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The GCKIII kinase Sps1 and the 14-3-3 isoforms, Bmh1 and Bmh2, cooperate to ensure proper sporulation in Saccharomyces cerevisiae.

Slubowski CJ, Paulissen SM, Huang LS - PLoS ONE (2014)

Bottom Line: This interaction is significant, as BMH1 and BMH2 are required during sporulation and genetically interact with SPS1 in sporulating cells.We identify a nuclear localization sequence in Sps1 at amino acids 411-415, and show that this sequence is necessary and sufficient for nuclear localization.Taken together, these data identify regions within Sps1 critical for its function and indicate that SPS1 and 14-3-3s act together to promote proper sporulation in S. cerevisiae.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Massachusetts Boston, Boston, Massachusetts, United States of America.

ABSTRACT
Sporulation in the budding yeast Saccharomyces cerevisiae is a developmental program initiated in response to nutritional deprivation. Sps1, a serine/threonine kinase, is required for sporulation, but relatively little is known about the molecular mechanisms through which it regulates this process. Here we show that SPS1 encodes a bona-fide member of the GCKIII subfamily of STE20 kinases, both through phylogenetic analysis of the kinase domain and examination of its C-terminal regulatory domain. Within the regulatory domain, we find Sps1 contains an invariant ExxxPG region conserved from plant to human GCKIIIs that we call the EPG motif; we show this EPG motif is important for SPS1 function. We also find that Sps1 is phosphorylated near its N-terminus on Threonine 12, and that this phosphorylation is required for the efficient production of spores. In Sps1, Threonine 12 lies within a 14-3-3 consensus binding sequence, and we show that the S. cerevisiae 14-3-3 proteins Bmh1 and Bmh2 bind Sps1 in a Threonine 12-dependent fashion. This interaction is significant, as BMH1 and BMH2 are required during sporulation and genetically interact with SPS1 in sporulating cells. Finally, we observe that Sps1, Bmh1 and Bmh2 are present in both the nucleus and cytoplasm during sporulation. We identify a nuclear localization sequence in Sps1 at amino acids 411-415, and show that this sequence is necessary and sufficient for nuclear localization. Taken together, these data identify regions within Sps1 critical for its function and indicate that SPS1 and 14-3-3s act together to promote proper sporulation in S. cerevisiae.

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Bmh1 and Bmh2 protein expression and localization during sporulation.(A) Immunoblot probed with anti-Bmh antibody, showing Bmh1 and Bmh2 expression during sporulation using LH177 (wild type). Pgk1 was used as a loading control. (B) Quantification of Bmh isoform ratio during log-phase growth and sporulation. LH971 (BMH1-GFP BMH2-GFP) was sampled during log-phase growth and at 8 hours into sporulation. Immunoblot was probed with anti-GFP antibody and band intensities were measured. (C) Localization of Bmh1-GFP in a wild type background (LH972; top) and in an sps1Δ background (LH974; bottom) during sporulation. Htb2-mCherry is used as a nuclear marker. PreM: Pre-meiosis, MI: Meiosis I, MII: Meiosis II, Spore: mature spore. Scale bar = 2 µ.
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pone-0113528-g005: Bmh1 and Bmh2 protein expression and localization during sporulation.(A) Immunoblot probed with anti-Bmh antibody, showing Bmh1 and Bmh2 expression during sporulation using LH177 (wild type). Pgk1 was used as a loading control. (B) Quantification of Bmh isoform ratio during log-phase growth and sporulation. LH971 (BMH1-GFP BMH2-GFP) was sampled during log-phase growth and at 8 hours into sporulation. Immunoblot was probed with anti-GFP antibody and band intensities were measured. (C) Localization of Bmh1-GFP in a wild type background (LH972; top) and in an sps1Δ background (LH974; bottom) during sporulation. Htb2-mCherry is used as a nuclear marker. PreM: Pre-meiosis, MI: Meiosis I, MII: Meiosis II, Spore: mature spore. Scale bar = 2 µ.

Mentions: We examined Bmh1 and Bmh2 expression during sporulation in wild type cells, and found that both are expressed throughout sporulation (Figure 5A). As Bmh1 was previously reported to be the major isoform in vegetative growth [35], [65], we compared the levels of expression of Bmh1 and Bmh2 during log-phase growth and sporulation. We used the strain BMH1-GFP BMH2-GFP and examined the protein levels using an anti-GFP antibody to avoid any bias in isoform detection by the anti-Bmh antibody. We found that the ratio of Bmh1 to Bmh2 in log-phase growth was 3.4±0.2 (mean ± S.D.), decreasing to 1.7±0.2 (mean ± S.D.) in sporulating cells. This suggests that Bmh1 is more abundant than Bmh2 during log-phase growth, but that Bmh2 levels increase relative to Bmh1 in sporulating cells (Figure 5B).


The GCKIII kinase Sps1 and the 14-3-3 isoforms, Bmh1 and Bmh2, cooperate to ensure proper sporulation in Saccharomyces cerevisiae.

Slubowski CJ, Paulissen SM, Huang LS - PLoS ONE (2014)

Bmh1 and Bmh2 protein expression and localization during sporulation.(A) Immunoblot probed with anti-Bmh antibody, showing Bmh1 and Bmh2 expression during sporulation using LH177 (wild type). Pgk1 was used as a loading control. (B) Quantification of Bmh isoform ratio during log-phase growth and sporulation. LH971 (BMH1-GFP BMH2-GFP) was sampled during log-phase growth and at 8 hours into sporulation. Immunoblot was probed with anti-GFP antibody and band intensities were measured. (C) Localization of Bmh1-GFP in a wild type background (LH972; top) and in an sps1Δ background (LH974; bottom) during sporulation. Htb2-mCherry is used as a nuclear marker. PreM: Pre-meiosis, MI: Meiosis I, MII: Meiosis II, Spore: mature spore. Scale bar = 2 µ.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4237420&req=5

pone-0113528-g005: Bmh1 and Bmh2 protein expression and localization during sporulation.(A) Immunoblot probed with anti-Bmh antibody, showing Bmh1 and Bmh2 expression during sporulation using LH177 (wild type). Pgk1 was used as a loading control. (B) Quantification of Bmh isoform ratio during log-phase growth and sporulation. LH971 (BMH1-GFP BMH2-GFP) was sampled during log-phase growth and at 8 hours into sporulation. Immunoblot was probed with anti-GFP antibody and band intensities were measured. (C) Localization of Bmh1-GFP in a wild type background (LH972; top) and in an sps1Δ background (LH974; bottom) during sporulation. Htb2-mCherry is used as a nuclear marker. PreM: Pre-meiosis, MI: Meiosis I, MII: Meiosis II, Spore: mature spore. Scale bar = 2 µ.
Mentions: We examined Bmh1 and Bmh2 expression during sporulation in wild type cells, and found that both are expressed throughout sporulation (Figure 5A). As Bmh1 was previously reported to be the major isoform in vegetative growth [35], [65], we compared the levels of expression of Bmh1 and Bmh2 during log-phase growth and sporulation. We used the strain BMH1-GFP BMH2-GFP and examined the protein levels using an anti-GFP antibody to avoid any bias in isoform detection by the anti-Bmh antibody. We found that the ratio of Bmh1 to Bmh2 in log-phase growth was 3.4±0.2 (mean ± S.D.), decreasing to 1.7±0.2 (mean ± S.D.) in sporulating cells. This suggests that Bmh1 is more abundant than Bmh2 during log-phase growth, but that Bmh2 levels increase relative to Bmh1 in sporulating cells (Figure 5B).

Bottom Line: This interaction is significant, as BMH1 and BMH2 are required during sporulation and genetically interact with SPS1 in sporulating cells.We identify a nuclear localization sequence in Sps1 at amino acids 411-415, and show that this sequence is necessary and sufficient for nuclear localization.Taken together, these data identify regions within Sps1 critical for its function and indicate that SPS1 and 14-3-3s act together to promote proper sporulation in S. cerevisiae.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Massachusetts Boston, Boston, Massachusetts, United States of America.

ABSTRACT
Sporulation in the budding yeast Saccharomyces cerevisiae is a developmental program initiated in response to nutritional deprivation. Sps1, a serine/threonine kinase, is required for sporulation, but relatively little is known about the molecular mechanisms through which it regulates this process. Here we show that SPS1 encodes a bona-fide member of the GCKIII subfamily of STE20 kinases, both through phylogenetic analysis of the kinase domain and examination of its C-terminal regulatory domain. Within the regulatory domain, we find Sps1 contains an invariant ExxxPG region conserved from plant to human GCKIIIs that we call the EPG motif; we show this EPG motif is important for SPS1 function. We also find that Sps1 is phosphorylated near its N-terminus on Threonine 12, and that this phosphorylation is required for the efficient production of spores. In Sps1, Threonine 12 lies within a 14-3-3 consensus binding sequence, and we show that the S. cerevisiae 14-3-3 proteins Bmh1 and Bmh2 bind Sps1 in a Threonine 12-dependent fashion. This interaction is significant, as BMH1 and BMH2 are required during sporulation and genetically interact with SPS1 in sporulating cells. Finally, we observe that Sps1, Bmh1 and Bmh2 are present in both the nucleus and cytoplasm during sporulation. We identify a nuclear localization sequence in Sps1 at amino acids 411-415, and show that this sequence is necessary and sufficient for nuclear localization. Taken together, these data identify regions within Sps1 critical for its function and indicate that SPS1 and 14-3-3s act together to promote proper sporulation in S. cerevisiae.

Show MeSH