1. In eukaryotic microorganisms, the PHO signaling pathway regulates the expression of certain genes. These genes, Pho target genes, encode proteins involved in regulating phosphate homeostasis. When the level of extracellular inorganic phosphate (Pi) is high, a transcriptional activator Pho4 is phosphorylated by a complex of two proteins, Pho80-Pho85. As a result, the Pho target genes are not expressed. When the level of extracellular $\mathrm{Pi}$ is low, the activity of the Pho80-Pho85 complex is inhibited by another protein, Pho81, enabling Pho4 to induce the expression of these target genes. A simplified model of this pathway is shown in Figure 1.
A High-Phosphate Environment
B
Low-Phosphate Environment
Figure 1. A simplified model of the regulation of expression of Pho target genes in (A) a high-phosphate (high-Pi) environment and (B) a low-phosphate (low-Pi) environment
To study the role of the different proteins in the PHO pathway, researchers used a wild-type strain of yeast to create a strain with a mutant form of Pho81 (pho81mt) and a strain with a mutant form of Pho4 (pho4mt). In each of these mutant strains, researchers measured the activity of a particular enzyme, APase, which removes phosphates from its substrates and is encoded by PHO1, a Pho target gene (Table 1). They then determined the level of PHO1 mRNA relative to that of the wild-type yeast strain, which was set to 10.
TABLE 1. APase ACTIVITY AND RELATIVE AMOUNTS OF PHO1 mRNA IN WILD-TYPE AND MUTANT STRAINS OF YEAST IN HIGH- AND LOW-PHOSPHATE ENVIRONMENTS
\begin{tabular}{|c|c|c|c|c|c|}
\hline Yeast Strain & Mutation & & \begin{tabular}{l}
APase Activity in \
Low-Pi Environment \
$\left(\mathrm{mU} / \mathrm{mL}^{\prime} \mathrm{OD}_{600} ight)$ \
$\pm 2 \mathrm{SE}_{\bar{x}}$
\end{tabular} & \begin{tabular}{l}
Relative Amounts of \
PHO1 mRNA in \
High-Pi Environment \
$\pm 2 \mathrm{SE}_{\bar{x}}$
\end{tabular} & \begin{tabular}{l}
Relative Amounts of \
PHO1 mRNA in \
Low-Pi Environment \
$\pm 2 \mathrm{SE}_{\bar{x}}$
\end{tabular} \
\hline Wild-type & None & $0.5 \pm 0.1$ & $17.3 \pm 0.9$ & $0.1 \pm 0.0$ & $10 \pm 2.0$ \
\hline pho81mt & \begin{tabular}{|c|}
\begin{tabular}{l}
Nonfunctional \
Pho81
\end{tabular} \
\end{tabular} & $0.4 \pm 0.1$ & $0.6 \pm 0.1$ & $0.7 \pm 0.2$ & $0.9 \pm 0.8$ \
\hline pho4mt & \begin{tabular}{|c|}
\begin{tabular}{c}
Nonfunctional \
Pho4
\end{tabular} \
\end{tabular} & $0.5 \pm 0.0$ & $0.8 \pm 0.2$ & $0.6 \pm 0.4$ & $0.3 \pm 0.1$ \
\hline
\end{tabular}
(a) Describe the effect that the addition of a charged phosphate group can have on a protein that would cause the protein to become inactive. Explain how a signal can be amplified during signal transduction in a pathway such as the $\mathrm{PHO}$ signaling pathway.
(b) Based on Table 1, identify a dependent variable in the researchers' experiment. Justify the researchers' using the wild-type strain for the creation of the mutant strains. Justify the researchers' using mutant strains in which only a single component of the pathway was mutated in each strain.
(c) Based on the data in Table 1, identify the yeast strain and growth conditions that lead to the highest relative amount of PHOI mRNA. Calculate the percent change in APase activity in wild-type yeast cells in a high-Pi environment compared with that of wild-type cells in a low-Pi environment.
(d) In a follow-up experiment, researchers created a strain of yeast with a mutation that resulted in a nonfunctional Pho85 protein. Based on Figure 1, predict the effects of this mutation on PHOI expression in the mutant strain in a high-Pi environment. Provide reasoning to justify your prediction.

Question

1. In eukaryotic microorganisms, the PHO signaling pathway regulates the expression of certain genes. These genes, Pho target genes, encode proteins involved in regulating phosphate homeostasis. When the level of extracellular inorganic phosphate (Pi) is high, a transcriptional activator Pho4 is phosphorylated by a complex of two proteins, Pho80-Pho85. As a result, the Pho target genes are not expressed. When the level of extracellular $\mathrm{Pi}$ is low, the activity of the Pho80-Pho85 complex is inhibited by another protein, Pho81, enabling Pho4 to induce the expression of these target genes. A simplified model of this pathway is shown in Figure 1.
A High-Phosphate Environment
B
Low-Phosphate Environment
Figure 1. A simplified model of the regulation of expression of Pho target genes in (A) a high-phosphate (high-Pi) environment and (B) a low-phosphate (low-Pi) environment
To study the role of the different proteins in the PHO pathway, researchers used a wild-type strain of yeast to create a strain with a mutant form of Pho81 (pho81mt) and a strain with a mutant form of Pho4 (pho4mt). In each of these mutant strains, researchers measured the activity of a particular enzyme, APase, which removes phosphates from its substrates and is encoded by PHO1, a Pho target gene (Table 1). They then determined the level of PHO1 mRNA relative to that of the wild-type yeast strain, which was set to 10.
TABLE 1. APase ACTIVITY AND RELATIVE AMOUNTS OF PHO1 mRNA IN WILD-TYPE AND MUTANT STRAINS OF YEAST IN HIGH- AND LOW-PHOSPHATE ENVIRONMENTS
\begin{tabular}{|c|c|c|c|c|c|}
\hline Yeast Strain & Mutation & & \begin{tabular}{l} 
APase Activity in \
Low-Pi Environment \
$\left(\mathrm{mU} / \mathrm{mL}^{\prime} \mathrm{OD}_{600}ight)$ \
$\pm 2 \mathrm{SE}_{\bar{x}}$
\end{tabular} & \begin{tabular}{l} 
Relative Amounts of \
PHO1 mRNA in \
High-Pi Environment \
$\pm 2 \mathrm{SE}_{\bar{x}}$
\end{tabular} & \begin{tabular}{l} 
Relative Amounts of \
PHO1 mRNA in \
Low-Pi Environment \
$\pm 2 \mathrm{SE}_{\bar{x}}$
\end{tabular} \
\hline Wild-type & None & $0.5 \pm 0.1$ & $17.3 \pm 0.9$ & $0.1 \pm 0.0$ & $10 \pm 2.0$ \
\hline pho81mt & \begin{tabular}{|c|}
\begin{tabular}{l} 
Nonfunctional \
Pho81
\end{tabular} \
\end{tabular} & $0.4 \pm 0.1$ & $0.6 \pm 0.1$ & $0.7 \pm 0.2$ & $0.9 \pm 0.8$ \
\hline pho4mt & \begin{tabular}{|c|}
\begin{tabular}{c} 
Nonfunctional \
Pho4
\end{tabular} \
\end{tabular} & $0.5 \pm 0.0$ & $0.8 \pm 0.2$ & $0.6 \pm 0.4$ & $0.3 \pm 0.1$ \
\hline
\end{tabular}
(a) Describe the effect that the addition of a charged phosphate group can have on a protein that would cause the protein to become inactive. Explain how a signal can be amplified during signal transduction in a pathway such as the $\mathrm{PHO}$ signaling pathway.
(b) Based on Table 1, identify a dependent variable in the researchers' experiment. Justify the researchers' using the wild-type strain for the creation of the mutant strains. Justify the researchers' using mutant strains in which only a single component of the pathway was mutated in each strain.
(c) Based on the data in Table 1, identify the yeast strain and growth conditions that lead to the highest relative amount of PHOI mRNA. Calculate the percent change in APase activity in wild-type yeast cells in a high-Pi environment compared with that of wild-type cells in a low-Pi environment.
(d) In a follow-up experiment, researchers created a strain of yeast with a mutation that resulted in a nonfunctional Pho85 protein. Based on Figure 1, predict the effects of this mutation on PHOI expression in the mutant strain in a high-Pi environment. Provide reasoning to justify your prediction.

Accepted Answer

∻a Answer∻ ⚹ The addition of a charged phosphate group can cause a protein to become inactive by inducing a conformational change that inhibits its function. Signal amplification occurs through a cascade of events where one activated molecule activates multiple downstream molecules. ⚹ ∻Solution∻ ‖ a ⋮ The addition of a charged phosphate group (phosphorylation) to a protein can alter its shape or charge, leading to a conformational change that can inhibit the protein's function by affecting its ability to interact with other molecules or substrates. Signal transduction pathways, like the PHO signaling pathway, can amplify a signal through a series of steps where each activated molecule activates several downstream molecules, leading to a large cellular response from a small initial signal. ‖ ∻Key Concept∻ ⚹ Phosphorylation and Signal Amplification ⚹ ∻Explanation∻ ⚹ Phosphorylation can inactivate proteins by changing their structure, and signal amplification in pathways occurs when one signal molecule activates multiple downstream molecules, creating a large-scale response. ⚹ ∻b Answer∻ ⚹ The dependent variable in the researchers' experiment is the APase activity and the relative amounts of PHO1 mRNA. The wild-type strain is used as a control to compare the effects of mutations, and single-component mutations allow for the specific study of each protein's role in the pathway. ⚹ ∻Solution∻ ‖ b ⋮ The dependent variables are the APase activity and the relative amounts of PHO1 mRNA, as these are the outcomes measured in response to the experimental conditions. Using the wild-type strain as a baseline for creating mutant strains ensures that any observed differences in APase activity or PHO1 mRNA levels are due to the introduced mutations rather than genetic variability. Mutating only a single component of the pathway in each strain allows researchers to isolate the specific contribution of that component to the pathway's overall function. ‖ ∻Key Concept∻ ⚹ Dependent Variable and Control in Experiments ⚹ ∻Explanation∻ ⚹ The dependent variable is what is measured in response to changes in the experiment, and using a wild-type control allows for comparison. Single mutations enable the study of individual pathway components. ⚹ ∻c Answer∻ ⚹ The wild-type yeast strain in a low-Pi environment leads to the highest relative amount of PHO1 mRNA. The percent change in APase activity in wild-type yeast cells in a high-Pi environment compared with a low-Pi environment is 3200%. ⚹ ∻Solution∻ ‖ c ⋮ The highest relative amount of PHO1 mRNA is observed in the wild-type strain under low-Pi conditions (10 ± 2.0). To calculate the percent change in APase activity, use the formula: $ ext{Percent Change} = \frac{ ext{New Value} - ext{Old Value}}{ ext{Old Value}} imes 100\%$. For APase activity, the new value is the activity in a low-Pi environment (17.3 mU/mL/OD600) and the old value is the activity in a high-Pi environment (0.5 mU/mL/OD600). Thus, the percent change is $\frac{17.3 - 0.5}{0.5} imes 100\% = 3200\%$. ‖ ∻Key Concept∻ ⚹ Highest PHO1 mRNA Expression and Percent Change Calculation ⚹ ∻Explanation∻ ⚹ The wild-type strain in low-Pi conditions shows the highest PHO1 mRNA expression, and the percent change in APase activity is calculated by comparing the enzyme activity in different Pi environments. ⚹ ∻d Answer∻ ⚹ A nonfunctional Pho85 protein in a high-Pi environment would likely lead to increased PHO1 expression, as Pho85 is part of the complex that inhibits Pho4, and without its function, Pho4 would remain active and promote gene expression. ⚹ ∻Solution∻ ‖ d ⋮ Based on Figure 1, in a high-Pi environment, Pho85 normally forms a complex with Pho80 to phosphorylate and inhibit Pho4, preventing the expression of PHO1. If Pho85 is nonfunctional, the Pho80-Pho85 complex cannot form, and Pho4 would not be phosphorylated and inhibited. As a result, Pho4 would remain active and could induce the expression of PHO1, leading to increased PHO1 mRNA and APase activity. ‖ ∻Key Concept∻ ⚹ Effects of Pho85 Mutation ⚹ ∻Explanation∻ ⚹ A mutation resulting in nonfunctional Pho85 disrupts the inhibition of Pho4, leading to increased expression of PHO1 in a high-Pi environment. ⚹◊What is the role of Pho81 in the PHO signaling pathway in regulating the expression of Pho target genes in yeast?⍭ Generate me a similar question◊

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