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Cytochrome P450 enzymes are heme-dependent monoxygenases that play a central role in human physiology. Despite the numerous physiological processes that P450 enzymes impact, the electron donors P450 oxidoreductase and cytochrome b5 are the only proteins known to interact with and modulate the activity of ER microsomal P450s. Here, we report that Dap1/PGRMC1 is required for ER P450 function in yeast and humans. We show that S. pombe Dap1 is a hemoprotein that binds and positively regulates Cyp51A1 and Cyp61A1, two P450s required for sterol biosynthesis. Similarly, loss of human PGRMC1 reduces activity of Cyp51A1, blocking cholesterol synthesis and increasing production of toxic sterol intermediates. PGRMC1 stably binds Cyp51A1 and human P450s from three additional families including Cyp3A4, which metabolizes pharmaceutical compounds. These findings demonstrate that PGRMC1 is required for P450 activity and suggest that interindividual variation in PGRMC1 function may impact multiple biochemical pathways and drug metabolism.

The anaphase-promoting complex/cyclosome (APC/C) is a conserved multisubunit ubiquitin ligase required for the degradation of key cell cycle regulators. The APC/C becomes active at the metaphase/anaphase transition and remains active during G(1) phase. One mechanism linked to activation of the APC/C is phosphorylation. Although many sites of mitotic phosphorylation have been identified in core components of the APC/C, the consequence of any individual phosphorylation event has not been elucidated in vivo. In this study, we show that Hcn1 is an essential core component of the fission yeast APC/C and is critical for maintaining complex integrity. Moreover, Hcn1 is a phosphoprotein in vivo. Phosphorylation of Hcn1 occurs at a single Cdk1 site in vitro and in vivo. Mutation of this site to alanine, but not aspartic acid, compromises APC/C function and leads to a specific defect in the completion of cell division.

Although generating large amounts of proteomic data using tandem mass spectrometry has become routine, there is currently no single set of comprehensive tools for the rigorous analysis of tandem mass spectrometry results given the large variety of possible experimental aims. Currently available applications are typically designed for displaying proteins and posttranslational modifications from the point of view of the mass spectrometrist and are not versatile enough to allow investigators to develop biological models of protein function, protein structure, or cell state. In addition, storage and dissemination of mass spectrometry-based proteomic data are problems facing the scientific community. To address these issues, we have developed a relational database model that efficiently stores and manages large amounts of tandem mass spectrometry results. We have developed an integrated suite of multifunctional analysis software for interpreting, comparing, and displaying these results. Our system, Bioinformatic Graphical Comparative Analysis Tools (BIGCAT), allows sophisticated analysis of tandem mass spectrometry results in a biologically intuitive format and provides a solution to many data storage and dissemination issues.

Translation regulation is a critical means by which cells control growth, division, and apoptosis. To gain further insight into translation and related processes, we performed multifaceted mass spectrometry-based proteomic screens of yeast ribosomal complexes and discovered an association of 77 uncharacterized yeast proteins with ribosomes. Immunoblotting revealed an EDTA-dependent cosedimentation with ribosomes in sucrose gradients for 11 candidate translation-machinery-associated (TMA) proteins. Tandem affinity purification linked one candidate, LSM12, to the RNA processing proteins PBP1 and PBP4. A second candidate, TMA46, interacted with RBG1, a GTPase that interacts with ribosomes. By adapting translation assays to high-throughput screening methods, we showed that null yeast strains harboring deletions for several of the TMA genes had alterations in protein synthesis rates (TMA7 and TMA19), susceptibility to drugs that inhibit translation (TMA7), translation fidelity (TMA20), and polyribosome profiles (TMA7, TMA19, and TMA20). TMA20 has significant sequence homology with the oncogene MCT-1. Expression of human MCT-1 in the Deltatma20 yeast mutant complemented translation-related defects, strongly implying that MCT-1 functions in translation-related processes. Together these findings implicate the TMA proteins and, potentially, their human homologs, in translation related processes.

PSF (PTB-associated splicing factor) is a large nuclear protein that has been implicated in numerous processes including transcription and RNA splicing. It has been shown to directly associate with U5 snRNA and has also been found within numerous purified splicing complexes. Here, we show that when HeLa nuclear extracts are adjusted to splicing conditions, PSF is found as part of a large complex that contains all five snRNPs and most known splicing factors. Formation of the complex does not require addition of exogenous pre-mRNA substrate and occurs at 4 degrees C but is salt sensitive. Sedimentation experiments and identification of individual components by mass spectrometry revealed association with multiple nuclear factors, most of which overlap with spliceosome components.

An important component of proteomic research is the high-throughput discovery of novel proteins and protein-protein interactions that control molecular events that contribute to critical cellular functions and human disease. The interactions of proteins are essential for cellular functions. Identifying perturbation of normal cellular protein interactions is vital for understanding the disease process and intervening to control the disease. A second area of proteomics research is the discovery of proteins that will serve as biomarkers for the early detection, diagnosis and drug treatment response for specific diseases. These studies have been referred to as clinical proteomics. To discover biomarkers, proteomics research employs the quantitative comparison of peptide and protein expression in body fluids and tissues from diseased individuals (case) versus normal individuals (control). Methods that couple 2D capillary liquid chromatography (LC) and tandem mass spectrometry (MS/MS) analysis have greatly facilitated this discovery science. Coupling 2D-LC/MS/MS analysis with automated genome-assisted spectra interpretation allows a direct, high-throughput and high-sensitivity identification of thousands of individual proteins from complex biological samples. The systematic comparison of experimental conditions and controls allows protein function or disease states to be modeled. This review discusses the different purification and quantification strategies that have been developed and used in combination with 2D-LC/MS/MS and computational analysis to examine regulatory protein networks and clinical samples.

In the last ten years, the field of proteomics has expanded at a rapid rate. A range of exciting new technology has been developed and enthusiastically applied to an enormous variety of biological questions. However, the degree of stringency required in proteomic data generation and analysis appears to have been underestimated. As a result, there are likely to be numerous published findings that are of questionable quality, requiring further confirmation and/or validation. This manuscript outlines a number of key issues in proteomic research, including those associated with experimental design, differential display and biomarker discovery, protein identification and analytical incompleteness. In an effort to set a standard that reflects current thinking on the necessary and desirable characteristics of publishable manuscripts in the field, a minimal set of guidelines for proteomics research is then described. These guidelines will serve as a set of criteria which editors of PROTEOMICS will use for assessment of future submissions to the Journal.

ARB1 is an essential yeast protein closely related to members of a subclass of the ATP-binding cassette (ABC) superfamily of proteins that are known to interact with ribosomes and function in protein synthesis or ribosome biogenesis. We show that depletion of ARB1 from Saccharomyces cerevisiae cells leads to a deficit in 18S rRNA and 40S subunits that can be attributed to slower cleavage at the A0, A1, and A2 processing sites in 35S pre-rRNA, delayed processing of 20S rRNA to mature 18S rRNA, and a possible defect in nuclear export of pre-40S subunits. Depletion of ARB1 also delays rRNA processing events in the 60S biogenesis pathway. We further demonstrate that ARB1 shuttles from nucleus to cytoplasm, cosediments with 40S, 60S, and 80S/90S ribosomal species, and is physically associated in vivo with TIF6, LSG1, and other proteins implicated previously in different aspects of 60S or 40S biogenesis. Mutations of conserved ARB1 residues expected to function in ATP hydrolysis were lethal. We propose that ARB1 functions as a mechanochemical ATPase to stimulate multiple steps in the 40S and 60S ribosomal biogenesis pathways.

Stimulated exocytosis of intracellular granules plays a critical role in conversion of inactive, circulating neutrophils to fully activated cells capable of chemotaxis, phagocytosis, and bacterial killing. The functional changes induced by exocytosis of each of the granule subsets, gelatinase (tertiary) granules, specific (secondary) granules, and azurophil (primary) granules, are poorly defined. To improve the understanding of the role of exocytosis of these granule subsets, a proteomic analysis of the azurophil, specific, and gelatinase granules from human neutrophils was performed. Two different methods for granule protein identification were applied. First, two-dimensional (2D) gel electrophoresis followed by MALDI-TOF MS analysis of peptides obtained by in-gel trypsin digestion of proteins was performed. Second, peptides from tryptic digests of granule membrane proteins were separated by two-dimensional microcapillary chromatography using strong cation exchange and reverse phase microcapillary high pressure liquid chromatography and analyzed with electrospray ionization tandem mass spectrometry (2D HLPC ESI-MS/MS). Our analysis identified 286 proteins on the three granule subsets, 87 of which were identified by MALDI MS and 247 were identified by 2D HPLC ESI-MS/MS. The increased sensitivity of 2D HPLC ESI-MS/MS, however, resulted in identification of over 500 proteins from subcellular organelles contaminating isolated granules. Defining the proteome of neutrophil granule subsets provides a basis for understanding the role of exocytosis in neutrophil biology. Additionally, the described methods may be applied to mobilizable compartments of other secretory cells.

A method for the rapid correlation of tandem mass spectra to a list of protein sequences in a database has been developed. The combination of the fast and accurate computational search algorithm, X!Tandem, and a Linux cluster parallel computing environment with PVM or MPI, significantly reduces the time required to perform the correlation of tandem mass spectra to protein sequences in a database. A file of tandem mass spectra is divided into a specified number of files, each containing an equal number of the spectra from the larger file. These files are then searched in parallel against a protein sequence database. The results of each parallel output file are collated into one file for viewing through a web interface. Thousands of spectra can be searched in an accurate, practical, and time effective manner. The source code for running Parallel Tandem utilizing either PVM or MPI on Linux operating system is available from http://www.thegpm.org. This source code is made available under Artistic License from the authors.

The norepinephrine transporter (NET) terminates noradrenergic signals by clearing released NE at synapses. NET regulation by receptors and intracellular signaling pathways is supported by a growing list of associated proteins including syntaxin1A, protein phosphatase 2A (PP2A) catalytic subunit (PP2A-C), PICK1, and Hic-5. In the present study, we sought evidence for additional partnerships by mass spectrometry-based analysis of proteins co-immunoprecipitated with human NET (hNET) stably expressed in a mouse noradrenergic neuroblastoma cell line. Our initial proteomic analyses reveal multiple peptides derived from hNET, peptides arising from the mouse PP2A anchoring subunit (PP2A-Ar) and peptides derived from 14-3-3 proteins. We verified physical association of NET with PP2A-Ar via co-immunoprecipitation studies using mouse vas deferens extracts and with 14-3-3 via a fusion pull-down approach, implicating specifically the hNET NH2-terminus for interactions. The transporter complexes described likely support mechanisms regulating transporter activity, localization, and trafficking.

Proteins control and mediate most of the biological activities in the cell. In most cases, proteins either interact with regulatory proteins or function in large molecular assemblies to carryout biological processes. Understanding the functions of individual proteins requires the identification of these interacting proteins. With its speed and sensitivity, mass spectrometry has become the dominant method for identifying components of protein complexes. This article reviews and discusses various approaches to purify protein complexes and analyze the proteins using mass spectrometry. As examples, methods to isolate and analyze protein complexes responsible for the translation of messenger RNAs into polypeptides are described.

Septins are GTP binding proteins important for cytokinesis in many eukaryotes. The Schizosaccaromyces pombe genome sequence predicts orthologues of four of five Saccharomyces cerevisiae septins involved in cytokinesis and these are named Spns1-4p. That spns1-4 are not essential genes permitted the application of a combined genetic and proteomics approach to determine their functional relationships. Our findings indicate that Spns1-4p are present throughout interphase as a diffusely localized approximately 8.5S complex containing two copies of each septin linked together as a chain in the order Spn3p-Spn4p-Spn1p-Spn2p. Septin recruitment to the medial region of the cell is genetically separable from ring formation, and whereas it is normally restricted to mitosis, it can be promoted without activation of the mitotic cell cycle machinery. Coalescence into ring structures requires Spn1p and Spn4p associate with at least one other septin subunit and the expression of Mid2p that is normally restricted to mitosis. This study establishes the functional requirements for septin complex organization in vivo.

The Drosophila Myb complex has roles in both activating and repressing developmentally regulated DNA replication. To further understand biochemically the functions of the Myb complex, we fractionated Drosophila embryo extracts relying upon affinity chromatography. We found that E2F2, DP, RBF1, RBF2, and the Drosophila homolog of LIN-52, a class B synthetic multivulva (synMuv) protein, copurify with the Myb complex components to form the Myb-MuvB complex. In addition, we found that the transcriptional repressor protein, lethal (3) malignant brain tumor protein, L(3)MBT, and the histone deacetylase, Rpd3, associated with the Myb-MuvB complex. Members of the Myb-MuvB complex were localized to promoters and were shown to corepress transcription of developmentally regulated genes. These and other data now link together the Myb and E2F2 complexes in higher-order assembly to specific chromosomal sites for the regulation of transcription.

Translation of mRNA into protein is a fundamental step in eukaryotic gene expression requiring the large (60S) and small (40S) ribosome subunits and associated proteins. By modern proteomic approaches, we previously identified a novel 40S-associated protein named Asc1p in budding yeast and RACK1 in mammals. The goals of this study were to establish Asc1p or RACK1 as a core conserved eukaryotic ribosomal protein and to determine the role of Asc1p or RACK1 in translational control. We provide biochemical, evolutionary, genetic, and functional evidence showing that Asc1p or RACK1 is indeed a conserved core component of the eukaryotic ribosome. We also show that purified Asc1p-deficient ribosomes have increased translational activity compared to that of wild-type yeast ribosomes. Further, we demonstrate that asc1Delta null strains have increased levels of specific proteins in vivo and that this molecular phenotype is complemented by either Asc1p or RACK1. Our data suggest that one of Asc1p's or RACK1's functions is to repress gene expression.

The SAGA histone acetyltransferase and TFIID complexes play key roles in eukaryotic transcription. Using hierarchical cluster analysis of mass spectrometry data to identify proteins that copurify with components of the budding yeast TFIID transcription complex, we discovered that an uncharacterized protein corresponding to the YPL047W open reading frame significantly associated with shared components of the TFIID and SAGA complexes. Using mass spectrometry and biochemical assays, we show that YPL047W (SGF11, 11-kDa SAGA-associated factor) is an integral subunit of SAGA. However, SGF11 does not appear to play a role in SAGA-mediated histone acetylation. DNA microarray analysis showed that SGF11 mediates transcription of a subset of SAGA-dependent genes, as well as SAGA-independent genes. SAGA purified from a sgf11 Delta deletion strain has reduced amounts of Ubp8p, and a ubp8 Delta deletion strain shows changes in transcription similar to those seen with the sgf11 Delta deletion strain. Together, these data show that Sgf11p is a novel component of the yeast SAGA complex and that SGF11 regulates transcription of a subset of SAGA-regulated genes. Our data suggest that the role of SGF11 in transcription is independent of SAGA's histone acetyltransferase activity but may involve Ubp8p recruitment to or stabilization in SAGA.

The general amino acid control (GAAC) enables yeast cells to overcome amino acid deprivation by activation of the alpha subunit of translation initiation factor 2 (eIF2alpha) kinase GCN2 and consequent induction of GCN4, a transcriptional activator of amino acid biosynthetic genes. Binding of GCN2 to GCN1 is required for stimulation of GCN2 kinase activity by uncharged tRNA in starved cells. Here we show that YIH1, when overexpressed, dampens the GAAC response (Gcn- phenotype) by suppressing eIF2alpha phosphorylation by GCN2. The overexpressed YIH1 binds GCN1 and reduces GCN1-GCN2 complex formation, and, consistent with this, the Gcn- phenotype produced by YIH1 overexpression is suppressed by GCN2 overexpression. YIH1 interacts with the same GCN1 fragment that binds GCN2, and this YIH1-GCN1 interaction requires Arg-2259 in GCN1 in vitro and in full-length GCN1 in vivo, as found for GCN2-GCN1 interaction. However, deletion of YIH1 does not increase eIF2alpha phosphorylation or derepress the GAAC, suggesting that YIH1 at native levels is not a general inhibitor of GCN2 activity. We discovered that YIH1 normally resides in a complex with monomeric actin, rather than GCN1, and that a genetic reduction in actin levels decreases the GAAC response. This Gcn- phenotype was partially suppressed by deletion of YIH1, consistent with YIH1-mediated inhibition of GCN2 in actin-deficient cells. We suggest that YIH1 resides in a YIH1-actin complex and may be released for inhibition of GCN2 and stimulation of protein synthesis under specialized conditions or in a restricted cellular compartment in which YIH1 is displaced from monomeric actin.

The conserved histone variant H2A.Z functions in euchromatin to antagonize the spread of heterochromatin. The mechanism by which histone H2A is replaced by H2A.Z in the nucleosome is unknown. We identified a complex containing 13 different polypeptides associated with a soluble pool of H2A.Z in Saccharomyces cerevisiae. This complex was designated SWR1-Com in reference to the Swr1p subunit, a Swi2/Snf2-paralog. Swr1p and six other subunits were found only in SWR1-Com, whereas six other subunits were also found in the NuA4 histone acetyltransferase and/or the Ino80 chromatin remodeling complex. H2A.Z and SWR1 were essential for viability of cells lacking the EAF1 component of NuA4, pointing to a close functional connection between these two complexes. Strikingly, chromatin immunoprecipitation analysis of cells lacking Swr1p, the presumed ATPase of the complex, revealed a profound defect in the deposition of H2A.Z at euchromatic regions that flank the silent mating type cassette HMR and at 12 other chromosomal sites tested. Consistent with a specialized role for Swr1p in H2A.Z deposition, the majority of the genome-wide transcriptional defects seen in swr1Delta cells were also found in htz1Delta cells. These studies revealed a novel role for a member of the ATP-dependent chromatin remodeling enzyme family in determining the region-specific histone subunit composition of chromatin in vivo and controlling the epigenetic state of chromatin. Metazoan orthologs of Swr1p (Drosophila Domino; human SRCAP and p400) may have analogous functions., Competing Interests: The authors have declared that no conflicts of interest exist.

The gamma-tubulin complex, via its ability to organize microtubules, is critical for accurate chromosome segregation and cytokinesis in the fission yeast, Schizosaccharomyces pombe. To better understand its roles, we have purified the S. pombe gamma-tubulin complex. Mass spectrometric analyses of the purified complex revealed known components and identified two novel proteins (i.e., Mbo1p and Gfh1p) with homology to gamma-tubulin-associated proteins from other organisms. We show that both Mbo1p and Gfh1p localize to microtubule organizing centers. Although cells deleted for either mbo1(+) or gfh1(+) are viable, they exhibit a number of defects associated with altered microtubule function such as defects in cell polarity, nuclear positioning, spindle orientation, and cleavage site specification. In addition, mbo1Delta and gfh1Delta cells exhibit defects in astral microtubule formation and anchoring, suggesting that these proteins have specific roles in astral microtubule function. This study expands the known roles of gamma-tubulin complex components in organizing different types of microtubule structures in S. pombe.

Multidimensional liquid chromatography of peptides produced by protease digestion of complex protein mixtures followed by tandem mass spectrometry can be coupled with automated database searching to identify large numbers of proteins in complex samples. These methods avoid the limitations of gel electrophoresis and in-gel digestions by directly identifying protein mixtures in solution.

SRrp86 is a unique member of the SR protein superfamily containing one RNA recognition motif and two serine-arginine (SR)-rich domains separated by an unusual glutamic acid-lysine (EK)-rich region. Previously, we showed that SRrp86 could regulate alternative splicing by both positively and negatively modulating the activity of other SR proteins and that the unique EK domain could inhibit both constitutive and alternative splicing. These functions were most consistent with the model in which SRrp86 functions by interacting with and thereby modulating the activity of target proteins. To identify the specific proteins that interact with SRrp86, we used a yeast two-hybrid library screen and immunoprecipitation coupled to mass spectrometry. We show that SRrp86 interacts with all of the core SR proteins, as well as a subset of other splicing regulatory proteins, including SAF-B, hnRNP G, YB-1, and p72. In contrast to previous results that showed activation of SRp20 by SRrp86, we now show that SAF-B, hnRNP G, and 9G8 all antagonize the activity of SRrp86. Overall, we conclude that not only does SRrp86 regulate SR protein activity but that it is, in turn, regulated by other splicing factors to control alternative splice site selection.

Multidimensional peptide separation will play an increasingly important role in the drive to identify and quantitate the proteome. By increasing the peak and load capacity, multidimensional approaches increase the number and dynamic range of peptides that can be analyzed in a complex biological organism. Separation methods using different physical properties of peptides have been combined with varying degrees of success. The ultimate goal is a rapid separation strategy that can be coupled with analytical methods, such as mass spectrometry, to provide comprehensive monitoring of the changing concentration, interactions, and structures of proteins in the proteome.

We are using biochemical and genetic approaches to study Rtf1 and the Spt4-Spt5 complex, which independently have been implicated in transcription elongation by RNA polymerase II. Here, we report a remarkable convergence of these studies. First, we purified Rtf1 and its associated yeast proteins. Combining this approach with genetic analysis, we show that Rtf1 and Leo1, a protein of unknown function, are members of the RNA polymerase II-associated Paf1 complex. Further analysis revealed allele-specific genetic interactions between Paf1 complex members, Spt4-Spt5, and Spt16-Pob3, the yeast counterpart of the human elongation factor FACT. In addition, we independently isolated paf1 and leo1 mutations in an unbiased genetic screen for suppressors of a cold-sensitive spt5 mutation. These genetic interactions are supported by physical interactions between the Paf1 complex, Spt4-Spt5 and Spt16-Pob3. Finally, we found that defects in the Paf1 complex cause sensitivity to 6-azauracil and diminished PUR5 induction, properties frequently associated with impaired transcription elongation. Taken together, these data suggest that the Paf1 complex functions during the elongation phase of transcription in conjunction with Spt4-Spt5 and Spt16-Pob3.