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PubMed2005

Structural and functional characterization of noncoding repetitive RNAs transcribed in stressed human cells.

Thermal and chemical stresses induce the formation in human cells of novel and transient nuclear structures called nuclear stress bodies (nSBs). These contain heat shock factor 1 (HSF-1) and a specific subset of pre-mRNA processing factors. Nuclear stress bodies are assembled on specific pericentromeric heterochromatic domains containing satellite III (SatIII) DNA. In response to stress, these domains change their epigenetic status from heterochromatin to euchromatin and are transcribed in poly-adenylated RNAs that remain associated with nSBs. In this article, we describe the cloning, sequencing, and functional characterization of these transcripts. They are composed of SatIII repeats and originate from the transcription of multiple sites within the SatIII arrays. Interestingly, the level of SatIII RNAs can be down-regulated both by antisense oligonucleotides and small interfering RNAs (siRNA). Knockdown of SatIII RNA by siRNAs requires the activity of Argonaute 2, a component of the RNA-induced silencing complex. Down-regulation of satellite III RNAs significantly affects the recruitment of RNA processing factors to nSBs without altering the association of HSF-1 with these structures nor the presence of acetylated histones within nSBs. Thus, satellite III RNAs have a major role in the formation of nSBs.

Animals↗

PubMed2005

Functional characterization of five novel CYP2C8 variants, G171S, R186X, R186G, K247R, and K383N, found in a Japanese population.

Cytochrome P450 2C8 is one of the primary enzymes responsible for the metabolism of a wide range of drugs such as paclitaxel, cerivastatin, and amiodarone. We have sequenced the CYP2C8 gene from 201 Japanese subjects and found five novel nonsynonymous single nucleotide polymorphisms (SNPs): 511G>A (G171S), 556C>T (R186X; X represents the translational stop codon), 556C>G (R186G), 740A>G (K247R), and 1149G>T (K383N), with the allele frequency of 0.0025. The CYP2C8 variants were heterologously expressed in COS-1 cells and functionally characterized in terms of expression level, paclitaxel 6alpha-hydroxylase activity, and intracellular localization. The prematurely terminated R186X variant was undetectable by Western blotting and inactive toward paclitaxel 6alpha-hydroxylation. The G171S, K247R, and K383N variants exhibited properties similar to those of the wild-type CYP2C8. Paclitaxel 6alpha-hydroxylase activity of the R186G transfectant was only 10 to 20% that of wild-type CYP2C8. Furthermore, the R186G variant displayed a lower level of protein expression in comparison to the wild type, which was restored by the addition of a proteasome inhibitor (MG-132; Z-Leu-Leu-Leu-aldehyde). The reduced CO-difference spectral analysis using recombinant proteins from an insect cell/baculovirus system revealed that the R186G variant has a minor peak at 420 nm in addition to the characteristic Soret peak at 450 nm, suggesting the existence of improperly folded protein. These results indicate that the novel CYP2C8 SNPs, 556C>T (R186X) and 556C>G (R186G), could influence the metabolism of CYP2C8 substrates such as paclitaxel and cerivastatin.

Animals↗

PubMed2006

Functional characterization of front-end desaturases from trypanosomatids depicts the first polyunsaturated fatty acid biosynthetic pathway from a parasitic protozoan.

A survey of the three kinetoplastid genome projects revealed the presence of three putative front-end desaturase genes in Leishmania major, one in Trypanosoma brucei and two highly identical ones (98%) in T. cruzi. The encoded gene products were tentatively annotated as Delta8, Delta5 and Delta6 desaturases for L. major, and Delta6 desaturase for both trypanosomes. After phylogenetic and structural analysis of the deduced proteins, we predicted that the putative Delta6 desaturases could have Delta4 desaturase activity, based mainly on the conserved HX(3)HH motif for the second histidine box, when compared with Delta4 desaturases from Thraustochytrium, Euglena gracilis and the microalga, Pavlova lutheri, which are more than 30% identical to the trypanosomatid enzymes. After cloning and expression in Saccharomyces cerevisiae, it was possible to functionally characterize each of the front-end desaturases present in L. major and T. brucei. Our prediction about the presence of Delta4 desaturase activity in the three kinetoplastids was corroborated. In the same way, Delta5 desaturase activity was confirmed to be present in L. major. Interestingly, the putative Delta8 desaturase turned out to be a functional Delta6 desaturase, being 35% and 31% identical to Rhizopus oryzae and Pythium irregulareDelta6 desaturases, respectively. Our results indicate that no conclusive predictions can be made about the function of this class of enzymes merely on the basis of sequence homology. Moreover, they indicate that a complete pathway for very-long-chain polyunsaturated fatty acid biosynthesis is functional in L. major using Delta6, Delta5 and Delta4 desaturases. In trypanosomes, only Delta4 desaturases are present. The putative algal origin of the pathway in kinetoplastids is discussed.

Amino Acid Sequence↗

PubMed2000

Functional characterization of the two alternative promoters of human p45 NF-E2 gene.

OBJECTIVE: The transcription factor NF-E2, a heterodimeric protein complex composed of p45 and small Maf family proteins, is considered crucial for the proper differentiation of erythrocytes and megakaryocytes in vivo. We report the results of studies aimed at understanding the regulatory mechanisms controlling p45 gene expression in erythroid cells. MATERIALS AND METHODS: Human p45 mRNAs have two alternative isoforms, aNF-E2 and fNF-E2, and these isoforms are transcribed from the alternative promoters. We investigated lineage-specific expression of both isomers in human erythroid and megakaryocytic cells by reverse transcriptase polymerase chain reaction or Northern blot analysis. For functional characterization of both promoters, plasmids in which reporter genes were placed under the control of a series of truncated or mutated promoter fragments were transfected to human hematopoietic cell lines. RESULTS: When CD34(+) cells isolated from human cord blood were induced to unilineage erythroid or megakaryocytic differentiation in liquid suspension culture, both transcripts, although barely detected at day 0, were induced in both erythroid and megakaryocytic cultures. fNF-E2 mRNA was found to be more abundant in erythroid cells than megakaryocytic cells at day 7 of culture. Although both isomers were expressed in human erythroid-megakaryocytic cell lines, megakaryocytic maturation with loss of erythroid phenotype induced by phorbol 12-myristate 13-acetate (PMA) resulted in exclusive downregulation of fNF-E2, suggesting that fNF-E2 promoter is more erythroid specific. Functional analysis of fNF-E2 promoter showed that the promoter is active only in erythroid-megakaryocytic cells and that the double GATA site in the proximal region is necessary for its efficient activity. CONCLUSION: These results suggest that GATA proteins, which govern the differentiation of erythroid lineage cells, are required for full promoter activity of the p45 gene.

Binding Sites↗

PubMed1997

Functional characterization of a chicken major histocompatibility complex class II B gene promoter.

A 0.7 kilobase (kb) DNA fragment from the 5' flanking region of a chicken major histocompatibility complex (MHC) class II B gene was cloned into chloramphenicol acetyltransferase (CAT) reporter vectors and was transfected into a chicken macrophage cell line that expresses a low level of MHC class II antigens. Positive orientation-dependent promoter activity of the chicken DNA was evident in a reporter construct containing an SV40 enhancer. Deletion analysis of this 0.7 kb DNA fragment revealed a short fragment in the 3' end that was crucial for the promoter function and negative regulatory elements (NRE) located further upstream. The conserved MHC class II X and Y boxes did not have a significant effect on promoter activity. Sequence analysis of the 0.7 kb class II B gene upstream region suggests possible involvement of interferon (IFN), E twenty-six specific (ETS)-related proteins, and other factors in regulating this promoter. A chicken T-cell line culture supernatant increased surface expression of MHC class II antigens, as well as class II promoter activity, in this macrophage cell line. This first functional characterization of a chicken MHC class II B gene promoter will aid in understanding the regulatory mechanisms that control the expression of these genes.

Animals↗

PubMed1997

Functional characterization of a distinct ryanodine receptor mutation in human malignant hyperthermia-susceptible muscle.

Malignant hyperthermia is an inherited autosomal disorder of skeletal muscle in which certain volatile anesthetics and depolarizing muscle relaxants trigger an abnormally high release of Ca2+ from the intracellular Ca2+ store, the sarcoplasmic reticulum. In about 50% of cases, malignant hyperthermia susceptibility is linked to the gene encoding the skeletal muscle ryanodine receptor/Ca2+ release channel (RYR1). To date, eight point mutations have been identified in human RYR1. Although these mutations are thought to lead to an increased caffeine and halothane sensitivity in the contractile response of skeletal muscle, their functional consequences have not been investigated on the molecular level. In the present study, we provide the first functional characterization of a point mutation located in the central part of RYR1, Gly2434 --> Arg. Using high affinity [3H]ryanodine binding as the experimental approach, we show that this mutation enhances the sensitivity of RYR1 to activating concentrations of Ca2+ and to the exogenous and diagnostically used ligands caffeine and 4-chloro-m-cresol. In parallel, the sensitivity to inhibiting concentrations of Ca2+ and calmodulin was reduced, transferring the mutant Ca2+ release channel into a hyperexcitable state.

Calcium↗

PubMed2003

Steroid hormone regulation of the human kallikrein 10 (KLK10) gene in cancer cell lines and functional characterization of the KLK10 gene promoter.

BACKGROUND: The human kallikrein 10 (KLK10) gene is a new member of the human tissue kallikrein gene family. It encodes for a secreted serine protease (hK10) with predicted trypsin-like enzymatic activity. KLK10 is highly expressed in the sex organs and its expression level changes in malignancy. METHODS: To determine the role of steroid hormones in KLK10 gene expression, we investigated its modulation by 17beta-estradiol, 5alpha-dihydrotestosterone, norgestrel, dexamethasone and aldosterone, at both the transcription and translation level, in a panel of cancer cell lines. After steroid hormone stimulation, the change of KLK10 mRNA was monitored with reverse transcriptase polymerase chain reaction and hK10 protein levels in the culture supernatant were quantified with an hK10-specific immunoassay. The presence of hormone response elements in the KLK10 gene promoter was examined with the chloramphenicol acetyltransferase reporter gene system. RESULTS: The KLK10 expression was mainly up-regulated by estrogens, androgens and progestins, and to a lesser extent by dexamethasone and aldosterone in the breast cancer cell lines BT-474, MCF-7 and T-47D, both at the mRNA and protein levels. The effect of stimulation of these steroids on KLK10 expression varied among the cell lines. Estrogens, androgens and progestins were most potent in the BT-474, T-47D and MCF-7 cells, respectively. The up-regulation effect of estrogens, androgens, and progestins on KLK10 expression can be blocked by their antagonists ICI-182, 780, RU-56,187, and mifepristone, respectively. Time course studies showed that hK10 protein started to increase 1 day after steroid hormone stimulation and this increase persisted for 7 days. These data suggest that steroid hormones up-regulate KLK10 gene expression through direct interaction between hormone-receptor complexes and their cognate hormone response elements. To search for hormone response elements, we functionally characterized the KLK10 promoter by placing it upstream of the chloramphenicol acetyltransferase reporter gene. We found that KLK10 promoter activity did not rely on the presence of functional estrogen and androgen receptors. Also, the presence of functional estrogen and androgen receptors did not increase its constitutive activity. We suggest that the hormone response elements that mediate the transcriptional regulation of KLK10 are unlikely to locate in the KLK10 promoter. CONCLUSIONS: Estrogens, androgens and progestins modulate KLK10 expression through their own receptors but this regulation is not mediated by steroid hormone response elements in the promoter of the KLK10 gene.

Aldosterone↗

PubMed1998

Molecular cloning, functional characterization and mRNA expression analysis of the murine chemokine receptor CCR6 and its specific ligand MIP-3alpha.

We have cloned the murine CCR6 receptor and its ligand, the beta-chemokine mMIP-3alpha. Calcium mobilization assays performed with mCCR6 transfectants showed significant responses upon addition of mMIP-3alpha. Murine MIP-3alpha RNA is expressed in thymus, small intestine and colon, whereas mCCR6 RNA is expressed in spleen and lymph nodes. RT-PCR analysis of FACS-sorted lymphoid and antigen presenting cell subsets showed mCCR6 expression mainly in B cells, CD8- splenic dendritic cells and CD4+ T cells. The cloning and functional characterization of the mCCR6 and mMIP-3alpha will allow the study of the role of these proteins in mouse models of inflammation and immunity.

Amino Acid Sequence↗

PubMed2005

The rat Na+-sulfate cotransporter rNaS2: functional characterization, tissue distribution, and gene (slc13a4) structure.

Inorganic sulfate is essential for numerous functions in mammalian physiology. In the present study, we characterized the functional properties of the rat Na+-sulfate cotransporter NaS2 (rNaS2), determined its tissue distribution, and identified its gene (slc13a4) structure. Expression of rNaS2 protein in Xenopus oocytes led to a Na+-dependent transport of sulfate that was inhibited by phosphate, thiosulfate, tungstate, selenate, oxalate, and molybdate, but not by citrate, succinate, or DIDS. Transport kinetics of rNaS2 determined a K(M) for sulfate of 1.26 mM. Na+ kinetics determined a Hill coefficient of n=3.0+/-0.7, suggesting a Na+:SO4 (2-) stoichiometry of 3:1. rNaS2 mRNA was highly expressed in placenta, with lower levels found in the brain and liver. slc13a4 maps to rat chromosome 4 and contains 17 exons, spanning over 46 kb in length. This gene produces two alternatively spliced transcripts, of which the transcript lacking exon 2 is the most abundant form. Its 5' flanking region contains CAAT- and GC-box motifs and a number of putative transcription factor binding sites, including GATA-1, SP1, and AP-2 consensus sequences. This is the first study to characterize rNaS2 transport kinetics, define its tissue distribution, and resolve its gene (slc13a4) structure and 5' flanking region.

Animals↗

PubMed1999

Isolation and functional characterization of two distinct sexual-stage-specific promoters of the human malaria parasite Plasmodium falciparum.

Transmission of malaria depends on the successful development of the sexual stages of the parasite within the midgut of the mosquito vector. The differentiation process leading to the production of the sexual stages is delineated by several developmental switches. Arresting the progression through this sexual differentiation pathway would effectively block the spread of the disease. The successful development of such transmission-blocking agents is hampered by the lack of a detailed understanding of the program of gene expression that governs sexual differentiation of the parasite. Here we describe the isolation and functional characterization of the Plasmodium falciparum pfs16 and pfs25 promoters, whose activation marks the developmental switches executed during the sexual differentiation process. We have studied the differential activation of the pfs16 and pfs25 promoters during intraerythrocytic development by transfection of P. falciparum and during gametogenesis and early sporogonic development by transfection of the related malarial parasite P. gallinaceum. Our data indicate that the promoter of the pfs16 gene is activated at the onset of gametocytogenesis, while the activity of the pfs25 promoter is induced following the transition to the mosquito vector. Both promoters have unusual DNA compositions and are extremely A/T rich. We have identified the regions in the pfs16 and pfs25 promoters that are essential for high transcriptional activity. Furthermore, we have identified a DNA-binding protein, termed PAF-1, which activates pfs25 transcription in the mosquito midgut. The data presented here shed the first light on the details of processes of gene regulation in the important human pathogen P. falciparum.

Animals↗

PubMed2007

Functional characterization of small CTD phosphatases.

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase (RNAP) II undergoes reversible phosphorylation with each round of transcription essential for the regulation of gene expression. A family of small CTD phosphatases (SCPs) was identified based on a homology search to TFIIF-associating CTD phosphatase 1 (FCP1). Unlike FCP1, SCP preferentially catalyze the dephosphorylation of Ser5 within the CTD and is especially active toward RNAP II phosphorylated by TFIIH (1). Recently, SCP1 was demonstrated as a transcriptional regulator that acts to silence neuronal genes (2). This chapter describes the procedures for various assays involved in the discovery and functional characterization of SCPs.

Amino Acid Sequence↗

PubMed2002

Quantitation and functional characterization of neural cells derived from ES cells using nestin enhancer-mediated targeting in vitro.

To gain insight into early events of neurogenesis, transgenic embryonic stem (ES) cells were generated using the enhanced green fluorescence protein (EGFP) reporter gene under the regulatory control of the neural stem cell marker, nestin. The expression of EGFP in undifferentiated ES cells suggested that the onset of endogenous nestin occurred before neurulation. Upon differentiation of ES cells, the EGFP expression became confined to the neural lineage and asynchrony in ES-cell-derived neural differentiation was evident. The EGFP intensity was prominent in the proliferative progenitors and unipolar neurons, whereas downregulation occurred in differentiating bi- and multipolar neurons. This was corroborated quantitatively using flow cytometry where maximal generation of neural progenitors was observed 4-12 days post-plating. The proliferative potential of neural progenitors as well as glia, in contrast to post-mitotic neurons, was also evident by time-lapse microscopy. The functional characterization of progenitors revealed an absence of voltage-activated inward currents, whereas the Na+ current (INa) was detected prior to Ca2+ currents (ICa) in differentiating neurons. Additionally, inhibitory receptor-operated channels could be detected at these early stages of development in bi- and multipolar neurons suggesting that the pre-committed progenitors had retained their intrinsic ability to give rise to functional neurons. This study sheds new light on early events of neurogenesis defining the quantitative and qualitative aspects and demarcating the functional neural cell types from ES cells in vitro.

Animals↗

PubMed1998

Components of a calmodulin-dependent protein kinase cascade. Molecular cloning, functional characterization and cellular localization of Ca2+/calmodulin-dependent protein kinase kinase beta.

Ca2+/calmodulin-dependent protein kinases I and IV (CaMKI and CaMKIV, respectively) require phosphorylation on an equivalent single Thr in the activation loop of subdomain VIII for maximal activity. Two distinct CaMKI/IV kinases, CaMKKalpha and CaMKKbeta, were purified from rat brain and partially sequenced (Edelman, A. M., Mitchelhill, K., Selbert, M. A., Anderson, K. A., Hook, S. S., Stapleton, D., Goldstein, E. G., Means, A. R., and Kemp, B. E. (1996) J. Biol. Chem. 271, 10806-10810). We report here the cloning and sequencing of cDNAs for human and rat CaMKKbeta, tissue and regional brain localization of CaMKKbeta protein, and mRNA and functional characterization of recombinant CaMKKbeta in vitro and in Jurkat T cells. The sequences of human and rat CaMKKbeta demonstrate 65% identity and 80% similarity with CaMKKalpha and 30-40% identity with CaMKI and CaMKIV themselves. CaMKKbeta is broadly distributed among rat tissues with highest levels in CaMKIV-expressing tissues such as brain, thymus, spleen, and testis. In brain, CaMKKbeta tracks more closely with CaMKIV than does CaMKKalpha. Bacterially expressed CaMKKbeta undergoes intramolecular autophosphorylation, is regulated by Ca2+/CaM, and phosphorylates CaMKI and CaMKIV on Thr177 and Thr200, respectively. CaMKKbeta activates both CaMKI and CaMKIV when coexpressed in Jurkat T cells as judged by phosphorylated cAMP response element-binding protein-dependent reporter gene expression. CaMKKbeta activity is enhanced by elevation of intracellular Ca2+, although substantial activity is observed at the resting Ca2+ concentration. The strict Ca2+ requirement of CaMKIV-dependent phosphorylation of cAMP response element-binding protein, is therefore controlled at the level of CaMKIV rather than CaMKK.

Amino Acid Sequence↗

PubMed2003

Overexpression, purification, and functional characterization of ATP-binding cassette transporters in the yeast, Pichia pastoris.

The ATP-binding cassette (ABC) transporter superfamily is a large gene family that has been highly conserved throughout evolution. The physiological importance of these membrane transporters is highlighted by the large variety of substrates they transport, and by the observation that mutations in many of them cause heritable diseases in human. Likewise, overexpression of certain ABC transporters, such as P-glycoprotein and members of the multidrug resistance associated protein (MRP) family, is associated with multidrug resistance in various cells and organisms. Understanding the structure and molecular mechanisms of transport of the ABC transporters in normal tissues and their possibly altered function in human diseases requires large amounts of purified and active proteins. For this, efficient expression systems are needed. The methylotrophic yeast Pichia pastoris has proven to be an efficient and inexpensive experimental model for high-level expression of many proteins, including ABC transporters. In the present review, we will summarize recent advances on the use of this system for the expression, purification, and functional characterization of P-glycoprotein and two members of the MRP subfamily.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed1999

Functional characterization of the Opitz syndrome gene product (midin): evidence for homodimerization and association with microtubules throughout the cell cycle.

Opitz syndrome (OS) is a multiple congenital anomaly manifested by abnormal closure of midline structures. The gene responsible for the X-linked form of this disease, MID1, encodes a protein (midin) that contains a RING, two B-boxes, a coiled-coil (the so-called tripartite motif) and an RFP-like domain. The tripartite motif is characteristic of a family of proteins, named the B-box family, involved in cell proliferation and development. Since the subcellular compartmentalization and the ability to form multiprotein structures both appear to be crucial for the function of this family of proteins, we have studied these properties on the wild-type and mutated forms of midin. We found that endogenous midin is associated with microtubules throughout the cell cycle, co-localizing with cytoplasmic fibres in interphase and with the mitotic spindle and midbodies during mitosis and cytokinesis. Immunoprecipitation experiments demonstrated the ability of the tripartite motif to mediate midin homodimerization, consistent with the evidence, obtained by gel filtration analysis, that midin exists in the form of large protein complexes. Functional characterization of altered forms of midin, resulting from mutations found in OS patients, revealed that association with microtubules is compromised, while the ability to homodimerize and form multiprotein complexes is retained. We suggest that midin is involved in the formation of multiprotein structures acting as anchor points to microtubules and that impaired association with these cytoskeletal structures causes OS developmental defects.

Animals↗

PubMed2004

Molecular cloning and functional characterization of fatty acyl desaturase and elongase cDNAs involved in the production of eicosapentaenoic and docosahexaenoic acids from alpha-linolenic acid in Atlantic salmon (Salmo salar).

Fish are the only major dietary source for humans of omega-3 highly unsaturated fatty acids (HUFAs) and with declining fisheries farmed fish such as Atlantic salmon (Salmo salar) constitute an increasing proportion of the fish in the human diet. However, the current high use of fish oils, derived from wild capture marine fisheries, in aquaculture feeds is not sustainable in the longer term and will constrain continuing growth of aquaculture activities. Greater understanding of how fish metabolize and biosynthesize HUFA may lead to more sustainable aquaculture diets. The study described here contributes to an effort to determine the molecular genetics of the HUFA biosynthetic pathway in salmon, with the overall aim being to determine mechanisms for optimizing the use of vegetable oils in Atlantic salmon culture. In this paper we describe the cloning and functional characterization of 2 genes from salmon involved in the biosynthesis of HUFA. A salmon desaturase complementary DNA, SalDes, was isolated that include an open reading frame of 1362 bp specifying a protein of 454 amino acids. The protein sequence includes all the characteristics of microsomal fatty acid desaturases, including 3 histidine boxes, 2 transmembrane regions, and an N-terminal cytochrome b(5) domain containing a heme-binding motif similar to that of other fatty acid desaturases. Functional expression in the yeast Saccharomyces cerevisiae showed SalDes is predominantly an omega-3 delta5 desaturase, a key enzyme in the synthesis of eicosapentaenoic acid (20:5n-3) from alpha-linolenic acid (18:3n-3). The desaturase showed only low levels of delta6 activity toward C(18) polyunsaturated fatty acids. In addition, a fatty acid elongase cDNA, SalElo, was isolated that included an open reading frame of 888 bp, specifying a protein of 295 amino acids. The protein sequence of SalElo included characteristics of microsomal fatty acid elongases, including a histidine box and a transmembrane region. Upon expression in yeast SalElo showed broad substrate specificity for polyunsaturated fatty acids with a range of chain lengths, with the rank order being C(18) > C(20) > C(22). Thus this one polypeptide product displays all fatty acid elongase activities required for the biosynthesis of docosahexaenoic acid (22:6n-3) from 18:3n-3.

Acetyltransferases↗

PubMed1999

Functional characterization of Chlamydomonas mutants defective in cytochrome f maturation.

We have altered the N terminus of cytochrome f by site-directed mutagenesis of the chloroplast petA gene in Chlamydomonas reinhardtii. We have replaced the tyrosine residue, Tyr(32), located immediately downstream of the processing site Ala(29)-Gln(30)-Ala(31) by a proline. Tyr(32) is the N terminus of the mature protein and serves as the sixth axial ligand to the heme iron. This mutant, F32P, accumulated different forms of holocytochrome f and assembled them into the cytochrome b(6)f complex. The strain was able to grow phototrophically. Our results therefore contradict a previous report (Zhou, J., Fernandez-Velasco, J. G., and Malkin, R. (1996) J. Biol. Chem. 271, 1-8) that a mutation, considered to be identical to the mutation described here, prevented cytochrome b(6)f assembly. A comparative functional characterization of F32P with F29L-31L, a site-directed processing mutant in which we had replaced the processing site by a Leu(29)-Gln(30)-Leu(31) sequence (2), revealed that both mutants accumulate high spin cytochrome f, with an unusual orientation of the heme and low spin cytochrome f with an alpha-band peak at 552 nm. Both hemes have significantly lower redox potentials than wild type cytochrome f. We attribute the high spin form to uncleaved pre-holocytochrome f and the low spin form to misprocessed forms of cytochrome f that were cleaved at a position different from the regular Ala(29)-Gln-Ala(31) motif. In contrast to F29L-31L, F32P displayed a small population of functional cytochrome f, presumably cleaved at Ala(29), with characteristics close to those of wild type cytochrome f. The latter form would account for cytochrome b(6)f turnover and photosynthetic electron transfer that sustain phototrophic growth of F32P.

Amino Acid Sequence↗

PubMed1996

Cloning and functional characterization of a system ASC-like Na+-dependent neutral amino acid transporter.

A cDNA was isolated from mouse testis which encodes a Na+-dependent neutral amino acid transporter. The encoded protein, designated ASCT2, showed amino acid sequence similarity to the mammalian glutamate transporters (40-44% identity), Na+-dependent neutral amino acid transporter ASCT1 (57% identity; Arriza, J. L., Kavanaugh, M. P., Fairman, W. A., Wu, Y.-N., Murdoch, G. H., North, R. A., and Amara, S. G.(1993) J. Biol. Chem. 268, 15329-15332; Shafqat, S., Tamarappoo, B. K., Kilberg, M. S., Puranam, R. S., McNamara, J. O., Guadano-Ferraz, A., and Fremeau, T., Jr. (1993) J. Biol. Chem. 268, 15351-15355) and a mouse adipocyte differentiation-associated gene product AAAT (94% identity; Liao, K., and Lane, D.(1995) Biochem. Biophys. Res. Commun. 208, 1008-1015). When expressed in Xenopus laevis oocytes, ASCT2 exhibited Na+-dependent uptakes of neutral amino acids such as L-alanine, L-serine, L-threonine, L-cysteine, and L-glutamine at high affinity with Km values around 20 microM. L-Methionine, L-leucine, L-glycine, and L-valine were also transported by ASCT2 but with lower affinity. The substrate selectivity of ASCT2 was typical of amino acid transport system ASC, which prefers neutral amino acids without bulky or branched side chains. ASCT2 also transported L-glutamate at low affinity (Km = 1.6 mM). L-Glutamate transport was enhanced by lowering extracellular pH, suggesting that L-glutamate was transported as protonated form. In contrast to electrogenic transport of glutamate transporters and the other ASC isoform ASCT1, ASCT2-mediated amino acid transport was electroneutral. Na+ dependence of L-alanine uptake fits to the Michaelis-Menten equation, suggesting a single Na+ cotransported with one amino acid, which was distinct from glutamate transporters coupled to two Na+. Northern blot hybridization revealed that ASCT2 was mainly expressed in kidney, large intestine, lung, skeletal muscle, testis, and adipose tissue. Functional characterization of ASCT2 provided fruitful information on the properties of substrate binding sites and the mechanisms of transport of Na+-dependent neutral and acidic amino acid transporter family, which would facilitate the structure-function analyses based on the comparison of the primary structures of ASCT2 and the other members of the family.

Amino Acid Sequence↗