Yeast Bax Inhibitor, Bxi1p, Is an ER-Localized Protein that Links the Unfolded Protein Response and Programmed Cell Death in \u3cem\u3eSaccharomyces cerevisiae\u3c/em\u3e

Bax inhibitor-1 (BI-1) is an anti-apoptotic gene whose expression is upregulated in a wide range of human cancers. Studies in both mammalian and plant cells suggest that the BI-1 protein resides in the endoplasmic reticulum and is involved in the unfolded protein response (UPR) that is triggered by ER stress. It is thought to act via a mechanism involving altered calcium dynamics. In this paper, we provide evidence that the Saccharomyces cerevisiae protein encoded by the open reading frame, YNL305C, is a bona fide homolog for BI-1. First, we confirm that yeast cells from two different strain backgrounds lacking YNL305C, which we have renamed BXI1, are more sensitive to heat-shock induced cell death than wildtype controls even though they have indistinguishable growth rates at 30°C. They are also more susceptible both to ethanol-induced and to glucose-induced programmed cell death. Significantly, we show that Bxi1p-GFP colocalizes with the ER localized protein Sec63p-RFP. We have also discovered that Δbxi1 cells are not only more sensitive to drugs that induce ER stress, but also have a decreased unfolded protein response as measured with a UPRE-lacZ reporter. Finally, we have discovered that deleting BXI1 diminishes the calcium signaling response in response to the accumulation of unfolded proteins in the ER as measured by a calcineurin-dependent CDRE-lacZ reporter. In toto, our data suggests that the Bxi1p, like its metazoan homologs, is an ER-localized protein that links the unfolded protein response and programmed cell death

Yeast Bax Inhibitor, Bxi1p, Is an ER-Localized Protein That Links the Unfolded Protein Response and Programmed Cell Death in Saccharomyces cerevisiae

Bax inhibitor-1 (BI-1) is an anti-apoptotic gene whose expression is upregulated in a wide range of human cancers. Studies in both mammalian and plant cells suggest that the BI-1 protein resides in the endoplasmic reticulum and is involved in the unfolded protein response (UPR) that is triggered by ER stress. It is thought to act via a mechanism involving altered calcium dynamics. In this paper, we provide evidence that the Saccharomyces cerevisiae protein encoded by the open reading frame, YNL305C, is a bona fide homolog for BI-1. First, we confirm that yeast cells from two different strain backgrounds lacking YNL305C, which we have renamed BXI1, are more sensitive to heat-shock induced cell death than wildtype controls even though they have indistinguishable growth rates at 30°C. They are also more susceptible both to ethanol-induced and to glucose-induced programmed cell death. Significantly, we show that Bxi1p-GFP colocalizes with the ER localized protein Sec63p-RFP. We have also discovered that Δbxi1 cells are not only more sensitive to drugs that induce ER stress, but also have a decreased unfolded protein response as measured with a UPRE-lacZ reporter. Finally, we have discovered that deleting BXI1 diminishes the calcium signaling response in response to the accumulation of unfolded proteins in the ER as measured by a calcineurin-dependent CDRE-lacZ reporter. In toto, our data suggests that the Bxi1p, like its metazoan homologs, is an ER-localized protein that links the unfolded protein response and programmed cell death

Yeast Bax Inhibitor, Bxi1p, Is an ER-Localized Protein that Links the Unfolded Protein Response and Programmed Cell Death in \u3cem\u3eSaccharomyces cerevisiae\u3c/em\u3e

Bax inhibitor-1 (BI-1) is an anti-apoptotic gene whose expression is upregulated in a wide range of human cancers. Studies in both mammalian and plant cells suggest that the BI-1 protein resides in the endoplasmic reticulum and is involved in the unfolded protein response (UPR) that is triggered by ER stress. It is thought to act via a mechanism involving altered calcium dynamics. In this paper, we provide evidence that the Saccharomyces cerevisiae protein encoded by the open reading frame, YNL305C, is a bona fide homolog for BI-1. First, we confirm that yeast cells from two different strain backgrounds lacking YNL305C, which we have renamed BXI1, are more sensitive to heat-shock induced cell death than wildtype controls even though they have indistinguishable growth rates at 30°C. They are also more susceptible both to ethanol-induced and to glucose-induced programmed cell death. Significantly, we show that Bxi1p-GFP colocalizes with the ER localized protein Sec63p-RFP. We have also discovered that Δbxi1 cells are not only more sensitive to drugs that induce ER stress, but also have a decreased unfolded protein response as measured with a UPRE-lacZ reporter. Finally, we have discovered that deleting BXI1 diminishes the calcium signaling response in response to the accumulation of unfolded proteins in the ER as measured by a calcineurin-dependent CDRE-lacZ reporter. In toto, our data suggests that the Bxi1p, like its metazoan homologs, is an ER-localized protein that links the unfolded protein response and programmed cell death

Wildtype and mutant <i>Δbxi1</i> cells have indistinguishable growth kinetics at 30°C.

<p> Wildtype and <i>Δbxi1</i> cells from the (A) BY4742 and (B) Σ1278b strain backgrounds were streaked on rich YPD plates and cultured at 30°C and at 37°C for two days.</p

Yeast cells lacking <i>BXI1</i> are sensitive to β-mercaptoethanol and tunicamycin.

<p>Wildtype and <i>Δbxi1</i> cells from the BY4742 and Σ1278b strain backgrounds were cultured in rich YPD media overnight. They were then resuspended in fresh media and allowed to reach exponential phase (an approximate OD₆₀₀ value of 0.4). For each strain, a series of 10-fold dilutions was then prepared in water over a range of concentrations from 10⁻¹ to 10⁻⁵ relative to the initial culture. Spots of 5 µl from each dilution series were then plated on the indicated media and cultured at 30°C for 2 days. Plates supplemented with drugs were poured and used on the same day. All spot assays were repeated at least three times and a representative experiment is shown.</p

Deleting <i>BXI1</i> diminishes the unfolded protein response induced by tunicamycin.

<p>Cells of the indicated genotype were transformed with a UPR-lacZ reporter, grown overnight in selective media at 30°C. They were then resuspended in fresh media and allowed to reach exponential phase (an approximate OD₆₀₀ value of 0.4). Next, they were resuspended in fresh media or in fresh media containing 0.6 µg/ml TM and allowed to grow at 30°C for three hours. β-galactosidase assays with the colorimetric substrate <i>o</i>-nitrophenyl-β-D-galactopyranoside (ONPG) were then performed using the permeabilized cell method and quantified as previously described. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020882#pone.0020882-Amberg1" target="_blank">[37]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020882#pone.0020882-Guarente1" target="_blank">[41]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020882#pone.0020882-Guarente2" target="_blank">[42]</a> The differences in β-galactosidase levels were deemed statistically significant by the Student's t-test (p<0.05). Error bars indicate standard deviations for trials with at least three independent transformants.</p

Yeast cells lacking <i>BXI1</i> are more susceptible to ethanol-induced and glucose-induced programmed cell death.

<p>Wildtype and <i>Δbxi1</i> cells from the BY4742 strain background were cultured in rich YPD media overnight and resuspended in fresh media and allowed to reach exponential phase (an approximate OD₆₀₀ value of 0.4). (A) Exponential growing yeast cells were then resuspended in fresh media or in fresh media containing 22% ethanol, allowed to grow at 30°C for the indicated times, serially diluted onto YPD plates, and cultured at 30°C for 2 days. (B) Exponential growing yeast cells were then resuspended in water or in 2% glucose, allowed to grow at 30°C for the indicated times, serially diluted onto YPD plates, and cultured at 30°C for 2 days. The differences in viabilities were deemed statistically significant by the Student's t-test (p<0.05). Error bars indicate standard deviations for trials with at least three independent cultures.</p

Deleting <i>BXI1</i> diminishes calcium signaling in the absence and presence of unfolded proteins in the ER.

<p>Cells of the indicated genotype in the Σ1278b strain background were transformed with a 4XCDRE-lacZ reporter, grown overnight in selective media at 30°C. They were then resuspended in fresh media and allowed to reach exponential phase (an approximate OD₆₀₀ value of 0.4). Next, they were resuspended in fresh media or in fresh media containing 0.6 µg/ml TM and allowed to grow at 30°C for three hours. β-galactosidase assays with the colorimetric substrate <i>o</i>-nitrophenyl-β-D-galactopyranoside (ONPG) were then performed and quantified using the permeabilized cell method as previously described. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020882#pone.0020882-Amberg1" target="_blank">[37]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020882#pone.0020882-Guarente1" target="_blank">[41]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020882#pone.0020882-Guarente2" target="_blank">[42]</a> The differences in β-galactosidase levels were deemed statistically significant by the Student's t-test (p<0.05). Error bars indicate standard deviations for trials with at least three independent transformants.</p

Yeast cells lacking <i>BXI1</i> are sensitive to heat shock.

<p>Wildtype and <i>Δbxi1</i> cells from the BY4742 and Σ1278b strain backgrounds were cultured in rich YPD media overnight. They were then resuspended in fresh media and allowed to reach exponential phase (an approximate OD₆₀₀ value of 0.4). Liquid cultures were then heat shocked at 50°C in a water bath for the indicated times, serially diluted onto YPD plates, and cultured at 30°C for 2 days. The difference in viabilities was deemed statistically significant by the Student's t-test (p<0.05). Error bars indicate standard deviations for trials with at least three independent cultures.</p

Belize: A Modern Example of a Mixed Carbonate-Siliciclastic Shelf

The Belize shelf is located on the eastern side of the Yucatan Peninsula (Fig. 3.1). It extends along approximately 300 km in a north-south direction and 10–40 km in an east-west direction. The Belize shelf lagoon is a mixed carbonate-siliciclastic rimmed platform developed under a humid tropical climate. The southern shelf lagoon depositional system is attached to a mountainous mainland of the Yucatan peninsula. The main relief features are the Maya Mountains that culminate at a height approximating 1,000 m above sea level.Fig. 3.