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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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The interaction between Mks1p and Bmh1p is altered in cells expressing Rtg2p homologs. Immunoprecipitation of Bmh1p coprecipitates Mks1p. Pull down assays were carried out using cell extracts isolated from cells grown in the presence (a) or absence (b) of glutamate. The specificity of Bmh1p–Mks1p interaction was confirmed using cells expressing an untagged version of Bmh1p (c). Cells were processed by glass bead lysis followed by immunoprecipitation of Bmh1p using anti-FLAG affinity beads. Total (T), unbound (U), and immunoprecipitated (B) Bmh1p and the corresponding Mks1p protein levels were detected by immunoblotting using antibodies to the FLAG and Myc epitopes to recognize Bmh1p and Mks1p, respectively. All data were taken from the same autoradiograph under the same exposure conditions. Total and unbound samples represent 10% of the total lysates, while bound is 87% of the total lysates.
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fig05: The interaction between Mks1p and Bmh1p is altered in cells expressing Rtg2p homologs. Immunoprecipitation of Bmh1p coprecipitates Mks1p. Pull down assays were carried out using cell extracts isolated from cells grown in the presence (a) or absence (b) of glutamate. The specificity of Bmh1p–Mks1p interaction was confirmed using cells expressing an untagged version of Bmh1p (c). Cells were processed by glass bead lysis followed by immunoprecipitation of Bmh1p using anti-FLAG affinity beads. Total (T), unbound (U), and immunoprecipitated (B) Bmh1p and the corresponding Mks1p protein levels were detected by immunoblotting using antibodies to the FLAG and Myc epitopes to recognize Bmh1p and Mks1p, respectively. All data were taken from the same autoradiograph under the same exposure conditions. Total and unbound samples represent 10% of the total lysates, while bound is 87% of the total lysates.

Mentions: The current model for activation of retrograde signaling proposes that a dynamic interaction exists between Mks1p and Bmh1p and Mks1p and Rtg2p (Liu et al., 2003; Ferreira et al., 2005). To assess whether the decreased interaction between Mks1p and Rtg2p homologs was paralleled by an increased interaction between Mks1p and Bmh1p, a co-immunoprecipitation approach was taken. Mks1p–Bmh1p interaction was measured under both basal (+ glutamate) and inducing (- glutamate) conditions. Cells grown in the presence of glutamate showed a similar interaction affinity between Mks1p and Bmh1p irrespective of the expressed Rtg2p homolog (Fig. 5 and Table S4). Given the inverse relationship dynamics between Mks1p–Rtg2p and Mks1p–Bmh1p complex formation, cells grown under inducing conditions should decrease Mks1p–Bmh1p complex formation to facilitate an increased Mks1p–Rtg2p interaction (Liu et al., 2003). Interestingly, all Rtg2p homologs showed a stronger interaction between Mks1p and Bmh1p compared with that detected for S. cerevisiae RTG2 expressing cells under inducing conditions.


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

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

The interaction between Mks1p and Bmh1p is altered in cells expressing Rtg2p homologs. Immunoprecipitation of Bmh1p coprecipitates Mks1p. Pull down assays were carried out using cell extracts isolated from cells grown in the presence (a) or absence (b) of glutamate. The specificity of Bmh1p–Mks1p interaction was confirmed using cells expressing an untagged version of Bmh1p (c). Cells were processed by glass bead lysis followed by immunoprecipitation of Bmh1p using anti-FLAG affinity beads. Total (T), unbound (U), and immunoprecipitated (B) Bmh1p and the corresponding Mks1p protein levels were detected by immunoblotting using antibodies to the FLAG and Myc epitopes to recognize Bmh1p and Mks1p, respectively. All data were taken from the same autoradiograph under the same exposure conditions. Total and unbound samples represent 10% of the total lysates, while bound is 87% of the total lysates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig05: The interaction between Mks1p and Bmh1p is altered in cells expressing Rtg2p homologs. Immunoprecipitation of Bmh1p coprecipitates Mks1p. Pull down assays were carried out using cell extracts isolated from cells grown in the presence (a) or absence (b) of glutamate. The specificity of Bmh1p–Mks1p interaction was confirmed using cells expressing an untagged version of Bmh1p (c). Cells were processed by glass bead lysis followed by immunoprecipitation of Bmh1p using anti-FLAG affinity beads. Total (T), unbound (U), and immunoprecipitated (B) Bmh1p and the corresponding Mks1p protein levels were detected by immunoblotting using antibodies to the FLAG and Myc epitopes to recognize Bmh1p and Mks1p, respectively. All data were taken from the same autoradiograph under the same exposure conditions. Total and unbound samples represent 10% of the total lysates, while bound is 87% of the total lysates.
Mentions: The current model for activation of retrograde signaling proposes that a dynamic interaction exists between Mks1p and Bmh1p and Mks1p and Rtg2p (Liu et al., 2003; Ferreira et al., 2005). To assess whether the decreased interaction between Mks1p and Rtg2p homologs was paralleled by an increased interaction between Mks1p and Bmh1p, a co-immunoprecipitation approach was taken. Mks1p–Bmh1p interaction was measured under both basal (+ glutamate) and inducing (- glutamate) conditions. Cells grown in the presence of glutamate showed a similar interaction affinity between Mks1p and Bmh1p irrespective of the expressed Rtg2p homolog (Fig. 5 and Table S4). Given the inverse relationship dynamics between Mks1p–Rtg2p and Mks1p–Bmh1p complex formation, cells grown under inducing conditions should decrease Mks1p–Bmh1p complex formation to facilitate an increased Mks1p–Rtg2p interaction (Liu et al., 2003). Interestingly, all Rtg2p homologs showed a stronger interaction between Mks1p and Bmh1p compared with that detected for S. cerevisiae RTG2 expressing cells under inducing conditions.

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