Restriction mapping and localization of the lactose-metabolizing genes of Streptococcus cremoris pDI-21

Summary The conjugative plasmid pDI-21 (63 kb) from Streptococcus cremoris encodes genes which are responsible for lactose utilization and protein degradation. Restriction mapping and hybridization using heterologous probes have localized these genes to regions of 12.5 kb and 4.4 kb. The tagatose-6-phosphate pathway genes were closely linked and were assigned in the order; galactose-6-phosphate isomerase, d-tagatose-6-phosphate kinase and tagatose-1,6-bisphosphate aldolase. The Lac-PTS genes and the phospho--galactosidase gene were mapped approximately 1 kb from the tagatose-6-phosphate genes.     

Predicting evolutionary potential. I. Predicting the evolution of a lactose-PTS system in Escherichia coli.

Genomes contain not only information for current biological functions, but also information for potential novel functions that may allow the host to adapt to new environments. The field of experimental evolution studies that potential by selecting for novel functions and deducing the means by which the function evolved, but until now it has not attempted to predict the outcomes of such experiments. Here I present a model system that is being developed specifically to examine the issue of what kind of information is most useful in predicting how novel functions will evolve. The system is the evolution of a Lac-PTS transport system and a phospho-beta-galactosidase hydrolase system as a novel pathway for metabolism of lactose in Escherichia coli. Two kinds of information, sequence-based phylogenetic inference and biochemical activity, are considered as predictors of which E. coli genes will evolve the required new functions. Both biochemical data and phylogenetic inference predict that the cryptic celABC genes, which currently specify a PTS-beta-glucoside transport system, are most likely to evolve into a PTS-lactose transport system. Phylogenetic inference predicts that the bglA gene, which currently specifies a phospho-beta-glucosidase, is most likely to evolve into a phospho-beta-galactosidase. In contrast, biochemical data predict that the cryptic bglB gene, which also currently specifies a phospho-beta-glucosidase, is most likely to evolve into a phospho-beta-galactosidase.     

Plant receptor kinases bind and phosphorylate 14-3-3 proteins

14-3-3 proteins are pSer/pThr-binding proteins that interact with a wide array of cellular ‘client’ proteins. The plant brassinosteroids (BRs) receptor, BRASSINOSTEROID INSENSITIVE 1 (BRI1), is a member of the large family of leucine-rich repeat receptor-like kinases (LRR-RLKs) that contain cytoplasmic protein kinase domains. At least two LRR-RLKs are involved in BR perception and signal transduction: BRI1 and BRI1-associated receptor kinase 1 (BAK1). We determined that several 14-3-3 proteins bind to BRI1-CD and are phosphorylated by BRI1, BAK1 and At3g21430 receptor kinases in vitro. Moreover, we observed14-3-3 s are phosphorylated on threonine residue(s) with BR-dependent manner. To reveal the function of 14-3-3 proteins interacting with LRR-RLKs, we treated tyrosine phosphatase (PTP1B) to the BRI1-CD recombinant protein, which is autophosphorylated on tyrosine residue(s). Tyrosine autophosphorylation signal was disappeared, suggesting that 14-3-3 proteins cannot protect BRI1 tyrosine phosphorylation from PTP1B phosphatase. Our study suggests that 14-3-3 proteins may be important for plant growth and development through BR signaling.     

Multiple Skp1-related proteins in Caenorhabditis elegans: diverse patterns of interaction with Cullins and F-box proteins

BACKGROUND: The ubiquitin-proteasome pathway of proteolysis controls the abundance of specific regulatory proteins. The SCF complex is a type of ubiquitin-protein ligase (E3) that contributes to this pathway in many biological systems. In yeast and mammals, the SCF complex consists of common components, including Skp1, Cdc53/Cul1, and Rbx1, as well as variable components known as F-box proteins. Whereas only one functional Skp1 gene is present in the human genome, the genome of Caenorhabditis elegans has now been shown to contain at least 21 Skp1-related (skr) genes. The biochemical properties, expression, and function of the C. elegans SKR proteins were examined. RESULTS: Of the 17 SKR proteins examined, eight (SKR-1, -2, -3, -4, -7, -8, -9, and -10) were shown to interact with C. elegans CUL1 by yeast two-hybrid analysis or a coimmunoprecipitation assay in mammalian cells. Furthermore, SKR proteins exhibited diverse binding specificities for C. elegans F-box proteins. The tissue specificity of expression of the CUL1-interacting SKR proteins was also varied. Suppression of skr-1 or skr-2 genes by double-stranded RNA interference resulted in embryonic death, whereas that of skr-7, -8, -9, or -10 was associated with slow growth and morphological abnormalities. CONCLUSIONS: The multiple C. elegans SKR proteins exhibit marked differences in their association with Cullins and F-box proteins, in tissue specificity of expression, and in phenotypes associated with functional suppression by RNAi. At least eight of the SKR proteins may, like F-box proteins, act as variable components of the SCF complex in C. elegans.     

Studies on the high-mobility-group non-histone proteins from hen oviduct.

Nuclear high-mobility-group (HMG) proteins were isolated from hen oviduct. These were proteins HMG-1, -2, -3, -14 and -17, which are equivalent to the classification of calf thymus HMG proteins. Hen oviduct proteins HMG-1 and -2 were individually isolated by HCIO4.extraction and CM-Sephadex chromatographic separation. Their mol.wts. were determined as 28 000 and 27 000, respectively. The proteins have a high content of acidic and basic amino acids. The association of proteins HMG-1 and -2 with the genome of hen oviduct nuclei was probed by a limited digestion with nucleases. Hen oviduct nuclei were incubated with deoxyribonuclease I or micrococcal nuclease until 10% of the DNA was digested. The nuclear suspension was centrifuged and the contents of proteins HMG-1 and -2 in the supernatant and sediment fractions were analysed by polyacrylamide-gel electrophoresis. HMG proteins were found to be preferentially released by micrococcal-nuclease digestion rather than by deoxyribonuclease I.     

The potassium channel KAT1 is activated by plant and animal 14-3-3 proteins

14-3-3 proteins modulate the plant inward rectifier K+ channel KAT1 heterologously expressed in Xenopus oocytes. Injection of recombinant plant 14-3-3 proteins into oocytes shifted the activation curve of KAT1 by +11 mV and increased the tau(on). KAT1 was also modulated by 14-3-3 proteins of Xenopus oocytes. Titration of the endogenous 14-3-3 proteins by injection of the peptide Raf 621p resulted in a strong decrease in KAT1 current (approximately 70% at -150 mV). The mutation K56E performed on plant protein 14-3-3 in a highly conserved recognition site prevented channel activation. Because the maximal conductance of KAT1 was unaffected by 14-3-3, we can exclude that they act by increasing the number of channels, thus ruling out any effect of these proteins on channel trafficking and/or insertion into the oocyte membrane. 14-3-3 proteins also increased KAT1 current in inside-out patches, suggesting a direct interaction with the channel. Direct interaction was confirmed by overlay experiments with radioactive 14-3-3 on oocyte membranes expressing KAT1.     

Quantitative secretome analysis reveals that COL6A1 is a metastasis-associated protein using stacking gel-aided purification combined with iTRAQ labeling

In cancer metastasis, secreted proteins play an important role in promoting cancer cell migration and invasion and thus also in the increase of cancer metastasis in the extracellular microenvironment. In this study, we developed a strategy that combined a simple gel-aided protein purification with iTRAQ labeling to quantify and discover the metastasis-associated proteins in the lung cancer cell secretome. Secreted proteins associated with lung cancer metastasis were produced using CL1-0 and CL1-5 cells with different metastatic abilities. Quantitative secretomics analysis identified a total of 353 proteins, 7 of which were considered to be metastasis-associated proteins. These included TIMP1, COL6A1, uPA, and AAT, all of which were higher in CL1-5, and AL1A1, PRDX1, and NID1, which were higher in CL1-0. Six of these metastasis-associated proteins were validated with Western blot analysis. In addition, pathway analysis was performed in building the interaction network between the identified metastasis-associated proteins. Further functional analysis of COL6A1 on the metastatic abilities of CL1 cells was also carried out. An RNA interference-based knock-down of COL6A1 suppressed the metastatic ability of CL1-5 cells; in contrast, a plasmid-transfected overexpression of COL6A1 increased the metastatic ability of CL1-0 cells. This study describes a simple and high throughput sample purification method that can be used for the quantitative secretomics analysis of metastasis-associated proteins.     

Novel 150- and 180-kDa glycoproteins that bind transforming growth factor (TGF)-beta 1 but not TGF-beta 2 are present in several cell lines.

We identified transforming growth factor-beta (TGF-beta)-binding proteins which are distinct from previously described TGF-beta receptors or TGF-beta-binding proteins. These TGF-beta-binding proteins migrate as 150- and 180-kDa 125I-TGF-beta 1 affinity-labeled complexes which are consistently co-expressed in A549, Mv1Lu, MG-63, and BS-C-1 cells. They differ from the types I, II, and III TGF-beta receptors in their electrophoretic mobilities, their lack of binding to TGF-beta 2, and their failure to undergo the marked down-regulation seen with types I, II, and III receptors following a 16-h incubation with TGF-beta 1. The 150- and 180-kDa TGF-beta-binding proteins also are distinct from the recently described disulfide-linked TGF-beta 1-binding proteins which are present in rat glomeruli. In contrast to the glomerular TGF-beta 1-binding proteins, the electrophoretic mobilities of the 150- and 180-kDa binding proteins are unchanged following reduction. In addition, the 150- and 180-kDa TGF-beta-binding proteins are present in the detergent-rich phase during Triton X-114 phase separation, whereas the glomerular TGF-beta-binding proteins partition exclusively into the detergent-poor phase.     

Role for the L1-52/55K protein in the serotype specificity of adenovirus DNA packaging

The packaging of adenovirus (Ad) DNA into virions is dependent upon cis-acting sequences and trans-acting proteins. We studied the involvement of Ad packaging proteins in the serotype specificity of packaging. Both Ad5 and Ad17 IVa2 and L4-22K proteins complemented the growth of Ad5 IVa2 and L4-22K mutant viruses, respectively. In contrast, the Ad5 L1-52/55K protein complemented an Ad5 L1-52/55K mutant virus, but the Ad17 L1-52/55K protein did not. The analysis of chimeric proteins demonstrated that the N-terminal half of the Ad5 L1-52/55K protein mediated this function. Finally, we demonstrate that the L4-33K and L4-22K proteins have distinct functions during infection.     

Synaptotagmin-like protein 1-3: a novel family of C-terminal-type tandem C2 proteins

Synaptotagmins (Syt), rabphilin-3A, and Doc2 belong to a family of carboxyl terminal type (C-type) tandem C2 proteins and are thought to be involved in vesicular trafficking. We have cloned and characterized a novel family of C-type tandem C2 proteins, designated Slp1-3 (synaptotagmin-like protein 1-3). The Slp1-3 C2 domains show high homology to granuphilin-a C2 domains, but the amino-terminal domain of Slp1-3 does not contain any known protein motifs or a transmembrane domain. A subcellular fractionation study indicated that Slp1-3 proteins are peripheral membrane proteins. Phospholipid binding experiments indicated that Slp3 is a Ca(2+)-dependent isoform, but Slp1 and Slp2 are Ca(2+)-independent isoforms, because only the Slp3 C2A domain showed Ca(2+)-dependent phospholipid binding activity. The C-terminus of Slp1-3 also bound neurexin Ialpha in vitro, in the same manner as Syt family proteins, which may be important for the membrane association of Slp1-3. In addition, Slp family proteins are differentially distributed in different mouse tissues and at different developmental stages.     

Immunological Characterization of Trichocyst Proteins In the Ciliate Pseudomicrothorax Dubius

ABSTRACT. Ejectable trichocysts were isolated from the ciliate Pseudomicrothorax dubius. Polyclonal antibodies were raised against three groups of trichocyst proteins: G1 (30-31 kDa), G2 (26-27 kDa) and G3 (15-20 kDa). By indirect immunofluorescence, the three antisera strongly label the shafts of ejected trichocysts and the proximal ends of condensed trichocysts within the cells. By immunogold labeling for electron microscopy, the three sera specifically recognize the shafts of both extended and condensed trichocysts and shaft precursors in pretrichocysts as well. On one-dimensional immunoblots of isolated trichocysts, anti-G1 serum recognizes the G1 proteins, anti-G2 serum detects G2 proteins and some G1 proteins, and anti-G3 serum reacts with 15 bands, mainly the G3 and (30-41)-kDa proteins. In cells with and without trichocysts, the sera recognize non-ejectable trichocyst proteins at 41-42 kDa and 47 kDa. On two-dimensional immunoblots of isolated trichocysts, anti-G1 serum labels proteins with a pI of 4.75-5.7, anti-G2 serum labels proteins with a pI of 4.75-6.25 and anti-G3 serum labels proteins with a pI of 4.7-6.6. Analyses of cells with and without trichocysts allow identification of possible precursors between 41 and 47 kDa. Some are in the same pI range as their putative products, but others, labeled by anti-G3 serum, are less acidic than most of their mature products.     

Dimerization and protease resistance: New insight into the function of PR-1

The group 1 pathogenesis-related (PR-1) proteins have long been considered hallmarks of hypersensitive response/defense pathways in plants, but their biochemical functions are still obscure despite resolution of the NMR/X-ray structures of several PR-1-like proteins, including P14a (the prototype PR-1). We report here the characterization of two basic PR-1 proteins (PR-1-1 and PR-1-5) recently identified from hexaploid wheat (Triticum aestivum). Both proteins were expressed in Pichia pastoris as a single major species of ∼15 kDa. Sequence identity of the expressed PR-1 proteins was verified by MALDI-TOF/TOF analysis. Accumulation of the native PR-1-5 protein in pathogen-challenged wheat was confirmed by protein gel blot analysis. Low-temperature SDS-PAGE and yeast two-hybrid assays revealed that PR-1-1 exists primarily as a monomer whereas PR-1-5 forms homodimers. Both PR-1 proteins are resistant to proteases compared to bovine serum albumin, but PR-1-1 shows resistance mainly to subtilisin and protease K (serine proteases) whereas PR-1-5 shows resistance to subtilisin, protease K and papain (a cysteine protease). Site-specific mutations at the five putative active sites in the PR-1 domain all affected dimerization, with the mutations at Glu-72 and Glu-102 (in the PR-1-5 numeration) also diminishing protease resistance. Sequence analysis revealed that the Glu-72 and Glu-102 residues are located in motif-like sequences that are conserved in both PR-1 and the human apoptosis-related caspase proteins. These findings prompt us to examine the function of PR-1 for a role in protease-mediated programmed cell death pathways in plants.     

The plant U1 small nuclear ribonucleoprotein particle 70K protein interacts with two novel serine/arginine-rich proteins.

The U1 small nuclear ribonucleoprotein particle (U1 snRNP) 70K protein (U1-70K), one of the three U1 snRNP-specific proteins, is implicated in basic and alternative splicing of nuclear pre-mRNAs. We have used the Arabidopsis U1-70K in the yeast two-hybrid system to isolate cDNAs encoding proteins that interact with it. This screening has resulted in the isolation of two novel plant serine/arginine-rich (SR) proteins, SRZ-22 and SRZ-21 (SRZ proteins). Neither the N-terminal region nor the arginine-rich C-terminal region of U1-70K alone interact with the SRZ proteins. The interaction of U1-70K with the SRZ proteins is confirmed further in vitro using a blot overlay assay. The plant SRZ proteins are highly similar to each other and contain conserved modular domains unique to different groups of splicing factors in the SR family of proteins. SRZ proteins are similar to human 9G8 splicing factor because they contain a zinc knuckle, precipitate with 65% ammonium sulfate, and cross-react with the 9G8 monoclonal antibody. However, unlike the 9G8 splicing factor, SRZ proteins contain a glycine hinge, a unique feature in other splicing factors (SC35 and ASF/SF2), located between the RNA binding domain and the zinc knuckle. SRZ-22 and SRZ-21 are encoded by two distinct genes and are expressed in all tissues tested with varied levels of expression. Our results suggest that the plant SRZ proteins represent a new group of SR proteins. The interaction of plant U1-70K with the SRZ proteins may account for some differences in pre-mRNA splicing between plants and animals.     

Self-association of the spindle pole body-related intermediate filament protein Fin1p and its phosphorylation-dependent interaction with 14-3-3 proteins in yeast

The Fin1 protein of the yeast Saccharomyces cerevisiae forms filaments between the spindle pole bodies of dividing cells. In the two-hybrid system it binds to 14-3-3 proteins, which are highly conserved proteins involved in many cellular processes and which are capable of binding to more than 120 different proteins. Here, we describe the interaction of the Fin1 protein with the 14-3-3 proteins Bmh1p and Bmh2p in more detail. Purified Fin1p interacts with recombinant yeast 14-3-3 proteins. This interaction is strongly reduced after dephosphorylation of Fin1p. Surface plasmon resonance analysis showed that Fin1p has a higher affinity for Bmh2p than for Bmh1p (K(D) 289 versus 585 nm). Sequences in both the central and C-terminal part of Fin1p are required for the interaction with Bmh2p in the two-hybrid system. In yeast strains lacking 14-3-3 proteins Fin1 filament formation was observed, indicating that the 14-3-3 proteins are not required for this process. Fin1 also interacts with itself in the two-hybrid system. For this interaction sequences at the C terminus, containing one of two putative coiled-coil regions, are sufficient. Fin1p-Fin1p interactions were demonstrated in vivo by fluorescent resonance energy transfer between cyan fluorescent protein-labeled Fin1p and yellow fluorescent protein-labeled Fin1p.     

Characterization of functional domains of human EB1 family proteins

EB1 family proteins are evolutionarily conserved proteins that bind microtubule plus-ends and centrosomes and regulate the dynamics and organization of microtubules. Human EB1 family proteins, which include EB1, EBF3, and RP1, also associate with the tumor suppressor protein adenomatous polyposis coli (APC) and p150glued, a component of the dynactin complex. The structural basis for interaction between human EB1 family proteins and their associated proteins has not been defined in detail. EB1 family proteins have a calponin homology (CH) domain at their N terminus and an EB1-like C-terminal motif at their C terminus; the functional importance of these domains has not been determined. To better understand functions of human EB1 family proteins and to reveal functional similarities and differences among these proteins, we performed detailed characterizations of interactions between human EB1 family proteins and their associated proteins. We show that amino acids 1-133 of EB1 and EBF3 and the corresponding region of RP1, which contain a CH domain, are necessary and sufficient for binding microtubules, thus demonstrating for the first time that a CH domain contributes to binding microtubules. EB1 family proteins use overlapping but different regions that contain the EB1-like C-terminal motif to associate with APC and p150glued. Neither APC nor p150glued binding domain is necessary for EB1 or EBF3 to induce microtubule bundling, which requires amino acids 1-181 and 1-185 of EB1 and EBF3, respectively. We also determined that the EB1 family protein-binding regions are amino acids 2781-2820 and 18-111 of APC and p150glued, respectively.     

Partial purification and characterization of GDP dissociation stimulator (GDS) for the rho proteins from bovine brain cytosol

A novel type of regulatory proteins for the rho proteins (rhoA p21 and rhoB p20), ras p21-like small GTP-binding proteins (G proteins), are partially purified from bovine brain cytosol. These regulatory proteins, named rho GDP dissociation stimulator (GDS) 1 and -2, stimulate the dissociation of GDP from rhoA p21 and rhoB p20. rho GDS1 and -2 are inactive for other ras p21/ras p21-like small G proteins including c-Ha-ras p21, smg p21B, and smg p25A. Since we have previously shown that the rate limiting step for the GDP/GTP exchange reaction of the rho proteins is the dissociation of GDP from these proteins, the present results suggest that rho GDS1 and -2 stimulate the GDP/GTP exchange reaction of the rho proteins. rho GDS1 and -2 are distinct from the GAP- and GDI-types of regulatory proteins for the rho proteins previously purified from bovine brain cytosol. rho GAP stimulates the GTPase activity of the rho proteins and rho GDI inhibits the GDP/GTP exchange reaction of the rho proteins. The present results together with these earlier observations indicate that the rho proteins are regulated by at least three different types of regulatory proteins, GDS, GDI, and GAP.     

Five structural classes of major outer membrane proteins in Neisseria meningitidis

Group B Neisseria meningitidis is thus far subdivided into 15 protein serotypes based on antigenically different major outer membrane proteins. Most serotypes have three or four major proteins in their outer membranes. Comparative structural analysis by chymotryptic 125I-peptide mapping was performed on these major proteins from the prototype strains as well as from six non-serotypable strains. The major outer membrane proteins from each of the serotypes were first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using the Laemmli system. Individual proteins within the gel slices were radioiodinated and digested with chymotrypsin, and then their 125I-peptides were separated by electrophoresis and chromatography on cellulose thin-layer plates. The peptide maps obtained by autoradiography were categorized into five different structural classes which correlated with the apparent molecular weights of proteins, i.e., 46 +/- 1K, 41 +/- 1K, 38 +/- 1K, 33 +/- 1K, and 28 +/- 1K. Each of the major outer membrane proteins within a strain had a distinctly different chymotryptic peptide map, indicating significant differences in the primary structure of these proteins. In contrast, outer membrane proteins of the same or very similar molecular weight from different serotype strains had similar, occasionally identical peptide maps, indicating a high degree of structural homology. The unique peptides from proteins of the same structural classes were often hydrophilic, whereas common peptides were often hydrophobic, suggesting that the serotype determinants reside within the variable hydrophilic regions of major outer membrane proteins.