8. In genetic screens of mutagenized yeast, temperature sensitive mutants defective in thesecretory pathway (sec mutants) were identified. These mutants have helped scientistsstudy the protein secretion pathway that is necessary/essential for life in eukaryoticcells.To identify sec mutants: mutagenized yeast cells were grown for 12 hours at lowtemperature, shifted to higher temperature for two hours, and then the population ofcells were centrifuged in a density gradient.a. Mutagenized cells were first grown at the lower temperature and then shifted tothe higher temperature.i. Why was the shift in temperature an important step of the experimentalprotocol?ii. Why would the experiment have been unsuccessful if the cells weregrown exclusively at the higher temperature?b. After centrifugation, where would sec mutant cells be located compared to othercells in the population? Briefly explain why.

The shift from low temperature to high temperature is an important step in identifying secretory mutants in yeast because it can reveal defects in the yeast's ability to secrete proteins properly. This temperature shift is commonly used in yeast genetics and research for the following reasons:1. Temperature Sensitivity of Protein Folding: Yeast, like many other eukaryotic organisms, has specific temperature-sensitive mutants. These mutants have mutations that affect the stability and proper folding of proteins at high temperatures. When yeast is shifted from a permissive (lower) temperature to a restrictive (higher) temperature, these temperature-sensitive mutants may exhibit defects in protein folding and function.2. Secretory Pathway Disruption: Proteins destined for secretion, such as those meant to be released from the cell, are typically synthesized and folded in the endoplasmic reticulum (ER) before being transported through the secretory pathway to their final destination. The transition to a higher temperature can disrupt the function of temperature-sensitive proteins involved in the secretory pathway. This disruption can lead to the accumulation of misfolded or unfolded proteins within the ER, activating the unfolded protein response (UPR).3. Activation of Stress Responses: The shift to high temperature can induce stress responses in yeast cells. The ER stress response, for example, is triggered by the accumulation of unfolded or misfolded proteins in the ER. This response can be monitored by assessing the expression of specific reporter genes. Mutants that are defective in the secretory pathway may activate this response when subjected to a temperature shift, making it easier to identify them.4. Identification of Secretory Mutants: By observing the growth and behavior of yeast mutants at high temperature, researchers can identify those with defects in the secretory pathway or protein secretion. Temperature-sensitive secretory mutants may exhibit reduced growth, impaired protein secretion, or other observable phenotypic changes at the restrictive temperature.Overall, the temperature shift from low to high is a useful method for identifying yeast mutants with defects in the secretory pathway. It helps researchers study the cellular mechanisms involved in protein folding, secretion, and the response to stress, shedding light on fundamental biological processes and providing insights into potential therapeutic targets for various diseases.Identifying secretory mutants in yeast by growing cells exclusively at a high temperature can fail for several reasons:1. Masking of Mutant Phenotype: Mutations in yeast that affect the secretory pathway may be temperature-sensitive. This means that they exhibit their defective phenotype (e.g., impaired protein secretion) only at certain temperatures. At high temperatures, the yeast may be unable to grow or exhibit other non-specific stress responses, which could mask the specific defects in protein secretion that researchers are trying to identify.2. Lack of Temperature Discrimination: Using a temperature shift from low to high temperature is a specific and controlled way to activate potential secretory defects. It allows researchers to distinguish between the mutant's normal functioning at a lower temperature and its compromised function at the higher temperature. Growing yeast at a constant high temperature will not provide this separation.3. Accumulation of Misfolded Proteins: At a constant high temperature, cells may accumulate misfolded or aggregated proteins in the endoplasmic reticulum (ER) due to the increased stress. However, it may be challenging to determine whether this accumulation is due to defects in the secretory pathway or solely a result of the stress response to the high temperature.4. Loss of Cell Viability: High temperatures can be detrimental to yeast cells, leading to reduced viability and cell death. This could lead to the loss of the yeast culture, making it difficult to perform experiments over an extended period.5. Incomplete Understanding of the Mutation: If the mutation's temperature sensitivity is not well-characterized, growing yeast exclusively at a high temperature may not reveal the relevant phenotypic defects. The mutation may only affect specific processes at particular temperatures, and these effects would be missed in a constant high-temperature environment.To effectively identify secretory mutants in yeast, it is crucial to subject the cells to temperature shifts, allowing researchers to control the timing and conditions under which the defects are manifested. This approach provides a more accurate and controlled way to study the impact of mutations on the secretory pathway and ensures that the observed phenotypes are directly related to the mutation rather than non-specific effects of stress or temperature.b. Location of the sec-mutant cells after centrifugationThe location of sec-mutant cells after centrifugation can vary depending on the specific characteristics of the mutant and the experimental conditions. In a general context, if sec-mutant cells are subject to centrifugation, their location and behaviour can be explained as follows:1. Pellet and Supernatant: - After centrifugation, the sample typically separates into two distinct fractions: the pellet and the supernatant. - The pellet consists of heavier particles, such as intact cells or cell debris, that have sedimented to the bottom of the centrifuge tube. - The supernatant is the liquid portion above the pellet, containing smaller particles, molecules, and soluble substances.2. Location of sec-Mutant Cells: - Sec-mutant cells are yeast cells with mutations affecting the secretory pathway, which may result in impaired protein secretion. - Depending on the specific nature of the sec mutation, sec-mutant cells may exhibit various behaviors after centrifugation: - If the sec mutation leads to a severe defect in protein secretion, some sec-mutant cells may be present in the pellet due to their inability to secrete proteins properly. This could result in the aggregation of mutant cells or the retention of secretory vesicles within the cells. - If the sec mutation is milder and does not significantly impact cell viability or protein secretion, sec-mutant cells may remain in the supernatant along with the wild-type cells.3. Analysis and Interpretation: - The location of sec-mutant cells in the pellet or supernatant is determined by their specific properties and the severity of the secretory defect. - Researchers can analyze the distribution of sec-mutant cells to gain insights into the impact of the mutation on the secretory pathway. For example, if sec-mutant cells are predominantly found in the pellet, it may indicate a severe secretory defect. In contrast, if they are mainly in the supernatant, the mutation may have a milder effect.It's important to note that the behavior of sec-mutant cells after centrifugation is context-specific and depends on the particular mutation and experimental conditions. Researchers typically design experiments to specifically address the characteristics of the sec mutation and its impact on protein secretion. The centrifugation step helps separate different cellular components for further analysis, contributing to a better understanding of the secretory pathway in yeast cells.