeostasis through its control of the expression of the first three enzymes of the Krebs cycle (Liu & Butow, 1999). This RTG regulatory pathway functions to increase α-ketoglutarate production ensuring a sufficient supply of glutamate for various biosynthetic reactions. Like cells deleted for cit1 cit2, or aco1, rtg2 deletion mutants are strict glutamate auxotrophs (Gangloff et al., 1990; Kim et al., 1986; Liao & Butow, 1993). Therefore, to determine whether the putative fungal RTG2 homologs encode functional Rtg2p counterparts, we first tested their ability to complement the glutamate auxotrophy of rtg2Δ. When expressed from the endogenous RTG2 promoter, all Rtg2p homologs complemented growth on media lacking glutamate except for RTG2 from A. gossypii (Fig. 1a). Given that all homologs were expressed from the S. cerevisiae RTG2 promoter rather than their endogenous promoters, differences in promoter recognition is not likely a source of this noncomplementation. To determine whether enhanced expression improves complementation, we retested glutamate auxotrophy using RTG2 homologs expressed from the constitutive GPD promoter (Fig. 1b). Under these conditions, all Rtg2p homologs complemented the growth of rtg2Δ, suggesting that all homologs are functional for glutamate auxotrophy in S. cerevisiae. Western blot analysis confirmed the expression level of Rtg2p from A. gossypii was reduced relative to the levels of other Rtg2p homologs (Fig. S1). Quantitative PCR and protein half-life studies suggest that neither a reduction in mRNA levels nor increased protein turnover are likely explanations for decreased A. gossypii Rtg2p levels (data not shown). The potential of a translational mechanism of regulation influencing the expression of A. gossypii Rtg2p in the S. cerevisiae remains to be determined.

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