Frequently Asked Questions

1. What is GFP?
Green Fluorescent Protein S65T (GFP) is a fluorescent protein derived from Aequorea victoria. Its excitation peak is 489nm and its emission peak is 509nm.

2. What is an SGA?
Synthetic Genetic Array (SGA) technology was originally developed at Prof. Charlie Boone’s lab (Tong et al, 2001) and allows an easy insertion of any genetic trait into an entire collection of yeast strains.

3. What are the conditions for maintaining the GFP collection?
The strains are grown in 50 μl SD (0.67% yeast nitrogen base without amino acids [Conda Pronadisa] and 2% dextrose) containing the appropriate supplements for selection in 384-well plates (catalogue No. 781162; Greiner Bio-One).

4. What was the procedure for tagging the proteins?

please refer to:
Huh WK., Falvo JV., Gerke LC., Carroll AS., Howson RW., Weissman JS. & O’Shea EK. (2003) Global analysis of protein localization in budding yeast. Nature, 425(6959):686-91.

5. Which environmental stresses were used for the study?
The three environmental stresses utilized by Breker et al are: hydrogen peroxide (H2O2) treatment (catalogue No. 2186–01; J.T.Baker) at a final concentration of 1 mM for 3-5 hours; nitrogen starvation for which mid-log growing cells were moved into SD (0.17% yeast nitrogen base without amino acids and without ammonium sulfate [Conda Pronadisa] and 2% dextrose) for at least 15 h; and dithreothreitol DTT treatment at final concentration of 2mM for 3-5hours.

6. Which genetic mutations were used for the study?
In the study of the CCT complex, we have mutated Glu to Asp in the ATP binding site of subunits 3 (MA3) (See Nadler-Holy et al in References). We additionally provide unpublished data on the localization changes as a result of the hypomorphic allele of the proteosome subunit Pup2.

7. How was the automated imaging performed?
Liquid cultures were grown (384 well format) in SD medium in a shaking incubator (LiCONiC Instruments) at 30°C. A JANUS liquid handler (PerkinElmer), which is connected to the incubator, was used to back-dilute the strains to mid-log, followed by transferring the strains into glass-bottom 384-well microscope plates (Matrical Bioscience) coated with Concanavalin A (Sigma-Aldrich) to allow cell adhesion. Plates were then transferred into an automated inverted fluorescent microscopic ScanR system (Olympus) using a swap robotic arm (Hamilton). Imaging of plates was performed in 384-well format using a 60x air lens (NA 0.9) in SD medium at 24°C with a cooled charge-coupled device camera (ORCA-ER; Hamamatsu). Images were acquired at GFP (excitation at 490/20 nm, emission at 535/50 nm), mCherry (excitation at 572/35 nm, emission at 632/60 nm) and brightfield channels.

8. How was the image analysis performed?
Our screening assay was designed to explore yeast cell biology by assessing two cellular key features of interest: subcellular localization and fluorescence intensity. To analyze these images we have used an in-house script to browse manually and assign localization rapidly and efficiently. To extract proteomic abundance from images, we used the Olympus ScanR analysis software. This allows for the preprocessing of images by background subtraction, and segmentation of images to identify individual cells as separate objects. For further details please refer to “Methods and Statistics”.

9. How can I get the strains presented?
Please approach