Subcellular localization of the yeast proteome

Abstract

Protein localization data are a valuable information resource helpful in elucidating eukaryotic protein function. Here, we report the first proteome-scale analysis of protein localization within any eukaryote. Using directed topoisomerase I-mediated cloning strategies and genome-wide transposon mutagenesis, we have epitope-tagged 60% of the Saccharomyces cerevisiae proteome. By high-throughput immunolocalization of tagged gene products, we have determined the subcellular localization of 2744 yeast proteins. Extrapolating these data through a computational algorithm employing Bayesian formalism, we define the yeast localizome (the subcellular distribution of all 6100 yeast proteins). We estimate the yeast proteome to encompass ∼5100 soluble proteins and >1000 transmembrane proteins. Our results indicate that 47% of yeast proteins are cytoplasmic, 13% mitochondrial, 13% exocytic (including proteins of the endoplasmic reticulum and secretory vesicles), and 27% nuclear/nucleolar. A subset of nuclear proteins was further analyzed by immunolocalization using surface-spread preparations of meiotic chromosomes. Of these proteins, 38% were found associated with chromosomal DNA. As determined from phenotypic analyses of nuclear proteins, 34% are essential for spore viability-a percentage nearly twice as great as that observed for the proteome as a whole. In total, this study presents experimentally derived localization data for 955 proteins of previously unknown function: nearly half of all functionally uncharacterized proteins in yeast. To facilitate access to these data, we provide a searchable database featuring 2900 fluorescent micrographs at http://ygac.med.yale.edu. A global understanding of the molecular mechanisms underpinning cell biology necessitates an understanding not only of an organism's genome but also of the protein complement encoded within this genome (the proteome). In the past, data regarding an organism's proteome have typically been accumulated piecemeal through studies of a single protein or cell pathway. Genomic methodologies have altered this paradigm: a variety of approaches are now in place by which proteins may be directly analyzed on a proteome-wide scale. Chromatography-coupled mass spectrometry (Gygi et al. 1999;Washburn et al. 2001), large-scale two-hybrid screens (Uetz et al. 2000;Ito et al. 2001;Tong et al. 2002), immunoprecipitation/mass spectrometric analysis of protein complexes (Gavin et al. 2002;Ho et al. 2002), and protein microarray technologies (MacBeath and Schreiber 2000;Zhu et al. 2000Zhu et al. , 2001 are yielding unprecedented quantities of protein data. Recent genomic techniques combining microarray technologies with either chromatin immunoprecipitation (Ren et al. 2000;Iyer et al. 2001) or targeted DNA methylation (van Steensel et al. 2001) have been used to globally map binding sites of chromosomal proteins in vivo. Initiatives are even underway to automate and industrialize processes by which protein structures may be solved, potentially providing a library of structural...

Higher sensitivity secondary ion mass spectrometry of biological molecules for high resolution, chemically specific imaging

“…The heterogeneous structure of tissue and cells is apparent to even the most untrained eye. Furthermore, it has been established that the spatial distribution of biomolecules is intrinsically related to their role [1][2][3], and several diseases are associated with biomolecules having altered spatial distributions [4 -6].…”

Bayesian methods for proteomics

“…A year later, using Bayesian reasoning, Kumar et al (including Drawid and Gerstein) created a proteome localizome (localization of proteins) for yeast. They predicted that 47% of proteins were cytoplasmic, 13% mitochondrial, 13% exocytic, and 27% were nuclear [47].…”

Proteome imaging: A closer look at life's organization

“…Impressive advances have been made in this field, that now allow in vivo visualization of cellular biology and the use of clinically important staining techniques [23,24]. Such microscopy studies have already been used to substantiate the intuition that a protein's function is correlated with its localization in the cell [25,26] and to demonstrate that several diseases are associated with altered molecular distributions [27][28][29]. Using targeted fluorescent molecular probes or through the insertion of GFP or equivalent sequences in the organism's genome a particular protein or set of proteins can be labeled for high resolution imaging with confocal microscopy [30].…”