Multiple domains of repressor activator protein 1 contribute to facilitated binding of glycolysis regulatory protein 1

The function of repressor activator protein 1 (Rap1p) at glycolytic enzyme gene upstream activating sequence (UAS) elements in Saccharomyces cerevisiae is to facilitate binding of glycolysis regulatory protein 1 (Gcr1p) at adjacent sites. Rap1p has a modular domain structure. In its amino terminus there is an asymmetric DNA-bending domain, which is distinct from its DNA-binding domain, which resides in the middle of the protein. In the carboxyl terminus of Rap1p lie its silencing and putative activation domains. We carried out a molecular dissection of Rap1p to identify domains contributing to its ability to facilitate binding of Gcr1p. We prepared full-length and three truncated versions of Rap1p and tested their ability to facilitate binding of Gcr1p by gel shift assay. The ability to detect ternary complexes containing Rap1p⋅DNA⋅Gcr1p depended on the presence of binding sites for both proteins in the probe DNA. The DNA-binding domain of Rap1p, although competent to bind DNA, was unable to facilitate binding of Gcr1p. Full-length Rap1p and the amino- and carboxyl-truncated versions of Rap1p were each able to facilitate binding of Gcr1p at an appropriately spaced binding site. Under these conditions, Gcr1p displayed an approximately 4-fold greater affinity for Rap1p-bound DNA than for otherwise identical free DNA. When spacing between Rap1p- and Gcr1p-binding sites was altered by insertion of five nucleotides, the ability to form ternary Rap1p⋅DNA⋅Gcr1p complexes was inhibited by all but the DNA-binding domain of Rap1p itself; however, the ability of each individual protein to bind the DNA probe was unaffected.

Mice mutant for glucokinase regulatory protein exhibit decreased liver glucokinase: A sequestration mechanism in metabolic regulation

The importance of glucokinase (GK; EC 2.7.1.12) in glucose homeostasis has been demonstrated by the association of GK mutations with diabetes mellitus in humans and by alterations in glucose metabolism in transgenic and gene knockout mice. Liver GK activity in humans and rodents is allosterically inhibited by GK regulatory protein (GKRP). To further understand the role of GKRP in GK regulation, the mouse GKRP gene was inactivated. With the knockout of the GKRP gene, there was a parallel loss of GK protein and activity in mutant mouse liver. The loss was primarily because of posttranscriptional regulation of GK, indicating a positive regulatory role for GKRP in maintaining GK levels and activity. As in rat hepatocytes, both GK and GKRP were localized in the nuclei of mouse hepatocytes cultured in low-glucose-containing medium. In the presence of fructose or high concentrations of glucose, conditions known to relieve GK inhibition by GKRP in vitro, only GK was translocated into the cytoplasm. In the GKRP-mutant hepatocytes, GK was not found in the nucleus under any tested conditions. We propose that GKRP functions as an anchor to sequester and inhibit GK in the hepatocyte nucleus, where it is protected from degradation. This ensures that glucose phosphorylation is minimal when the liver is in the fasting, glucose-producing phase. This also enables the hepatocytes to rapidly mobilize GK into the cytoplasm to phosphorylate and store or metabolize glucose after the ingestion of dietary glucose. In GKRP-mutant mice, the disruption of this regulation and the subsequent decrease in GK activity leads to altered glucose metabolism and impaired glycemic control.

PAT1, a microtubule-interacting protein, recognizes the basolateral sorting signal of amyloid precursor protein

In epithelial cells, sorting of membrane proteins to the basolateral surface depends on the presence of a basolateral sorting signal (BaSS) in their cytoplasmic domain. Amyloid precursor protein (APP), a basolateral protein implicated in the pathogenesis of Alzheimer’s disease, contains a tyrosine-based BaSS, and mutation of the tyrosine residue results in nonpolarized transport of APP. Here we report identification of a protein, termed PAT1 (protein interacting with APP tail 1), that interacts with the APP-BaSS but binds poorly when the critical tyrosine is mutated and does not bind the tyrosine-based endocytic signal of APP. PAT1 shows homology to kinesin light chain, which is a component of the plus-end directed microtubule-based motor involved in transporting membrane proteins to the basolateral surface. PAT1, a cytoplasmic protein, associates with membranes, cofractionates with APP-containing vesicles, and binds microtubules in a nucleotide-sensitive manner. Cotransfection of PAT1 with a reporter protein shows that PAT1 is functionally linked with intracellular transport of APP. We propose that PAT1 is involved in the translocation of APP along microtubules toward the cell surface.

The Arabidopsis CHL1 protein plays a major role in high-affinity nitrate uptake

The CHL1 (NRT1) gene of Arabidopsis encodes a nitrate-inducible nitrate transporter that is thought to be a component of the low-affinity (mechanism II) nitrate-uptake system in plants. A search was performed to find high-affinity (mechanism I) uptake mutants by using chlorate selections on plants containing Tag1 transposable elements. Chlorate-resistant mutants defective in high-affinity nitrate uptake were identified, and one had a Tag1 insertion in chl1, which was responsible for the phenotype. Further analysis showed that chl1 mutants have reduced high-affinity uptake in induced plants and are missing a saturable component of the constitutive, high-affinity uptake system in addition to reduced low-affinity uptake. The contribution of CHL1 to constitutive high-affinity uptake is higher when plants are grown at more acidic pH, conditions that increase the level of CHL1 mRNA. chl1 mutants show reduced membrane depolarization in root epidermal cells in response to low (250 μM) and high (10 mM) concentrations of nitrate. Low levels of nitrate (100 μM) induce a rapid increase in CHL1 mRNA. These results show that CHL1 is an important component of both the high-affinity and the low-affinity nitrate-uptake systems and indicate that CHL1 may be a dual-affinity nitrate transporter.

Karyopherin β2 mediates nuclear import of a mRNA binding protein

We have cloned and sequenced cDNA for human karyopherin β2, also known as transportin. In a solution binding assay, recombinant β2 bound directly to recombinant nuclear mRNA-binding protein A1. Binding was inhibited by a peptide representing A1’s previously characterized M9 nuclear localization sequence (NLS), but not by a peptide representing a classical NLS. As previously shown for karyopherin β1, karyopherin β2 bound to several nucleoporins containing characteristic peptide repeat motifs. In a solution binding assay, both β1 and β2 competed with each other for binding to immobilized repeat nucleoporin Nup98. In digitonin-permeabilized cells, β2 was able to dock A1 at the nuclear rim and to import it into the nucleoplasm. At low concentrations of β2, there was no stimulation of import by the exogenous addition of the GTPase Ran. However, at higher concentrations of β2 there was marked stimulation of import by Ran. Import was inhibited by the nonhydrolyzable GTP analog guanylyl imidodiphosphate by a Ran mutant that is unable to hydrolyze GTP and also by wheat germ agglutinin. Consistent with the solution binding results, karyopherin β2 inhibited karyopherin α/β1-mediated import of a classical NLS containing substrate and, vice versa, β1 inhibited β2-mediated import of A1 substrate, suggesting that the two import pathways merge at the level of docking of β1 and β2 to repeat nucleoporins.

Stimulation of amyloid precursor protein synthesis by adrenergic receptors coupled to cAMP formation

Amyloid plaques in Alzheimer disease are primarily aggregates of Aβ peptides that are derived from the amyloid precursor protein (APP). Neurotransmitter agonists that activate phosphatidylinositol hydrolysis and protein kinase C stimulate APP processing and generate soluble, non-amyloidogenic APP (APPs). Elevations in cAMP oppose this stimulatory effect and lead to the accumulation of cell-associated APP holoprotein containing amyloidogenic Aβ peptides. We now report that cAMP signaling can also increase cellular levels of APP holoprotein by stimulating APP gene expression in astrocytes. Treatment of astrocytes with norepinephrine or isoproterenol for 24 h increased both APP mRNA and holoprotein levels, and these increases were blocked by the β-adrenergic antagonist propranolol. Treatment with 8-bromo-adenosine 3′,5′-cyclic monophosphate or forskolin for 24 h similarly increased APP holoprotein levels; astrocytes were also transformed into process-bearing cells expressing increased amounts of glial fibrillary acidic protein, suggesting that these cells resemble reactive astrocytes. The increases in APP mRNA and holoprotein in astrocytes caused by cAMP stimulation were inhibited by the immunosuppressant cyclosporin A. Our study suggests that APP overexpression by reactive astrocytes during neuronal injury may contribute to Alzheimer disease neuropathology, and that immunosuppressants can inhibit cAMP activation of APP gene transcription.

Mitochondrial stress protein recognition of inactivated dehydrogenases during mammalian cell death

The mammalian renal toxicant tetrafluoroethylcysteine (TFEC) is metabolized to a reactive intermediate that covalently modifies the lysine residues of a select group of mitochondrial proteins, forming difluorothioamidyl lysine protein adducts. Cellular damage is initiated by this process and cell death ensues. NH2-terminal sequence analysis of purified mitochondrial proteins containing difluorothioamidyl lysine adducts identified the lipoamide succinyltransferase and dihydrolipoamide dehydrogenase subunits of the α-ketoglutarate dehydrogenase complex (αKGDH), a key regulatory component of oxidative metabolism, as targets for TFEC action. Adduct formation resulted in marked inhibition of αKGDH enzymatic activity, whereas the related pyruvate dehydrogenase complex was unmodified by TFEC and its activity was not inhibited in vivo. Covalent modification of αKGDH subunits also resulted in interactions with mitochondrial chaperonin HSP60 in vivo and with HSP60 and mitochondrial HSP70 in vitro. These observations confirm the role of mammalian stress proteins in the recognition of abnormal proteins and provide supporting evidence for reactive metabolite-induced cell death by modification of critical protein targets.

Capping and methylation of mRNA by purified recombinant VP4 protein of bluetongue virus

The core of bluetongue virus (BTV) is a multienzyme complex composed of two major proteins (VP7 and VP3) and three minor proteins (VP1, VP4, and VP6) in addition to the viral genome. The core is transcriptionally active and produces capped mRNA from which all BTV proteins are translated, but the relative role of each core component in the overall reaction process remains unclear. Previously we showed that the 76-kDa VP4 protein possesses guanylyltransferase activity, a necessary part of the RNA capping reaction. Here, through the use of highly purified (>95%) VP4 and synthetic core-like particles containing VP4, we have investigated the extent to which this protein is also responsible for other activities associated with cap formation. We show that VP4 catalyzes the conversion of unmethylated GpppG or in vitro-produced uncapped BTV RNA transcripts to m7GpppGm in the presence of S-adenosyl-l-methionine. Analysis of the methylated products of the reaction by HPLC identified both methyltransferase type 1 and type 2 activities associated with VP4, demonstrating that the complete BTV capping reaction is associated with this one protein.

Chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein, subsequent folding, and development of fluorescence

The present paper describes the total chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein (GFP). The molecule is made up of 238 amino acid residues in a single polypeptide chain and is nonfluorescent. To carry out the synthesis, a procedure, first described in 1981 for the synthesis of complex peptides, was used. The procedure is based on performing segment condensation reactions in solution while providing maximum protection to the segment. The effectiveness of the procedure has been demonstrated by the synthesis of various biologically active peptides and small proteins, such as human angiogenin, a 123-residue protein analogue of ribonuclease A, human midkine, a 121-residue protein, and pleiotrophin, a 136-residue protein analogue of midkine. The GFP precursor molecule was synthesized from 26 fully protected segments in solution, and the final 238-residue peptide was treated with anhydrous hydrogen fluoride to obtain the precursor molecule of GFP containing two Cys(acetamidomethyl) residues. After removal of the acetamidomethyl groups, the product was dissolved in 0.1 M Tris⋅HCl buffer (pH 8.0) in the presence of DTT. After several hours at room temperature, the solution began to emit a green fluorescence (λmax = 509 nm) under near-UV light. Both fluorescence excitation and fluorescence emission spectra were measured and were found to have the same shape and maxima as those reported for native GFP. The present results demonstrate the utility of the segment condensation procedure in synthesizing large protein molecules such as GFP. The result also provides evidence that the formation of the chromophore in GFP is not dependent on any external cofactor.

A 55-kDa protein isolated from human cells shows DNA glycosylase activity toward 3,N4-ethenocytosine and the G/T mismatch

Etheno adducts in DNA arise from multiple endogenous and exogenous sources. Of these adducts we have reported that, 1,N6-ethenoadenine (ɛA) and 3,N4-ethenocytosine (ɛC) are removed from DNA by two separate DNA glycosylases. We later confirmed these results by using a gene knockout mouse lacking alkylpurine-DNA-N-glycosylase, which excises ɛA. The present work is directed toward identifying and purifying the human glycosylase activity releasing ɛC. HeLa cells were subjected to multiple steps of column chromatography, including two ɛC-DNA affinity columns, which resulted in >1,000-fold purification. Isolation and renaturation of the protein from SDS/polyacrylamide gel showed that the ɛC activity resides in a 55-kDa polypeptide. This apparent molecular mass is approximately the same as reported for the human G/T mismatch thymine-DNA glycosylase. This latter activity copurified to the final column step and was present in the isolated protein band having ɛC-DNA glycosylase activity. In addition, oligonucleotides containing ɛC⋅G or G/T(U), could compete for ɛC protein binding, further indicating that the ɛC-DNA glycosylase is specific for both types of substrates in recognition. The same substrate specificity for ɛC also was observed in a recombinant G/T mismatch DNA glycosylase from the thermophilic bacterium, Methanobacterium thermoautotrophicum THF.

Human fatty acid synthase: Assembling recombinant halves of the fatty acid synthase subunit protein reconstitutes enzyme activity

Our model of the native fatty acid synthase (FAS) depicts it as a dimer of two identical multifunctional proteins (Mr ≈ 272,000) arranged in an antiparallel configuration so that the active Cys-SH of the β-ketoacyl synthase of one subunit (where the acyl group is attached) is juxtaposed within 2 Å of the pantetheinyl-SH of the second subunit (where the malonyl group is bound). This arrangement generates two active centers for fatty acid synthesis and predicts that if we have two appropriate halves of the monomer, we should be able to reconstitute an active fatty acid-synthesizing site. We cloned, expressed, and purified catalytically active thioredoxin (TRX) fusion proteins of the NH2-terminal half of the human FAS subunit protein (TRX-hFAS-dI; residues 1–1,297; Mr ≈ 166) and of the C-terminal half (TRX-hFAS-dII-III; residues 1,296–2,504; Mr ≈ 155). Adding equivalent amounts of TRX-hFAS-dI and TRX-hFAS-dII-III to a reaction mixture containing acetyl-CoA, malonyl-CoA, and NADPH resulted in the synthesis of long-chain fatty acids. The rate of synthesis was dependent upon the presence of both recombinant proteins and reached a constant level when they were present in equivalent amounts, indicating that the reconstitution of an active fatty acid-synthesizing site required the presence of every partial activity associated with the subunit protein. Analyses of the product acids revealed myristate to be the most abundant with small amounts of palmitate and stearate, possibly because of the way the fused recombinant proteins interacted with each other so that the thioesterase hydrolyzed the acyl group in its myristoyl state. The successful reconstitution of the human FAS activity from its domain I and domains II and III fully supports our model for the structure–function relationship of FAS in animal tissues.

Prominin, a novel microvilli-specific polytopic membrane protein of the apical surface of epithelial cells, is targeted to plasmalemmal protrusions of non-epithelial cells

Using a new mAb raised against the mouse neuroepithelium, we have identified and cDNA-cloned prominin, an 858-amino acid-containing, 115-kDa glycoprotein. Prominin is a novel plasma membrane protein with an N-terminal extracellular domain, five transmembrane segments flanking two short cytoplasmic loops and two large glycosylated extracellular domains, and a cytoplasmic C-terminal domain. DNA sequences from Caenorhabditis elegans predict the existence of a protein with the same features, suggesting that prominin is conserved between vertebrates and invertebrates. Prominin is found not only in the neuroepithelium but also in various other epithelia of the mouse embryo. In the adult mouse, prominin has been detected in the brain ependymal layer, and in kidney tubules. In these epithelia, prominin is specific to the apical surface, where it is selectively associated with microvilli and microvilli-related structures. Remarkably, upon expression in CHO cells, prominin is preferentially localized to plasma membrane protrusions such as filopodia, lamellipodia, and microspikes. These observations imply that prominin contains information to be targeted to, and/or retained in, plasma membrane protrusions rather than the planar cell surface. Moreover, our results show that the mechanisms underlying targeting of membrane proteins to microvilli of epithelial cells and to plasma membrane protrusions of non-epithelial cells are highly related.

Phosphorylation of the immunosuppressant FK506-binding protein FKBP52 by casein kinase II: Regulation of HSP90-binding activity of FKBP52

FKBP52 (HSP56, p59, HBI) is the 59-kDa immunosuppressant FK506-binding protein and has peptidyl prolyl isomerase as well as a chaperone-like activity in vitro. FKBP52 associates with the heat shock protein HSP90 and is included in the steroid hormone receptor complexes in vivo. FKBP52 possesses a well conserved phosphorylation site for casein kinase II (CK2) that was previously shown to be associated with HSP90. Here we examined whether FKBP52 is phosphorylated by CK2 both in vivo and in vitro. Recombinant rabbit FKBP52 was phosphorylated by purified CK2. We expressed and purified deletion mutants of FKBP52 to determine the site(s) phosphorylated by CK2. Thr-143 in the hinge I region was identified as the major phosphorylation site for CK2. A synthetic peptide corresponding to this region was phosphorylated by CK2, and the peptide competitively inhibited the phosphorylation of other substrates by CK2. The [32P]phosphate labeling of FKBP52-expressing cells revealed that the same site is also phosphorylated in vivo. FK506 binding to FKBP52 did not affect the phosphorylation by CK2 and, conversely, the FK506-binding activity of FKBP52 was not affected by the phosphorylation. Most importantly, CK2-phosphorylated FKBP52 did not bind to HSP90. These results indicate that CK2 phosphorylates FKBP52 both in vitro and in vivo and thus may regulate the protein composition of chaperone-containing complexes such as those of steroid receptors and certain protein kinases.

Inhibition of ubiquitin/proteasome-dependent protein degradation by the Gly-Ala repeat domain of the Epstein–Barr virus nuclear antigen 1

The Epstein–Barr virus (EBV) encoded nuclear antigen (EBNA) 1 is expressed in latently infected B lymphocytes that persist for life in healthy virus carriers and is the only viral protein regularly detected in all EBV associated malignancies. The Gly-Ala repeat domain of EBNA1 was shown to inhibit in cis the presentation of major histocompatibility complex (MHC) class I restricted cytotoxic T cell epitopes from EBNA4. It appears that the majority of antigens presented via the MHC I pathway are subject to ATP-dependent ubiquitination and degradation by the proteasome. We have investigated the influence of the repeat on this process by comparing the degradation of EBNA1, EBNA4, and Gly-Ala containing EBNA4 chimeras in a cell-free system. EBNA4 was efficiently degraded in an ATP/ubiquitin/proteasome-dependent fashion whereas EBNA1 was resistant to degradation. Processing of EBNA1 was restored by deletion of the Gly-Ala domain whereas insertion of Gly-Ala repeats of various lengths and in different positions prevented the degradation of EBNA4 without appreciable effect on ubiquitination. Inhibition was also achieved by insertion of a Pro-Ala coding sequence. The results suggest that the repeat may affect MHC I restricted responses by inhibiting antigen processing via the ubiquitin/proteasome pathway. The presence of regularly interspersed Ala residues appears to be important for the effect.

WIP, a protein associated with Wiskott–Aldrich syndrome protein, induces actin polymerization and redistribution in lymphoid cells

Wiskott–Aldrich syndrome (WAS) is an X-linked immunodeficiency caused by mutations that affect the WAS protein (WASP) and characterized by cytoskeletal abnormalities in hematopoietic cells. By using the yeast two-hybrid system we have identified a proline-rich WASP-interacting protein (WIP), which coimmunoprecipitated with WASP from lymphocytes. WIP binds to WASP at a site distinct from the Cdc42 binding site and has actin as well as profilin binding motifs. Expression of WIP in human B cells, but not of a WIP truncation mutant that lacks the actin binding motif, increased polymerized actin content and induced the appearance of actin-containing cerebriform projections on the cell surface. These results suggest that WIP plays a role in cortical actin assembly that may be important for lymphocyte function.