Endoplasmic Reticulum Associated Degradation (ERAD) of Membrane Proteins in Yeast

点击翻译按钮获取中文摘要

Summary/Abstract Funds from NIGMS are requested for the purchase of an Amersham Typhoon IP phosphorimager along with 8 imaging plates and cassettes, an imaging tray to accommodate the plates, and software licenses for data analysis on remote computers. In turn, the University of Pittsburgh and Department of Biological Sciences have committed funds to directly off-set the cost of the imager ($6,000), to purchase the computer (~$2,500), and to provide a long-term maintenance contract. The imager rapidly scans gels and plates up to 34 x 46 cm, produces high resolution figures, yields quantitative data over a long dynamic range, and has state-of-the-art software to accommodate diverse applications. In contrast, results of radioisotope-based experiments in the Brodsky and Berman labs currently rely on an 8-year old GE Typhoon FLA 7000 phosphorimager. This imager uses an older software package that is unlinked from the scanning software and not user-friendly, suffers from mechanical and software failures, and produces lower quality images that restrict data analysis and quantitation. The imager also only accommodates gels that are smaller than 20 x 40 cm. The purchase of the Amersham Typhoon IP phosphorimager is linked to grant R01 GM075061 to Prof. Jeffrey Brodsky and grant R01 GM116889 to Prof. Andrea Berman. The most critical experiments outlined in grant R01 GM075061 to Prof. Brodsky require the use of radioisotopes, which allows for quantitative assessment of the selection, ubiquitination, and degradation of misfolded proteins in the endoplasmic reticulum. Thus, an optimally functioning phosphorimager is essential to achieve each of the project’s goals. Furthermore, an application for a MIRA (R35 GM131732) to Prof. Brodsky was approved for funding with a requested start date of May 1, 2019. The experiments proposed in this recently approved grant application also require the use of radioisotopes and thus a phosphorimager to achieve each of the project’s goals. Together, the procurement of the Amersham Typhoon IP phosphorimager is critical for both the immediate and longer-term goals of Prof. Brodsky’s research program.