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Updated: Aug 7, 2026
Mating-based Overexpression Library Screening in Yeast
Published on: July 6, 2018
Suppressor analysis of fimbrin (Sac6p) overexpression in yeast
T M Sandrock1, S M Brower, K A Toenjes
1Department of Molecular and Cellular Biology, Life Sciences South, University of Arizona, Tucson, Arizona 85721, USA.
Abstract:
Yeast fimbrin (Sac6p) is an actin filament-bundling protein that is lethal when overexpressed. To identify the basis for this lethality, we sought mutations that can suppress it. A total of 1326 suppressor mutations were isolated and analyzed. As the vast majority of mutations were expected to simply decrease the expression of Sac6p to tolerable levels, a rapid screen was devised to eliminate these mutations. A total of 1324 mutations were found to suppress by reducing levels of Sac6p in the cell. The remaining 2 mutations were both found to be in the actin gene and to make the novel changes G48V (act1-20) and K50E (act1-21). These mutations suppress the defect in cytoskeletal organization and cell morphology seen in ACT1 cells that overexpress SAC6. These findings indicate that the lethal phenotype caused by Sac6p overexpression is mediated through interaction with actin. Moreover, the altered residues lie in the region of actin previously implicated in the binding of Sac6p, and they result in a reduced affinity of actin for Sac6p. These results indicate that the two mutations most likely suppress by reducing the affinity of actin for Sac6p in vivo. This study suggests it should be possible to use this type of suppressor analysis to identify other pairs of physically interacting proteins and suggests that it may be possible to identify sites where such proteins interact with each other.
Insights
Overexpression of yeast fimbrin (Sac6p) is lethal, but two actin mutations (G48V and K50E) suppress this by reducing Sac6p binding. This reveals Sac6p
Area of Science:
- Cell Biology
- Protein Interactions
- Biochemistry
Background:
- Yeast fimbrin (Sac6p) is an essential actin filament-bundling protein.
- Overexpression of Sac6p leads to a lethal phenotype in yeast.
- Understanding the molecular basis of this lethality is crucial for cell biology research.
Purpose of the Study:
- To identify genetic suppressors of Sac6p overexpression lethality.
- To elucidate the interaction between Sac6p and actin.
- To characterize novel mutations in actin that affect Sac6p binding.
Main Methods:
- Isolation and analysis of suppressor mutations.
- Genetic screening to eliminate mutations reducing Sac6p expression.
- Characterization of actin mutations (act1-20 and act1-21) affecting Sac6p interaction.
Main Results:
- 1326 suppressor mutations were identified; 1324 reduced Sac6p levels.
- Two mutations, G48V (act1-20) and K50E (act1-21), were found in the actin gene.
- These actin mutations suppressed Sac6p overexpression lethality by reducing Sac6p-actin binding affinity.
Conclusions:
- The lethal phenotype of Sac6p overexpression is mediated through its interaction with actin.
- Specific residues in actin (G48 and K50) are critical for Sac6p binding.
- Suppressor analysis is a viable method for identifying interacting proteins and their binding sites.
