Russell W. Howson

Bio: Russell W. Howson is an academic researcher from University of California, San Francisco. The author has contributed to research in topics: Proteome & MAP2K7. The author has an hindex of 6, co-authored 7 publications receiving 8182 citations.

Global analysis of protein localization in budding yeast

Abstract: A fundamental goal of cell biology is to define the functions of proteins in the context of compartments that organize them in the cellular environment. Here we describe the construction and analysis of a collection of yeast strains expressing full-length, chromosomally tagged green fluorescent protein fusion proteins. We classify these proteins, representing 75% of the yeast proteome, into 22 distinct subcellular localization categories, and provide localization information for 70% of previously unlocalized proteins. Analysis of this high-resolution, high-coverage localization data set in the context of transcriptional, genetic, and protein-protein interaction data helps reveal the logic of transcriptional co-regulation, and provides a comprehensive view of interactions within and between organelles in eukaryotic cells.

Global analysis of protein expression in yeast

Abstract: The availability of complete genomic sequences and technologies that allow comprehensive analysis of global expression profiles of messenger RNA have greatly expanded our ability to monitor the internal state of a cell. Yet biological systems ultimately need to be explained in terms of the activity, regulation and modification of proteins--and the ubiquitous occurrence of post-transcriptional regulation makes mRNA an imperfect proxy for such information. To facilitate global protein analyses, we have created a Saccharomyces cerevisiae fusion library where each open reading frame is tagged with a high-affinity epitope and expressed from its natural chromosomal location. Through immunodetection of the common tag, we obtain a census of proteins expressed during log-phase growth and measurements of their absolute levels. We find that about 80% of the proteome is expressed during normal growth conditions, and, using additional sequence information, we systematically identify misannotated genes. The abundance of proteins ranges from fewer than 50 to more than 10(6) molecules per cell. Many of these molecules, including essential proteins and most transcription factors, are present at levels that are not readily detectable by other proteomic techniques nor predictable by mRNA levels or codon bias measurements.

Combining chemical genetics and proteomics to identify protein kinase substrates

Abstract: Phosphorylation is a ubiquitous protein modification important for regulating nearly every aspect of cellular biology. Protein kinases are highly conserved and constitute one of the largest gene families. Identifying the substrates of a kinase is essential for understanding its cellular role, but doing so remains a difficult task. We have developed a high-throughput method to identify substrates of yeast protein kinases that employs a collection of yeast strains each expressing a single epitope-tagged protein and a chemical genetic strategy that permits kinase reactions to be performed in native, whole-cell extracts. Using this method, we screened 4,250 strains expressing epitope-tagged proteins and identified 24 candidate substrates of the Pho85–Pcl1 cyclin-dependent kinase, including the known substrate Rvs167. The power of this method to identify true kinase substrates is strongly supported by functional overlap and colocalization of candidate substrates and the kinase, as well as by the specificity of Pho85–Pcl1 for some of the substrates compared with another Pho85-cyclin kinase complex. This method is readily adaptable to other yeast kinases.

Construction, verification and experimental use of two epitope-tagged collections of budding yeast strains.

Abstract: A major challenge in the post-genomic era is the development of experimental approaches to monitor the properties of proteins on a proteome-wide level. It would be particularly useful to systematically assay protein subcellular localization, post-translational modifications and protein-protein interactions, both at steady state and in response to environmental stimuli. Development of new reagents and methods will enhance our ability to do so efficiently and systematically. Here we describe the construction of two collections of budding yeast strains that facilitate proteome-wide measurements of protein properties. These collections consist of strains with an epitope tag integrated at the C-terminus of essentially every open reading frame (ORF), one with the tandem affinity purification (TAP) tag, and one with the green fluorescent protein (GFP) tag. We show that in both of these collections we have accurately tagged a high proportion of all ORFs (approximately 75% of the proteome) by confirming expression of the fusion proteins. Furthermore, we demonstrate the use of the TAP collection in performing high-throughput immunoprecipitation experiments. Building on these collections and the methods described in this paper, we hope that the yeast community will expand both the quantity and type of proteome level data available.

Pho86p, an endoplasmic reticulum (ER) resident protein in Saccharomyces cerevisiae, is required for ER exit of the high-affinity phosphate transporter Pho84p.

Abstract: In the budding yeast Saccharomyces cerevisiae, PHO84 and PHO86 are among the genes that are most highly induced in response to phosphate starvation. They are essential for growth when phosphate is limiting, and they function in the high-affinity phosphate uptake system. PHO84 encodes a high-affinity phosphate transporter, and mutations in PHO86 cause many of the same phenotypes as mutations in PHO84, including a phosphate uptake defect and constitutive expression of the secreted acid phosphatase, Pho5p. Here, we show that the subcellular localization of Pho84p is regulated in response to extracellular phosphate levels; it is localized to the plasma membrane in low-phosphate medium but quickly endocytosed and transported to the vacuole upon addition of phosphate to the medium. Moreover, Pho84p is localized to the endoplasmic reticulum (ER) and fails to be targeted to the plasma membrane in the absence of Pho86p. Utilizing an in vitro vesicle budding assay, we demonstrate that Pho86p is required for packaging of Pho84p into COPII vesicles. Pho86p is an ER resident protein, which itself is not transported out of the ER. Interestingly, the requirement of Pho86p for ER exit is specific to Pho84p, because other members of the hexose transporter family to which Pho84 belongs are not mislocalized in the absence of Pho86p.

MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.

Abstract: Efficient analysis of very large amounts of raw data for peptide identification and protein quantification is a principal challenge in mass spectrometry (MS)-based proteomics. Here we describe MaxQuant, an integrated suite of algorithms specifically developed for high-resolution, quantitative MS data. Using correlation analysis and graph theory, MaxQuant detects peaks, isotope clusters and stable amino acid isotope-labeled (SILAC) peptide pairs as three-dimensional objects in m/z, elution time and signal intensity space. By integrating multiple mass measurements and correcting for linear and nonlinear mass offsets, we achieve mass accuracy in the p.p.b. range, a sixfold increase over standard techniques. We increase the proportion of identified fragmentation spectra to 73% for SILAC peptide pairs via unambiguous assignment of isotope and missed-cleavage state and individual mass precision. MaxQuant automatically quantifies several hundred thousand peptides per SILAC-proteome experiment and allows statistically robust identification and quantification of >4,000 proteins in mammalian cell lysates.

Global analysis of protein localization in budding yeast

Abstract: A fundamental goal of cell biology is to define the functions of proteins in the context of compartments that organize them in the cellular environment. Here we describe the construction and analysis of a collection of yeast strains expressing full-length, chromosomally tagged green fluorescent protein fusion proteins. We classify these proteins, representing 75% of the yeast proteome, into 22 distinct subcellular localization categories, and provide localization information for 70% of previously unlocalized proteins. Analysis of this high-resolution, high-coverage localization data set in the context of transcriptional, genetic, and protein-protein interaction data helps reveal the logic of transcriptional co-regulation, and provides a comprehensive view of interactions within and between organelles in eukaryotic cells.

Global analysis of protein expression in yeast

Abstract: The availability of complete genomic sequences and technologies that allow comprehensive analysis of global expression profiles of messenger RNA have greatly expanded our ability to monitor the internal state of a cell. Yet biological systems ultimately need to be explained in terms of the activity, regulation and modification of proteins--and the ubiquitous occurrence of post-transcriptional regulation makes mRNA an imperfect proxy for such information. To facilitate global protein analyses, we have created a Saccharomyces cerevisiae fusion library where each open reading frame is tagged with a high-affinity epitope and expressed from its natural chromosomal location. Through immunodetection of the common tag, we obtain a census of proteins expressed during log-phase growth and measurements of their absolute levels. We find that about 80% of the proteome is expressed during normal growth conditions, and, using additional sequence information, we systematically identify misannotated genes. The abundance of proteins ranges from fewer than 50 to more than 10(6) molecules per cell. Many of these molecules, including essential proteins and most transcription factors, are present at levels that are not readily detectable by other proteomic techniques nor predictable by mRNA levels or codon bias measurements.

Lysine Acetylation Targets Protein Complexes and Co-Regulates Major Cellular Functions

Abstract: Lysine acetylation is a reversible posttranslational modification of proteins and plays a key role in regulating gene expression. Technological limitations have so far prevented a global analysis of lysine acetylation's cellular roles. We used high-resolution mass spectrometry to identify 3600 lysine acetylation sites on 1750 proteins and quantified acetylation changes in response to the deacetylase inhibitors suberoylanilide hydroxamic acid and MS-275. Lysine acetylation preferentially targets large macromolecular complexes involved in diverse cellular processes, such as chromatin remodeling, cell cycle, splicing, nuclear transport, and actin nucleation. Acetylation impaired phosphorylation-dependent interactions of 14-3-3 and regulated the yeast cyclin-dependent kinase Cdc28. Our data demonstrate that the regulatory scope of lysine acetylation is broad and comparable with that of other major posttranslational modifications.

Insights into the regulation of protein abundance from proteomic and transcriptomic analyses

Abstract: Recent advances in next-generation DNA sequencing and proteomics provide an unprecedented ability to survey mRNA and protein abundances. Such proteome-wide surveys are illuminating the extent to which different aspects of gene expression help to regulate cellular protein abundances. Current data demonstrate a substantial role for regulatory processes occurring after mRNA is made - that is, post-transcriptional, translational and protein degradation regulation - in controlling steady-state protein abundances. Intriguing observations are also emerging in relation to cells following perturbation, single-cell studies and the apparent evolutionary conservation of protein and mRNA abundances. Here, we summarize current understanding of the major factors regulating protein expression.