title: Adjustable under-expression of yeast mating pathway proteins in Saccharomyces cerevisiae using a programmed ribosomal frameshift.
abstract: Experimental research in molecular biology frequently relies on the promotion or suppression of gene expression, an important tool in the study of its functions. Although yeast is among the most studied model systems with the ease of maintenance and manipulation, current experimental methods are mostly limited to gene deletion, suppression or overexpression of genes. Therefore, the ability to reduce protein expressions and then observing the effects would promote a better understanding of the exact functions and their interactions. Reducing protein expression is mainly limited by the difficulties associated with controlling the reduction level, and in some cases, the initial endogenous abundance is too low. For the under-expression to be useful as an experimental tool, repeatability and stability of reduced expression is important. We found that cis-elements in programmed -1 ribosomal frameshifting (-1RFS) of beet western yellow virus (BWYV) could be utilized to reduced protein expression in Saccharomyces cerevisiae. The two main advantages of using -1RFS are adjustable reduction rates and ease of use. To demonstrate the utility of this under-expression system, examples of reduced protein abundance were shown using yeast mating pathway components. The abundance of MAP kinase Fus3 was reduced to approximately 28-75 % of the wild-type value. Other MAP kinase mating pathway components, including Ste5, Ste11, and Ste7, were also under-expressed to verify that the -1RFS system works with different proteins. Furthermore, reduced Fus3 abundance altered the overall signal transduction outcome of the mating pathway, demonstrating the potential for further studies of signal transduction adjustment via under-expression.
authors.affiliation: Department of Biological Sciences, Seoul National University, Seoul, Republic of Korea.
authors.forename: Min-Yeon
authors.initials: MY
authors.lastname: Choi
authors.affiliation: Department of Biological Sciences, Seoul National University, Seoul, Republic of Korea. biopark@snu.ac.kr.
authors.forename: Sang-Hyun
authors.initials: SH
authors.lastname: Park
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics* / Q000378:metabolism; D018965:Frameshifting, Ribosomal; D015966:Gene Expression Regulation, Fungal; D017793:Luteovirus / Q000235:genetics; D020930:MAP Kinase Kinase Kinases / Q000235:genetics* / Q000378:metabolism; D020929:Mitogen-Activated Protein Kinase Kinases / Q000235:genetics* / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics* / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C080321:STE5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; C075580:FUS3 protein, S cerevisiae; D020928:Mitogen-Activated Protein Kinases; D020930:MAP Kinase Kinase Kinases; C067018:Ste11 protein, S cerevisiae; D020929:Mitogen-Activated Protein Kinase Kinases; C119844:STE7 protein, S cerevisiae
keywords: Protein abundance; Ribosomal frameshift; Saccharomyces cerevisiae; Translational under-expression