Dataset Information

Improved Plasmids for Fluorescent Protein Tagging of Microtubules in Saccharomyces cerevisiae.

ABSTRACT: The ability to fluorescently label microtubules in live cells has enabled numerous studies of motile and mitotic processes. Such studies are particularly useful in budding yeast owing to the ease with which they can be genetically manipulated and imaged by live cell fluorescence microscopy. Because of problems associated with fusing genes encoding fluorescent proteins (FPs) to the native ?-tubulin (TUB1) gene, the FP-Tub1 fusion is generally integrated into the genome such that the endogenous TUB1 locus is left intact. Although such modifications have no apparent consequences on cell viability, it is unknown if these genome-integrated FP-tubulin fusions negatively affect microtubule functions. Thus, a simple, economical and highly sensitive assay of microtubule function is required. Furthermore, the current plasmids available for generation of FP-Tub1 fusions have not kept pace with the development of improved FPs. Here, we have developed a simple and sensitive assay of microtubule function that is sufficient to identify microtubule defects that were not apparent by fluorescence microscopy or cell growth assays. Using results obtained from this assay, we have engineered a new family of 30 FP-Tub1 plasmids that use various improved FPs and numerous selectable markers that upon genome integration have no apparent defect on microtubule function.

SUBMITTER: Markus SM

PROVIDER: S-EPMC4795465 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Improved Plasmids for Fluorescent Protein Tagging of Microtubules in Saccharomyces cerevisiae.

Markus Steven M SM Omer Safia S Baranowski Kaitlyn K Lee Wei-Lih WL

Traffic (Copenhagen, Denmark) 20150428 7

The ability to fluorescently label microtubules in live cells has enabled numerous studies of motile and mitotic processes. Such studies are particularly useful in budding yeast owing to the ease with which they can be genetically manipulated and imaged by live cell fluorescence microscopy. Because of problems associated with fusing genes encoding fluorescent proteins (FPs) to the native α-tubulin (TUB1) gene, the FP-Tub1 fusion is generally integrated into the genome such that the endogenous TU ...[more]

PMID: 25711127

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Project description:Protein folding is often considered the flux controlling process in protein synthesis and secretion. Here, two previously isolated Saccharomyces cerevisiae strains with increased α-amylase productivity were analyzed in chemostat cultures at different dilution rates using multi-omics data. Based on the analysis, we identified different routes of the protein folding pathway to improve protein production. In the first strain, the increased abundance of proteins working on the folding process, coordinated with upregulated glycogen metabolism and trehalose metabolism, helped increase α-amylase productivity 1.95-fold compared to the level in the original strain in chemostat culture at a dilution rate of 0.2/h. The second strain further strengthened the folding precision to improve protein production. More precise folding helps the cell improve protein production efficiency and reduce the expenditure of energy on the handling of misfolded proteins. As calculated using an enzyme-constrained genome-scale metabolic model, the second strain had an increased productivity of 2.36-fold with lower energy expenditure than that of the original under the same condition. Further study revealed that the regulation of N-glycans played an important role in the folding precision control and that overexpression of the glucosidase Cwh41p can significantly improve protein production, especially for the strains with improved folding capacity but lower folding precision. Our findings elucidated in detail the mechanisms in two strains having improved protein productivity and thereby provided novel insights for industrial recombinant protein production as well as demonstrating how multi-omics analysis can be used for identification of novel strain-engineering targets.IMPORTANCE Protein folding plays an important role in protein maturation and secretion. In recombinant protein production, many studies have focused on the folding pathway to improve productivity. Here, we identified two different routes for improving protein production by yeast. We found that improving folding precision is a better strategy. Dysfunction of this process is also associated with several aberrant protein-associated human diseases. Here, our findings about the role of glucosidase Cwh41p in the precision control system and the characterization of the strain with a more precise folding process could contribute to the development of novel therapeutic strategies.

Dataset Information

Improved Plasmids for Fluorescent Protein Tagging of Microtubules in Saccharomyces cerevisiae.

Publications

Improved Plasmids for Fluorescent Protein Tagging of Microtubules in Saccharomyces cerevisiae.

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