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Proteome-Wide Fluctuation Analysis Of S.cerevisiae

2009; Elsevier BV; Volume: 96; Issue: 3 Linguagem: Inglês

10.1016/j.bpj.2008.12.1535

1542-0086

Christopher J. Wood, Joseph Huff, Shiqiang Dai, Winfried Wiegraebe,

Biotin and Related Studies

We measured over 40.000 single live yeast S. cerevisiae cells to determine the concentration and diffusion constants of more than 4100 proteins. These proteins account for more than 75% of the yeast proteome. We used Fluorescence Correlation Spectroscopy (FCS), Photon Counting Histograms (PCH), and Brightness & Number analysis (B&N) to analyze the intensity fluctuations of single molecules fused to GFP. The data was collected using a commercial FCS setup attached to a confocal microscope (ConfoCor3 and LSM 510 META, Carl Zeiss Jena GmbH, Germany) controlled by custom software. The cells were imaged in transmitted light. We acquired fluorescence images, using the avalanche photo diodes of the FCS setup. This allows us to determine the localization of proteins, the cell cycle as well as cell health. We developed a software package to automate the measurements and data analysis. We use the Open Microscopy Environment (OME) to organize our images, fluctuation measurements, and analysis results. We calculated the protein copy number per cell, and compare the noise in concentration levels between different proteins in respect to localization and biochemical pathway. We find that the diffusion coefficient for GFP is identical in nucleus and cytosol. But interestingly, most of the proteins localized in the nucleus diffuse slower than proteins localized in the cytoplasm. We will present our data and compare them to information gathered with different methods like flow-cytometry and mass-spectroscopy. We will discuss conclusions derived by complementing our data with information collected in public databases like Saccharomyces Geneome Database (www.yeastgenome.org), Yeast GFP Fusion Localization Database (yeastgfp.ucsf.edu), and the General Repository for Interaction Datasets (www.thebiogrid.org).