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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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Cit2p and Aco1p protein profiles in mks1Δ cells expressing Rtg2p homologs. Each strain was grown in the presence or absence of glutamate as indicated. 5 × 106 cells from a mid-log culture were harvested by centrifugation and whole cell extracts prepared by alkaline lysis. Total protein extracts were separated by SDS-PAGE and analyzed by immunoblotting using antibodies against Aco1p, Pgk1p, and the triple-hemagglutin epitope to detect Cit2p. Pgk1p was included as a loading control. All RTG2 homologs were expressed from the native RTG2 promoter.
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fig03: Cit2p and Aco1p protein profiles in mks1Δ cells expressing Rtg2p homologs. Each strain was grown in the presence or absence of glutamate as indicated. 5 × 106 cells from a mid-log culture were harvested by centrifugation and whole cell extracts prepared by alkaline lysis. Total protein extracts were separated by SDS-PAGE and analyzed by immunoblotting using antibodies against Aco1p, Pgk1p, and the triple-hemagglutin epitope to detect Cit2p. Pgk1p was included as a loading control. All RTG2 homologs were expressed from the native RTG2 promoter.

Mentions: The ability of Rtg2p homologs to activate retrograde signaling was tested by measuring CIT2 and ACO1 transcript levels and Cit2p and Aco1p protein levels for cells grown in the absence of glutamate, conditions known to relieve inhibition of this pathway. A modest increase in CIT2 transcript levels over basal conditions was found for cells expressing RTG2 from S. cerevisiae. A similar trend was seen for all RTG2 homologs, except for cells expressing RTG2 from K. lactis, which showed little response to induction conditions. ACO1 transcript levels, on the other hand, showed no consistent response to inducing conditions for any of the strains tested. At the protein level, cells expressing Rtg2p homologs from C. glabrata and K. lactis showed increased expression of Cit2p and Aco1p over basal levels, similar to that detected for S. cerevisiae [Fig. 3; compare lanes 8–9 in (a) and lanes 4–5 in (b) with lanes 4–5 in (a)]. The increased protein levels for K. lactis were unexpected given the absence of a clear change at the transcript level. Although expression of Rtg2p from V. polyspora resulted in transcriptional activation of both Cit2p and Aco1p, the activated levels for these two proteins were lower than that of S. cerevisiae, consistent with the data obtained for CIT2 by qPCR [Fig. 3; compare lane 9 in (b) with lane 5 in (a)]. Note that CIT2 and ACO1 transcript and protein levels could not be determined for rtg2Δ cells and rtg2Δ cells expressing Rtg2p from A. gossypii as these two strains are unable to grow in the absence of glutamate. Mks1p is a negative regulator of retrograde signaling, and deletion of MKS1 has been shown to constitutively activate the pathway, independent of RTG2 expression (Dilova et al., 2002; Dilova et al., 2004). As expected, deletion of MKS1 resulted in elevated levels of Cit2p and Aco1p for all strains regardless of the RTG2 gene expressed.


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

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

Cit2p and Aco1p protein profiles in mks1Δ cells expressing Rtg2p homologs. Each strain was grown in the presence or absence of glutamate as indicated. 5 × 106 cells from a mid-log culture were harvested by centrifugation and whole cell extracts prepared by alkaline lysis. Total protein extracts were separated by SDS-PAGE and analyzed by immunoblotting using antibodies against Aco1p, Pgk1p, and the triple-hemagglutin epitope to detect Cit2p. Pgk1p was included as a loading control. All RTG2 homologs were expressed from the native RTG2 promoter.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig03: Cit2p and Aco1p protein profiles in mks1Δ cells expressing Rtg2p homologs. Each strain was grown in the presence or absence of glutamate as indicated. 5 × 106 cells from a mid-log culture were harvested by centrifugation and whole cell extracts prepared by alkaline lysis. Total protein extracts were separated by SDS-PAGE and analyzed by immunoblotting using antibodies against Aco1p, Pgk1p, and the triple-hemagglutin epitope to detect Cit2p. Pgk1p was included as a loading control. All RTG2 homologs were expressed from the native RTG2 promoter.
Mentions: The ability of Rtg2p homologs to activate retrograde signaling was tested by measuring CIT2 and ACO1 transcript levels and Cit2p and Aco1p protein levels for cells grown in the absence of glutamate, conditions known to relieve inhibition of this pathway. A modest increase in CIT2 transcript levels over basal conditions was found for cells expressing RTG2 from S. cerevisiae. A similar trend was seen for all RTG2 homologs, except for cells expressing RTG2 from K. lactis, which showed little response to induction conditions. ACO1 transcript levels, on the other hand, showed no consistent response to inducing conditions for any of the strains tested. At the protein level, cells expressing Rtg2p homologs from C. glabrata and K. lactis showed increased expression of Cit2p and Aco1p over basal levels, similar to that detected for S. cerevisiae [Fig. 3; compare lanes 8–9 in (a) and lanes 4–5 in (b) with lanes 4–5 in (a)]. The increased protein levels for K. lactis were unexpected given the absence of a clear change at the transcript level. Although expression of Rtg2p from V. polyspora resulted in transcriptional activation of both Cit2p and Aco1p, the activated levels for these two proteins were lower than that of S. cerevisiae, consistent with the data obtained for CIT2 by qPCR [Fig. 3; compare lane 9 in (b) with lane 5 in (a)]. Note that CIT2 and ACO1 transcript and protein levels could not be determined for rtg2Δ cells and rtg2Δ cells expressing Rtg2p from A. gossypii as these two strains are unable to grow in the absence of glutamate. Mks1p is a negative regulator of retrograde signaling, and deletion of MKS1 has been shown to constitutively activate the pathway, independent of RTG2 expression (Dilova et al., 2002; Dilova et al., 2004). As expected, deletion of MKS1 resulted in elevated levels of Cit2p and Aco1p for all strains regardless of the RTG2 gene expressed.

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