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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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Rtg2p homologs have varied affinity for Mks1p. Immunoprecipitation of Mks1p-Myc coprecipitates Rtg2p homologs. Pull down assays were carried out on total protein lysates isolated from cells grown in the presence (a) or absence (b) of glutamate. The specificity of Mks1p–Rtg2 interaction was confirmed using cells expressing an untagged version of Mks1p (c). Cells were processed by glass bead lysis followed by immunoprecipitation of Mks1p using anti-Myc affinity beads. Total (T), unbound (U), and immunoprecipitated (B) Mks1p and the corresponding Rtg2p protein levels were detected by immunoblotting using antibodies to the Myc and HA epitopes to detect Mks1p and Rtg2p, respectively. Total and unbound samples represent 10% of the total lysate, while bound is 87% of total lysate.
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fig04: Rtg2p homologs have varied affinity for Mks1p. Immunoprecipitation of Mks1p-Myc coprecipitates Rtg2p homologs. Pull down assays were carried out on total protein lysates isolated from cells grown in the presence (a) or absence (b) of glutamate. The specificity of Mks1p–Rtg2 interaction was confirmed using cells expressing an untagged version of Mks1p (c). Cells were processed by glass bead lysis followed by immunoprecipitation of Mks1p using anti-Myc affinity beads. Total (T), unbound (U), and immunoprecipitated (B) Mks1p and the corresponding Rtg2p protein levels were detected by immunoblotting using antibodies to the Myc and HA epitopes to detect Mks1p and Rtg2p, respectively. Total and unbound samples represent 10% of the total lysate, while bound is 87% of total lysate.

Mentions: The ability of fungal Rtg2p homologs to associate with the negative regulator Mks1p was tested taking a co-immunoprecipitation approach. When compared with Rtg2p from S. cerevisiae, Rtg2p homologs from C. glabrata, K. lactis, and V. polyspora showed a 50–80% lower affinity for Mks1p (Fig. 4a and Table S3). This reduction in Rtg2p–Mks1p interaction was seen even with induction of retrograde signaling, although the data does suggest a minor enhancement in interaction between the two proteins for all strains (Fig. 4b and Table S3). Interestingly, expression of Rtg2p from V. polyspora showed a similar binding affinity for Mks1p as other Rtg2p homologs despite being unable to complement Cit2p expression (Fig. 2). This suggests that Rtg2p–Mks1p interaction alone may not be sufficient for transmitting a retrograde response. We also found that a significant pool of Rtg2p was not associated with Mks1p. A role of ‘free’ Rtg2p in retrograde signaling is unclear although a number of additional functions for Rtg2p have been uncovered. For example, Rtg2p has been shown to be a component of the nuclear-localized SLIK complex, a SAGA-like histone acetyltransferase; has been implicated in suppressing the formation of extra chromosomal ribosomal circles; has been linked to genome stability through its role as a suppressor of trinucleotide repeat expansion; and has been shown to function as a molecular player in the TOR nutrient signaling pathway (Pray-Grant et al., 2002; Borghouts et al., 2004; Bhattacharyya et al., 2002).


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

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

Rtg2p homologs have varied affinity for Mks1p. Immunoprecipitation of Mks1p-Myc coprecipitates Rtg2p homologs. Pull down assays were carried out on total protein lysates isolated from cells grown in the presence (a) or absence (b) of glutamate. The specificity of Mks1p–Rtg2 interaction was confirmed using cells expressing an untagged version of Mks1p (c). Cells were processed by glass bead lysis followed by immunoprecipitation of Mks1p using anti-Myc affinity beads. Total (T), unbound (U), and immunoprecipitated (B) Mks1p and the corresponding Rtg2p protein levels were detected by immunoblotting using antibodies to the Myc and HA epitopes to detect Mks1p and Rtg2p, respectively. Total and unbound samples represent 10% of the total lysate, while bound is 87% of total lysate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig04: Rtg2p homologs have varied affinity for Mks1p. Immunoprecipitation of Mks1p-Myc coprecipitates Rtg2p homologs. Pull down assays were carried out on total protein lysates isolated from cells grown in the presence (a) or absence (b) of glutamate. The specificity of Mks1p–Rtg2 interaction was confirmed using cells expressing an untagged version of Mks1p (c). Cells were processed by glass bead lysis followed by immunoprecipitation of Mks1p using anti-Myc affinity beads. Total (T), unbound (U), and immunoprecipitated (B) Mks1p and the corresponding Rtg2p protein levels were detected by immunoblotting using antibodies to the Myc and HA epitopes to detect Mks1p and Rtg2p, respectively. Total and unbound samples represent 10% of the total lysate, while bound is 87% of total lysate.
Mentions: The ability of fungal Rtg2p homologs to associate with the negative regulator Mks1p was tested taking a co-immunoprecipitation approach. When compared with Rtg2p from S. cerevisiae, Rtg2p homologs from C. glabrata, K. lactis, and V. polyspora showed a 50–80% lower affinity for Mks1p (Fig. 4a and Table S3). This reduction in Rtg2p–Mks1p interaction was seen even with induction of retrograde signaling, although the data does suggest a minor enhancement in interaction between the two proteins for all strains (Fig. 4b and Table S3). Interestingly, expression of Rtg2p from V. polyspora showed a similar binding affinity for Mks1p as other Rtg2p homologs despite being unable to complement Cit2p expression (Fig. 2). This suggests that Rtg2p–Mks1p interaction alone may not be sufficient for transmitting a retrograde response. We also found that a significant pool of Rtg2p was not associated with Mks1p. A role of ‘free’ Rtg2p in retrograde signaling is unclear although a number of additional functions for Rtg2p have been uncovered. For example, Rtg2p has been shown to be a component of the nuclear-localized SLIK complex, a SAGA-like histone acetyltransferase; has been implicated in suppressing the formation of extra chromosomal ribosomal circles; has been linked to genome stability through its role as a suppressor of trinucleotide repeat expansion; and has been shown to function as a molecular player in the TOR nutrient signaling pathway (Pray-Grant et al., 2002; Borghouts et al., 2004; Bhattacharyya et al., 2002).

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