Functional study of the Saccharomyces cerevisiae Nha1p C-terminus

Saccharomyces cerevisiae cells possess an alkali metal cation antiporter encoded by the NHA1 gene. Nha1p is unique in the family of yeast Na+/H+ antiporters on account of its broad substrate specificity (Na+, Li+, K+) and its long C-terminus (56% of the whole protein). In order to study the role of the C-terminus in Nha1p function, we constructed a series of 13 truncated NHA1 versions ranging from the complete one (2958 nucleotides, 985 amino acids) down to the shortest version (1416 nucleotides, 472 amino acids), with only 41 amino acid residues after the last putative transmembrane domain. Truncated NHA1 versions were expressed in an S. cerevisiae alkali metal cation-sensitive strain (B31; ena1-4Delta nha1Delta). We found that the entire Nha1p C-terminus domain is not necessary for either the proper localization of the antiporter in the plasma membrane or the transport of all four substrates (we identified rubidium as the fourth Nha1p substrate). Partial truncation of the C-terminus of about 70 terminal amino acids improves the tolerance of cells to Na+, Li+ and Rb+ compared with cells expressing the complete Nha1p. The presence of the neighbouring part of the C-terminus (amino acids 883-928), rich in aspartate and glutamate residues, is necessary for the maintenance of maximum Nha1p activity towards sodium and lithium. In the case of potassium, the participation of the long C-terminus in the regulation of intracellular potassium content is demonstrated. We also present evidence that the Nha1p C-terminus is involved in the cell response to sudden changes in environmental osmolarity.     

A truncation of 2B subfamily cytochromes P450 yields increased expression levels, increased solubility, and decreased aggregation while retaining function.

The hydrophobic membrane-spanning domain in four cytochromes P450 2B was removed (Delta3-21) and several positive charges were substituted at the N-terminus to increase expression and solubility. Histidine residues were appended to the C-terminus to simplify purification. The truncated proteins were highly expressed in Escherichia coli, could be released from the membrane using high salt conditions, and were purified from this fraction to specific contents up to 19 nmol P450/mg protein using a single Ni(2+)-agarose column. Gel filtration revealed that truncated P450 2B1 forms a monodisperse solution of hexamers in the absence of detergent and >95% monomers in 0.25% sodium cholate. All truncated proteins, including human 2B6, were active with 7-ethoxy-4-trifluoromethylcoumarin, and truncated 2B1 was shown to retain the native regio- and stereospecificity of testosterone hydroxylation. These data demonstrate that modification of the N-terminus yields high levels of properly folded P450s 2B with increased solubility, which are suitable for functional and structural analysis.     

cDNA cloning, genomic organization and expression of the novel human metallophosphoesterase gene MPPE1 on chromosome 18p11.2

We have isolated a 1,926-bp cDNA that encodes a novel polypeptide of 396 amino acid residues with a calculated molecular mass of 45.2 kDa. This MPPE1 polypeptide consists of a predicted signal sequence of 45 residues at the N-terminus, a 240-amino acid metallo-phosphoesterase domain, and a 24-amino acid transmembrane domain at the C-terminus. The genomic organization of the human MPPE1 gene proved to consist of 14 exons and to span about 27 kb. The gene was located on chromosome 18p11.2, adjacent to the G protein Golf alpha gene (GNAL), in tail-to-tail orientation, partially overlapping with the 3' UTR of the latter gene. MPPE1 is expressed as an mRNA of 2.2 kb in the brain, but not in any other tissues studied here. 3' RACE analysis defined a single functional polyadenylation site within the 3' UTR of the GNAL gene, while RT-PCR analysis revealed an alternatively spliced form of MPPE1, which included an additional exon located within the last intron. The alternatively spliced form encoded a truncated variant of MPPE1 with a calculated molecular mass of 38.8 kDa that lacks the C-terminal transmembrane domain.     

New sites of proviral integration associated with murine promonocytic leukemias and evidence for alternate modes of c-myb activation.

Murine promonocytic leukemias involving insertional mutagenesis of the c-myb locus can be induced by replication-competent retroviruses. In previously studied promonocytic leukemic cells induced by Moloney murine leukemia virus (called MML), the provirus has been invariably integrated upstream of exons 3 or 4 and the leukemic cells expressed aberrant RNAs with fused virus-myb sequences. Furthermore, Myb expressed by these cells has been shown to be truncated by 47 or 71 amino acids. The present report examines the mechanisms of myb activation in leukemias induced by two other retroviruses, amphotropic virus 4070A and Friend strain FB29 (the leukemias are called AMPH-ML and FB-ML, respectively). This study revealed two additional c-myb proviral insertion sites in these promonocytic leukemias. One FB-ML had a proviral integration in exon 9, and expressed a C-terminally truncated Myb protein of 47 kDa similar to that previously demonstrated to be expressed in the myelomonocytic cell lines NFS60 and VFL-2. However, a sequence of reverse-transcribed and amplified RNA from this leukemia demonstrated that the truncation involved a loss of 248 amino acids compared with a loss of 240 amino acids in the myelomonocytic cell lines. Another leukemia had a provirus integrated in the 5' end of c-myb upstream of exon 2 (in the first intron) and produced a Myb protein that was indistinguishable on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from normal Myb. This latter leukemia (FB-ML R1-4-10) expressed Myb with the smallest N-terminal truncation observed so far in promonocytic leukemias; translation begins at an ATG within c-myb exon 2, leading to loss of only 20 amino acids from the N terminus. Unlike the proteins produced in Moloney murine leukemia virus-induced promonocytic leukemias (MML) that have larger truncations, this protein has an intact DNA binding region and does not contain N-terminal amino acids encoded by gag. However, this protein is similar to all N-terminally truncated Mybs so far studied, in that the truncation resulted in deletion of a casein kinase II phosphorylation site which has been proposed to be involved in regulation of DNA binding.     

The ubiquitin-like protein Smt3p is activated for conjugation to other proteins by an Aos1p/Uba2p heterodimer.

SMT3 is an essential Saccharomyces cerevisiae gene encoding a 11.5 kDa protein similar to the mammalian ubiquitin-like protein SUMO-1. We have found that Smt3p, like SUMO-1 and ubiquitin, can be attached to other proteins post-translationally and have characterized the processes leading to the activation of the Smt3p C-terminus for conjugation. First, the SMT3 translation product is cleaved endoproteolytically to expose Gly98, the mature C-terminus. The presence of Gly98 is critical for Smt3p's abilities to be conjugated to protein substrates and to complement the lethality of a smt3Delta strain. Smt3p undergoes ATP-dependent activation by a novel heterodimeric enzyme consisting of Uba2p, a previously identified 71 kDa protein similar to the C-terminus of ubiquitin-activating enzymes (E1s), and Aos1p (activation of Smt3p), a 40 kDa protein similar to the N-terminus of E1s. Experiments with conditional uba2 mutants showed that Uba2p is required for Smt3p conjugation in vivo. Furthermore, UBA2 and AOS1 are both essential genes, providing additional evidence that they act in a distinct pathway whose role in cell viability is to conjugate Smt3p to other proteins.     

Identification and characterization of two novel splice forms of GRIP1 in the rat brain

We cloned two novel alternatively-spliced mRNA isoforms of glutamate receptor interacting protein 1 (GRIP1) which we named GRIP1d and GRIP1e 4-7. GRIP1d is a 135 kDa, 7-PDZ-domain variant of GRIP1, containing the 12 amino acid C-terminus originally described for the 4-PDZ-domain GRIP1c 4-7. GRIP1e 4-7 is a 75 kDa 4-PDZ-domain variant of GRIP1, containing the 12 amino acid C-terminus originally described for the 7-PDZ-domain GRIP1a/b. Northern blots indicated that GRIP1d mRNA is 5.1 kb long and abundant in brain. An antibody to the C-terminus of the 75 kDa GRIP1c 4-7 also recognized an abundant 135 kDa protein, consistent with the predicted size of GRIP1d. Similarly, an antibody to the C-terminus of the 135 kDa GRIP1a/b also recognized a low abundance 75 kDa protein, consistent with the predicted size of GRIP1e 4-7. Immunocytochemistry of hippocampal cultures and intact brain using these antibodies showed that (i) these isoforms are present in both GABAergic and glutamatergic synapses, and (ii) the isoforms co-localize in individual synapses. While GRIP1a/b isoforms are abundant in interneurons and highly concentrated in GABAergic presynaptic terminals, the isoforms recognized by the antibody to the C-terminus common to GRIP1c 4-7 and GRIP1d are much less abundant in interneurons and preferentially concentrate at the postsynaptic complex.     

Calpain-mediated truncation of dihydropyrimidinase-like 3 protein (DPYSL3) in response to NMDA and H2O2 toxicity

Dihydropyrimidinase-like protein 3 (DPYSL3), a member of TUC (TOAD-64/Ulip/CRMP), is believed to play a role in neuronal differentiation, axonal outgrowth and, possibly, neuronal regeneration. In primary cortical cultures, glutamate (NMDA) excitotoxicity and oxidative stress (H2O2) caused the cleavage of DPYSL3, resulting in the appearance of a doublet of 62 kDa and 60 kDa. Pre-treatment of cell cultures with calpain inhibitors, but not caspase 3 inhibitor, before exposure to NMDA or H2O2 completely blocked the appearance of the doublet, suggesting calpain-mediated truncation. Furthermore, in vitro digestion of DPYSL3 in cell lysate with purified calpain revealed a cleavage product identical to that observed in NMDA- and H2O2-treated cells, and its appearance was blocked by calpain inhibitors. Analysis of the DPYSL3 protein sequence revealed a possible cleavage site for calpain (Val-Arg-Ser) on the C-terminus of DPYSL3. Collectively, these studies demonstrate for the first time that DPYSL3 is a calpain substrate. The physiological relevance of the truncated DPYSL3 protein remains to be determined.     

Expression of human cytochrome P450 46A1 in Escherichia coli: effects of N- and C-terminal modifications

Heterologous expression in Escherichia coli, subcellular distribution, solubility, and catalytic and substrate-binding properties of four truncated cytochromes P450 46A1 were investigated in the present study. All four lacked the N-terminal transmembrane region (residues 3-27), and, in addition, Delta 46A1H had a 4x His-tag fused to the C-terminus; H Delta 46A1 had the N-terminal 4x His-tag; H Delta 46A1 Delta had a 4x His-tag at the N-terminus and did not contain a proline-rich region at the C-terminus (residues 494-499); and Delta 46A1 Delta lacked the C-terminal proline-rich region. The truncated enzymes were expressed at 390-650 nmol/L culture levels, distributed at about a 1:1 ratio between the membrane fraction and the cytosol in low ionic strength buffer, and were predominantly monomers in detergent-free buffer. They had moderately decreased catalytic efficiencies for either cholesterol or 24S-hydroxycholesterol or both, whereas their substrate-binding constants were either unchanged or decreased 2-fold. The two forms, Delta 46A1 Delta and H Delta 46A1 Delta, both lacking the C-terminal proline-rich region seem to be good candidates for future crystallographic studies because they contain only 0.3-0.8% of high molecular weight aggregates and their catalytic efficiencies are decreased no more than 2.3-fold.     

Molecular and functional analyses of two new calcium-activated chloride channel family members from mouse eye and intestine

Two new calcium-activated chloride channel (CLCA) family members, mCLCA5 and mCLCA6, have been cloned from mouse eye and intestine, respectively. mCLCA5 is highly homologous to hCLCA2, and mCLCA6 is highly homologous to hCLCA4. mCLCA5 is widely expressed with strong expression in eye and spleen, whereas mCLCA6 is primarily expressed in intestine and stomach. mCLCA6 is also expressed as a splice variant lacking exon 8 and part of exon 10 in intestine and stomach. Transfection of tsA201 cells with enhanced green fluorescent protein-tagged versions of the three cDNAs reveals protein products of 155 and 65 kDa for mCLCA5 and mCLCA6 and 145 and 65 kDa for the mCLCA6 splice variant. In vitro translation of mCLCA5 generates a 90-kDa protein that does not appear to be glycosylated. mCLCA6 also generates a 90-kDa protein that is glycosylated to a 110-kDa product, whereas the mCLCA6 splice variant generates an 80-kDa product that is 100 kDa after glycosylation. Treatment of enhanced green fluorescent protein-tagged mCLCA6 with PNGase F (peptide: N-glycosidase F) to remove N-linked glycosyl groups shows a reduction in size of the 65 kDa product to 60 kDa. Consistent with the hypothesis that mCLCA5, mCLCA6, and its splice variant encode calcium-activated chloride channels, in HEK293 cells expressing CLCAs ionomycin-evoked increases in intracellular calcium stimulated a current that reversed near Cl(-) equilibrium potential, E(Cl). Furthermore, these currents were inhibited by the chloride channel blocker niflumic acid. Given the prominent role of hCLCA2 in cancer cell adhesion and the unique high level of expression of hCLCA4 in brain, the identification of their murine counterparts presents the opportunity to clarify the role of CLCAs in disease and normal cell physiology.     

Two Plasmodium falciparum merozoite proteins binding to erythrocyte band 3 form a direct complex

Erythrocyte invasion by malaria parasites requires multiple protein interactions. Our earlier studies showed that erythrocyte band 3 is an invasion receptor binding Plasmodium falciparum merozoite surface protein 1 and 9 (MSP1, MSP9) existing as a co-ligand complex. In this study, we have used biochemical approaches to identify the binding sites within MSP1 and MSP9 involved in the co-ligand complex formation. A major MSP9-binding site is located within the 19kDa C-terminal domain of MSP1 (MSP1(19)). Two specific regions of MSP9 defined as Delta1a and Delta2 interacted with native MSP1(19). The 42 kDa domain of MSP1 (MSP1(42)) bearing MSP1(19) in the C-terminus bound directly to both MSP9/Delta1a and Delta2. Thus, the regions of MSP1 and MSP9 interacting with the erythrocyte band 3 receptor are also responsible for assembling the co-ligand complex. Our evidence suggests a ternary complex is formed between MSP1, MSP9, and band 3 during erythrocyte invasion by P. falciparum.     

Identification of a novel splice variant of X-linked inhibitor of apoptosis-associated factor 1

XAF1 (XIAP-associated factor 1) binds to XIAP and blocks its anti-apoptotic activity. It has been reported that XAF1 is mainly expressed in normal tissues but is missing or present at low levels in most cancer cell lines, which implies a tumor-suppressing function. In the present study we describe the identification of a novel splice variant of human XAF1, designated XAF1C, which contains a cryptic exon. Incorporation of this exon (exon 4b) into the mRNA introduces an in-frame stop codon, resulting in a shortened open-reading frame (ORF) of 495 nucleotides. This ORF is predicted to encode a 164 amino acid (AA) protein lacking the C-terminal domain of the previously described XAF1(A), but containing a unique 24 AA carboxy terminus. Like XAF1(A), XAF1C mRNA expression was detected in a variety of human cancer cell lines and also in normal human tissues. The ratio of XAF1(A) and XAF1C mRNA expression differs amongst the cell lines tested, suggesting differential mRNA stabilities and/or the existence of tissue- or cell type-specific splicing regulation. In transfected cells, xaf1c encodes a truncated protein of 18kDa, which is distributed primarily in the nucleus.