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PubMed2004

Diego interacts with Prickle and Strabismus/Van Gogh to localize planar cell polarity complexes.

Planar cell polarity (PCP) in the Drosophila eye is established by the distinct fate specifications of photoreceptors R3 and R4, and is regulated by the Frizzled (Fz)/PCP signaling pathway. Before the PCP proteins become asymmetrically localized to opposite poles of the cell in response to Fz/PCP signaling, they are uniformly apically colocalized. Little is known about how the apical localization is maintained. We provide evidence that the PCP protein Diego (Dgo) promotes the maintenance of apical localization of Flamingo (Fmi), an atypical Cadherin-family member, which itself is required for the apical localization of the other PCP factors. This function of Dgo is redundant with Prickle (Pk) and Strabismus (Stbm), and only appreciable in double mutant tissue. We show that the initial membrane association of Dgo depends on Fz, and that Dgo physically interacts with Stbm and Pk through its Ankyrin repeats, providing evidence for a PCP multiprotein complex. These interactions suggest a positive feedback loop initiated by Fz that results in the apical maintenance of other PCP factors through Fmi.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Herpes simplex virus VP16 forms a complex with the virion host shutoff protein vhs.

Herpes simplex virus (HSV) virions contain at least two regulatory proteins that modulate gene expression in infected cells: the transcriptional activator VP16 and the virion host shutoff protein vhs. VP16 stimulates transcription of the HSV immediate-early genes, and vhs suppresses host protein synthesis and induces accelerated turnover of cellular and viral mRNAs. We report here that vhs binds directly to VP16: vhs and VP16 were coprecipitated from infected cells by an anti-vhs antiserum, and vhs and VP16 protein A fusions each bound intact versions of the other protein in a solid-phase capture assay. In addition, vhs and VP16 interacted in the two-hybrid activator system when coexpressed in Saccharomyces cerevisiae. vhs residues 238 to 344 were sufficient for the interaction, and the VP16 acidic transcriptional activation domain was not required. vhs blocked the ability of VP16 to enter a multiprotein complex on an immediate-early TAATGARATTC consensus sequence, indicating that vhs interacts with one or more regions of VP16 required for promoter recognition. We suggest that this interaction may play a structural role in the assembly of HSV virions and modulate the activity of vhs during infection.

Animals↗

PubMed2006

Nuclear suppressors define three factors that participate in both 5' and 3' end processing of mRNAs in Chlamydomonas chloroplasts.

Chloroplast RNA processing and degradation are orchestrated by nucleus-encoded factors. Although several transcript-specific factors have been identified, those involved in global RNA metabolism have mostly remained elusive. Using Chlamydomonas reinhardtii, we have identified three pleiotropic nuclear mutations, mcd3, mcd4 and mcd5, which cause quantitative variation between polycistronic transcripts and accumulation of transcripts with novel 3' ends. The mcd3, mcd4 and mcd5 mutants were initially isolated as photoautotrophic suppressors of the petD 5' mutants LS2 and LS6, which harbour four nucleotide linker-scanning mutations near the 5' end of the mature transcript. The LS mutants accumulate 1-3% of the wild-type (WT) petD mRNA level and no cytochrome b6/f complex subunit IV, which is the petD gene product and required for photosynthesis. Each suppressor restores approximately 15% of the WT petD mRNA and subunit IV levels. Genetic analysis showed mcd4 to be recessive, and suggested that MCD4 interacts with the petD mRNA stability factor MCD1. To assess the specificity of mcd3, mcd4 and mcd5, transcripts from 32 chloroplast genes were analysed by RNA filter hybridizations. mcd3 and mcd4 displayed aberrant transcript patterns for 17 genes, whereas only three were altered in mcd5. Since the mutations affect multiple RNAs in a variety of ways, our data suggest that MCD3, MCD4 and MCD5 may participate in a series of multiprotein complexes responsible for RNA maturation and degradation in Chlamydomonas chloroplasts.

Algal Proteins↗

PubMed2001

MRG15 activates the B-myb promoter through formation of a nuclear complex with the retinoblastoma protein and the novel protein PAM14.

The MORF4-Related Gene on chromosome 15 (MRG15) is a member of a novel family of genes originally identified in studies to reveal cell senescence-inducing factors. MRG15 contains several predicted protein motifs, including a nuclear localization signal, a helix-loop-helix region, a leucine zipper, and a chromodomain. These motifs are commonly associated with transcription factors, suggesting that MRG15 may likewise function as a transcriptional regulator. To examine the potential function(s) of MRG15, we sought to identify cellular factors associated with this MRG family member. In this regard, we have found that both the retinoblastoma tumor suppressor (Rb) and a novel nuclear protein PAM14 (Protein Associated with MRG, 14 kDa) specifically associate with MRG15. We have further demonstrated that these interactions require the helix-loop-helix and leucine zipper domains of MRG15. Interestingly we have found all three proteins present in a multiprotein complex, suggesting that at least some of their functions may be interdependent. Although the functions of PAM14 have yet to be elucidated, Rb has several well characterized activities, including repression of E2F-activated promoters such as that of B-myb. Significantly we have demonstrated that MRG15 blocks the Rb-induced repression of this promoter, leading to B-myb promoter activation. Collectively these results suggest that MRG15 regulates transcription through interactions with a cellular protein complex containing Rb and PAM14.

Amino Acid Sequence↗

PubMed1999

Transcriptional autorepression of Msx1 gene is mediated by interactions of Msx1 protein with a multi-protein transcriptional complex containing TATA-binding protein, Sp1 and cAMP-response-element-binding protein-binding protein (CBP/p300).

The TATA-less murine Msx1 promoter contains two Msx1-binding motifs, located at -568 to -573 and +25 to +30, and is subject to potent autorepression [Takahashi, Guron, Shetty, Matsui and Raghow (1997) J. Biol. Chem. 272, 22667-22678]. To investigate the molecular mechanism by which Msx1 represses the activity of its own promoter, we transfected C2C12 myoblasts with Msx1-promoter-luciferase constructs and assessed reporter gene activity, with and without the exogenous expression of Msx1. We demonstrate that Msx1-mediated autorepression remained unaffected, regardless of the presence or absence of the Msx1 recognition motifs on the promoter. Furthermore, graded exogenous expression of TATA-binding protein (TBP), Sp1 or cAMP-response-element-binding protein-binding protein (CBP/p300) could counteract the autoinhibitory activity of Msx1. Finally, we demonstrate that Msx1 protein can be immunoprecipitated in a multiprotein complex containing TBP, Sp1 and CBP/p300. We hypothesize that the interaction of Msx1 protein with one or more ubiquitous or tissue-restricted transcription factors mediates transcriptional autorepression of the Msx1 gene.

Animals↗

PubMed1995

Chaperone function of a Grp 94-related protein for folding and transport of the pancreatic bile salt-dependent lipase.

In its fundamental attributes, the secretion pathway of the pancreatic bile salt-dependent lipase (BSDL) followed that described for all enzymes involved in regulated secretion. This route was inhibited by drugs that affect protein synthesis and intracellular transport. In the presence of monensin, BSDL was solely detected in microsome membrane fractions. The association of BSDL with intracellular membranes involved a protein complex, formed by at least two proteins of 94 and 56 kDa. In cells experiencing the metabolic stress due to azetidine-2-carboxylic acid, BSDL was additionally associated with a protein of 46 kDa. Affinity blotting showed that BSDL bound directly to the 94-kDa protein (p94). It was suggested that p94 could be a molecular chaperone, further identified as related to the 94-kDa glucose regulated protein (Grp 94). The membrane-associated BSDL (i.e. BSDL bound to the Grp 94-related p94) was O- and N-glycosylated and consequently appeared released from membranes in the trans-Golgi compartment. Therefore and for the first time, it is suggested that a multiprotein complex including the chaperone Grp 94-related p94 protein may play an essential role in the folding and transport of BSDL. One hypothesis is that the association of BSDL with membrane via the Grp 94-related p94 along its secretion pathway is required for its complete O-glycosylation, which occurs on the extended mucin-like structures present on the C-terminal part of the protein.

Animals↗

PubMed1998

Pontin52, an interaction partner of beta-catenin, binds to the TATA box binding protein.

beta-catenin, the vertebrate homolog of the Drosophila Armadillo protein, has been shown to have dual cellular functions, as a component of both the cadherin-catenin cell adhesion complex and the Wnt signaling pathway. At Wnt signaling, beta-catenin becomes stabilized in the cytoplasm and subsequently available for interaction with transcription factors of the lymphocyte enhancer factor-1/T-cell factor family, resulting in a nuclear localization of beta-catenin. Although beta-catenin does not bind DNA directly, its carboxyl- and amino-terminal regions exhibit a transactivating activity still not well understood molecularly. Here we report the identification of an interaction partner of beta-catenin, a nuclear protein designated Pontin52. Pontin52 binds beta-catenin in the region of Armadillo repeats 2-5 and, more importantly, also binds the TATA box binding protein. We provide evidence for an in vivo multiprotein complex composed of Pontin52, beta-catenin, and lymphocyte enhancer factor-1/T-cell factor. Our results suggest involvement of Pontin52 in the nuclear function of beta-catenin.

Amino Acid Sequence↗

PubMed2006

Versatile reporter systems show that transactivation by human T-cell leukemia virus type 1 Tax occurs independently of chromatin remodeling factor BRG1.

Potent activation of human T-cell leukemia virus type 1 (HTLV-1) gene expression is mediated by the virus-encoded transactivator protein Tax and three imperfect 21-bp repeats in the viral long terminal repeats. Each 21-bp repeat contains a cAMP-responsive-element core flanked by 5' G-rich and 3' C-rich sequences. Tax alone does not bind DNA. Rather, it interacts with basic domain-leucine zipper transcription factors CREB and ATF-1 to form ternary complexes with the 21-bp repeats. In the context of the ternary complexes, Tax contacts the G/C-rich sequences and recruits transcriptional coactivators CREB-binding protein (CBP)/p300 to effect potent transcriptional activation. Using an easily transduced and chromosomally integrated reporter system derived from a self-inactivating lentivirus vector, we showed in a BRG1- and BRM1-deficient adrenal carcinoma cell line, SW-13, that Tax- and 21-bp repeat-mediated transactivation does not require BRG1 or BRM1 and is not enhanced by BRG1. With a similar reporter system, we further demonstrated that Tax- and tumor necrosis factor alpha-induced NF-kappaB activation occurs readily in SW-13 cells in the absence of BRG1 and BRM1. These results suggest that the assembly of stable multiprotein complexes containing Tax, CREB/ATF-1, and CBP/p300 on the 21-bp repeats is the principal mechanism employed by Tax to preclude nucleosome formation at the HTLV-1 enhancer/promoter. This most likely bypasses the need for BRG1-containing chromatin-remodeling complexes. Likewise, recruitment of CBP/p300 by NF-kappaB may be sufficient to disrupt histone-DNA interaction for the initiation of transcription.

Activating Transcription Factors↗

PubMed2006

A domain-oriented approach to the reduction of combinatorial complexity in signal transduction networks.

BACKGROUND: Receptors and scaffold proteins possess a number of distinct domains and bind multiple partners. A common problem in modeling signaling systems arises from a combinatorial explosion of different states generated by feasible molecular species. The number of possible species grows exponentially with the number of different docking sites and can easily reach several millions. Models accounting for this combinatorial variety become impractical for many applications. RESULTS: Our results show that under realistic assumptions on domain interactions, the dynamics of signaling pathways can be exactly described by reduced, hierarchically structured models. The method presented here provides a rigorous way to model a large class of signaling networks using macro-states (macroscopic quantities such as the levels of occupancy of the binding domains) instead of micro-states (concentrations of individual species). The method is described using generic multidomain proteins and is applied to the molecule LAT. CONCLUSION: The presented method is a systematic and powerful tool to derive reduced model structures describing the dynamics of multiprotein complex formation accurately.

Binding Sites↗

PubMed1992

Transfection of CD14 into 70Z/3 cells dramatically enhances the sensitivity to complexes of lipopolysaccharide (LPS) and LPS binding protein.

Bacterial endotoxin (lipopolysaccharide [LPS]) causes fatal shock in humans and experimental animals. The shock is mediated by cytokines released by direct LPS stimulation of cells of monocytic origin (monocyte/macrophage [MO]). Recent studies have supported the concept that the plasma protein, LPS binding protein (LBP), plays an important role in controlling MO responses to LPS. Specifically, evidence has been presented to suggest that CD14, a membrane protein present in MO, serves as a receptor for complexes of LPS and the plasma protein LPS binding protein (LBP). In this function CD14 mediates attachment of LPS-bearing particles opsonized with LBP and appears to play an important role in regulating cytokine production induced by complexes of LPS and LBP. The CD14-, murine pre-B cell line 70Z/3 responds to LPS by synthesis of kappa light chains and consequent expression of surface IgM. To better understand the role of CD14 in controlling cellular responses to LPS, we investigated the effect of transfection of CD14 into 70Z/3 cells on LPS responsiveness. We report here that transfection of human or rabbit CD14 cDNA into 70Z/3 cells results in membrane expression of a glycosyl-phosphatidylinositol-anchored CD14. When LPS is complexed with LBP, CD14-bearing 70Z/3 cells bind more LPS than do the parental or 70Z/3 cells transfected with vector only. Remarkably, the expression of CD14 lowers the amount of LPS required to stimulate surface IgM expression by up to 10,000-fold when LPS dose-response curves in the CD14-, parental and CD14-bearing, transfected 70Z/3 cells are compared. In contrast, the response of CD14-bearing 70Z/3 cells and the parental 70Z/3 cell line (CD14-) to interferon gamma is indistinguishable. LPS stimulation of the parental and CD14-bearing 70Z/3 cells results in activation of NF-kB. These data provide evidence to support the concept that the LPS receptor in cells that constitutively express CD14 may be a multiprotein complex containing CD14 and membrane protein(s) common to a diverse group of LPS-responsive cells.

Acute-Phase Proteins↗

PubMed2005

RHL1 is an essential component of the plant DNA topoisomerase VI complex and is required for ploidy-dependent cell growth.

How cells achieve their final sizes is a pervasive biological question. One strategy to increase cell size is for the cell to amplify its chromosomal DNA content through endoreduplication cycles. Although endoreduplication is widespread in eukaryotes, we know very little about its molecular mechanisms. Successful progression of the endoreduplication cycle in Arabidopsis requires a plant homologue of archaeal DNA topoisomerase (topo) VI. To further understand how DNA is endoreduplicated and how this process is regulated, we isolated a dwarf Arabidopsis mutant, hyp7 (hypocotyl 7), in which various large cell types that in the wild type normally endoreduplicate multiple times complete only the first two rounds of endoreduplication and stall at 8C. HYP7 encodes the RHL1 (ROOT HAIRLESS 1) protein, and sequence analysis reveals that RHL1 has similarity to the C-terminal domain of mammalian DNA topo IIalpha, another type II topo that shares little sequence homology with topo VI. RHL1 shows DNA binding activity in vitro, and we present both genetic and in vivo evidence that RHL1 forms a multiprotein complex with plant topo VI. We propose that RHL1 plays an essential role in the topo VI complex to modulate its function and that the two distantly related topos, topo II and topo VI, have evolved a common domain that extends their function. Our data suggest that plant topo II and topo VI play distinct but overlapping roles during the mitotic cell cycle and endoreduplication cycle.

Amino Acid Sequence↗

PubMed1986

Organization and function of cytochrome b and ubiquinone in the cristae membrane of beef heart mitochondria.

The arrangement and function of the redox centers of the mammalian bc1 complex is described on the basis of structural data derived from amino acid sequence studies and secondary structure predictions and on the basis of functional studies (i.e., EPR data, inhibitor studies, and kinetic experiments). Two ubiquinone reaction centers do exist--a QH2 oxidation center situated at the outer, cytosolic surface of the cristae membrane (Q0 center), and a Q reduction center (Qi center) situated more to the inner surface of the cristae membrane. The Q0 center is formed by the b-566 domain of cytochrome b, the FeS protein, and maybe an additional small subunit, whereas the Qi center is formed by the b-562 domain of cytochrome b and presumably the 13.4 kDa protein ("QP-C"). The "Q binding proteins" are proposed to be protein subunits of the Q reaction centers of various multiprotein complexes. The path of electron flow branches at the Q0 center, half of the electrons flowing via the high-potential cytochrome chain to oxygen and half of the electrons cycling back into the Q pool via the cytochrome b path connecting the two Q reaction centers. During oxidation of QH2, 2H+ are released to the cytosolic space and during reduction of Q, 2H+ are taken up from the matrix side, resulting in a net transport across the membrane of 2H+ per e- flown from QH2 to cytochrome c, the H+ being transported across the membrane as H (H+ + e-) by the mobile carrier Q. The authors correct their earlier view of cytochrome b functioning as a H+ pump, proposing that the redox-linked pK changes of the acidic groups of cytochrome b are involved in the protonation/deprotonation processes taking place during the reduction and oxidation of Q. The reviewers stress that cytochrome b is in equilibrium with the Q pool via the Qi center, but not via the Q0 center. Their view of the mechanisms taking place at the reductase is a Q cycle linked to a Q-pool where cytochrome b is acting as an electron pump.

Animals↗

PubMed2005

Bromodomain analysis of Brd2-dependent transcriptional activation of cyclin A.

Cyclin A is regulated primarily through transcription control during the mammalian cell cycle. A dual mechanism of cyclin A transcriptional repression involves, on the one hand, promoter-bound inhibitory complexes of E2F transcription factors and RB (retinoblastoma) family proteins, and on the other, chromatin-directed histone deacetylase activity that is recruited to the cyclin A promoter early in the cell cycle in association with these RB proteins. This dual regulation maintains transcriptional silence of the cyclin A locus until its transcription is required in S-phase. At that time, RB family members dissociate from E2F proteins and nucleosomal restructuring of the locus takes place, to permit transcriptional activation and resultant S-phase progression to proceed. We have identified a double bromo-domain-containing protein Brd2, which exhibits apparent 'scaffold' or transcriptional adapter functions and mediates recruitment of both E2F transcription factors and chromatin-remodelling activity to the cyclin A promoter. We have shown previously that Brd2-containing nuclear, multiprotein complexes contain E2F-1 and -2. In the present study, we show that, in S-phase, they also contain histone H4-directed acetylase activity. Overexpression of Brd2 in fibroblasts accelerates the cell cycle through increased expression of cyclin A and its associated cyclin-dependent kinase activity. Chromatin immunoprecipitation studies show that Brd2 is physically present at the cyclin A promoter and its overexpression promotes increased histone H4 acetylation at the promoter as it becomes transcriptionally active, suggesting a new model for the dual regulation of cyclin A.

Acetyltransferases↗

PubMed2006

Preformed reggie/flotillin caps: stable priming platforms for macrodomain assembly in T cells.

T cell activation after contact with an antigen-presenting cell depends on the regulated assembly of the T cell receptor signaling complex, which involves the polarized assembly of a stable, raft-like macrodomain surrounding engaged T cell receptors. Here we show that the preformed reggie/flotillin caps present in resting T cells act as priming platforms for macrodomain assembly. Preformed reggie-1/flotillin-2 caps are exceptionally stable, as shown by fluorescence recovery after photobleaching (FRAP). Upon T cell stimulation, signaling molecules are recruited to the stable reggie/flotillin caps. Importantly, a trans-negative reggie-1/flotillin-2 deletion mutant, which interferes with assembly of the preformed reggie/flotillin cap, impairs raft polarization and macrodomain formation after T cell activation. Accordingly, expression of the trans-negative reggie-1 mutant leads to the incorrect positioning of the guanine nucleotide exchange factor Vav, resulting in defects in cytoskeletal reorganization. Thus, the preformed reggie/flotillin caps are stable priming platforms for the assembly of multiprotein complexes controlling actin reorganization during T cell activation.

Actins↗

PubMed2003

C. elegans PAT-6/actopaxin plays a critical role in the assembly of integrin adhesion complexes in vivo.

BACKGROUND: The novel focal adhesion protein actopaxin includes tandem unconventional calponin homology (CH) domains and a less well-conserved N-terminal stretch. Dominant-negative studies have implicated actopaxin in focal adhesion formation. RESULTS: PAT-6/actopaxin, the sole actopaxin homolog in C. elegans, is located in body wall muscle attachments that are in vivo homologs of focal adhesions. We show using pat-6 protein null alleles that PAT-6/actopaxin has critical nonredundant roles during attachment maturation. It is required to recruit UNC-89 and myofilaments to newly forming attachments, and also to reposition the attachments so that they form the highly ordered array of dense body and M line attachments that are characteristic of mature muscle cells. PAT-6/actopaxin is not required for the deposition of UNC-52/perlecan in the basal lamina, nor for the initiation of attachment assembly, including the clustering of integrin into foci and the recruitment of attachment proteins PAT-4/ILK, UNC-112, and DEB-1/vinculin from the cytosol. PAT-6/actopaxin, PAT-4/ILK, and UNC-112 are each required for the same steps during attachment assembly in vivo, consistent with the notion that they work together in multiprotein complex. Supporting this idea, PAT-4/ILK can simultaneously bind to PAT-6/actopaxin and UNC-112, forming a ternary complex, in yeast three-hybrid assays. Finally, we show that both calponin homology domains are required for PAT-6/actopaxin's critical functions during attachment assembly in vivo. CONCLUSIONS: We show directly by loss-of-function genetics that PAT-6/actopaxin plays essential roles during the maturation of integrin-mediated muscle attachments in vivo.

Actinin↗

PubMed1999

Role of HU and DNA supercoiling in transcription repression: specialized nucleoprotein repression complex at gal promoters in Escherichia coli.

Efficient repression of the two promoters P1 and P2 of the gal operon requires the formation of a DNA loop encompassing the promoters. In vitro, DNA looping-mediated repression involves binding of the Gal repressor (GalR) to two gal operators (OE and OI) and binding of the histone-like protein HU to a specific locus (hbs) about the midpoint between OE and OI, and supercoiled DNA. Without DNA looping, GalR binding to OE partially represses P1 and stimulates P2. We investigated the requirement for DNA supercoiling and HU in repression of the gal promoters in vivo in strains containing a fusion of a reporter gene, gusA or lacZ, to each promoter individually. While the P1 promoter was found to be repressible in the absence of DNA supercoiling and HU, the repression of P2 was entirely dependent upon DNA supercoiling in vivo. The P2 promoter was fully derepressed when supercoiling was inhibited by the addition of coumermycin in cells. P2, but not P1, was also totally derepressed by the absence of HU or the OI operator. From these results, we propose that the repression of the gal promoters in vivo is mediated by the formation of a higher order DNA-multiprotein complex containing GalR, HU and supercoiled DNA. In the absence of this complex, P1 but not P2 is still repressed by GalR binding to OE. The specific nucleoprotein complexes involving histone-like proteins, which repress promoter activity while remaining sensitive to inducing signals, as discussed, may occur more generally in bacterial nucleoids.

Aminocoumarins↗

PubMed2003

Estrogen receptor corepressors -- a role in human breast cancer?

Estrogen receptor alpha (ERalpha) has an established role in promoting breast cancer. Transcriptional activation by ERalpha is a complex and multistep process, and it is influenced by coactivator and corepressor proteins that can either positively or negatively modulate ERalpha-mediated transcriptional activity. Corepressors are proposed to provide a counterbalance to the estrogen-induced transactivation, and represent a potential mechanism employed by the cell to regulate hormonal responses. In this review, we present evidence from tissue culture, animal and clinical studies, supporting the hypothesis that corepressors are crucial regulators of ERalpha-mediated action, and that their loss could promote breast cancer development and resistance to endocrine therapy. We propose that ERalpha corepressors play an important biological role by controlling the magnitude of the estrogen response, mediating antiestrogen inhibition of ERalpha, repressing DNA-bound ERalpha in the absence of the ligand, and conferring active repression of ERalpha-downregulated genes. Different ERalpha corepressors regulate steroid receptor activity through a variety of mechanisms, including formation of multiprotein complexes that are able to affect chromatin remodeling, histone deacetylation, or basal transcription. Other mechanisms include competition with coactivators, interference with DNA binding and ERalpha homodimerization, alteration of ERalpha stability, sequestration of ERalpha in the cytoplasm, and effects on RNA processing. Most ERalpha corepressors can control the receptor's activity through more than one mechanism, and it is possible that the synergy between different pathways cooperates to fully inhibit ERalpha transcriptional activity, and create an integrated response to a variety of different cellular signaling pathways. We will discuss the role of corepressors in tumor suppression and the link they might present between ERalpha regulation and DNA repair. Finally, we will discuss major challenges in the field and speculate on the exciting findings that await us in the next few years.

Breast Neoplasms↗

PubMed1997

Identification of the proteins of the yeast U1 small nuclear ribonucleoprotein complex by mass spectrometry.

Here we report the rapid identification of the proteins of the spliceosomal U1 small nuclear ribonucleoprotein (snRNP) from the yeast Saccharomyces cerevisiae by searching mass spectrometric data in genomic sequence databases. The U1 snRNP, containing a histidine-tagged 70K protein, was isolated from cell extracts by anti m3G-cap immunoaffinity and subsequent nickel nitrilotriacetic acid chromatography. A U1 snRNP fraction containing 20 proteins was obtained. Further purification by glycerol gradient centrifugation identified nine U1 snRNP specific and six common proteins. The U1 snRNP proteins were partially sequenced by nanoelectrospray mass spectrometry, and their genes were identified in the data base via multiple peptide sequence tags. Apart from the already known common proteins D1, D3, F, and G, the D2 and E homologs were also identified. The same six common proteins were detected in core U2 snRNP, which was purified and analyzed separately. The biochemical association of these six proteins with yeast snRNPs is shown here for the first time. Intriguingly, the Sm B/B' homolog was not detected. In addition to the well characterized yeast U1 specific proteins [U1-70K (Snp1p), U1-A (Mud1p), Prp39p, and Prp40p] the homolog of the U1-C protein was identified together with four additional novel U1 specific proteins, which are not found in mammalian U1. This is the first time that the components of a multiprotein complex from an organism with a sequenced genome have been characterized by mass spectrometry. The technique should be applicable to any protein complex that can be biochemically purified from an organism whose genome is known.

Amino Acid Sequence↗