The type 4 subfamily of P-type ATPases, putative aminophospholipid translocases with a role in human disease

The maintenance of phospholipid asymmetry in membrane bilayers is a paradigm in cell biology. However, the mechanisms and proteins involved in phospholipid translocation are still poorly understood. Members of the type 4 subfamily of P-type ATPases have been implicated in the translocation of phospholipids from the outer to the inner leaflet of membrane bilayers. In humans, several inherited disorders have been identified which are associated with loci harboring type 4 P-type ATPase genes. Up to now, one inherited disorder, Byler disease or progressive familial intrahepatic cholestasis type 1 (PFIC1), has been directly linked to mutations in a type 4 P-type ATPase gene. How the absence of an aminophospholipid translocase activity relates to this severe disease is, however, still unclear. Studies in the yeast Saccharomyces cerevisiae have recently identified important roles for type 4 P-type ATPases in intracellular membrane- and protein-trafficking events. These processes require an (amino)phospholipid translocase activity to initiate budding or fusion of membrane vesicles from or with other membranes. The studies in yeast have greatly contributed to our cell biological insight in membrane dynamics and intracellular-trafficking events; if this knowledge can be translated to mammalian cells and organs, it will help to elucidate the molecular mechanisms which underlie severe inherited human diseases such as Byler disease.     

Arabidopsis amidase 1, a member of the amidase signature family

Amidase 1 (AMI1), a specific indole-3-acetamide amidohydrolase, is an Arabidopsis thaliana amidase signature enzyme that catalyzes the synthesis of indole-3-acetic acid from indole-3-acetamide. Amidase signature family members catalyze a diverse range of enzymatic reactions and are found widespread in nature, for instance in bacteria, mammals, and plants. At the protein level, the family members share a conserved stretch of approximately 50-130 amino acids, the name-giving amidase signature. Elucidation of the crystal structures of a mammalian fatty acid amide hydrolase and the bacterial malonamidase E2 revealed an unusual Ser-cisSer-Lys catalytic triad in proteins of this family. In addition, other members, such as the amidase from Rhodococcus rhodochrous strain J1 or Sulfolobus solfataricus, seem to use an accessory Cys-cisSer-Lys center. AMI1 possesses all conserved amino-acid residues of the Ser-cisSer-Lys triad, but lacks the CX(3)C motif and therefore the Cys-cisSer-Lys catalytic site. Using a set of point-mutated variants of AMI1 and chemical modifications, we analyzed the relative importance of single amino-acid residues of AMI1 with respect to substrate conversion. These experiments revealed that a specific serine residue, Ser137, is essential for AMI1 enzymatic activity. We also report structural and functional differences of AMI1 from other amidase signature enzymes.     

Molecular interactions of yeast Neo1p, an essential member of the Drs2 family of aminophospholipid translocases, and its role in membrane trafficking within the endomembrane system

Neo1p is an essential yeast member of the highly conserved Drs2 family of P-type ATPases with proposed aminophospholipid translocase activity. Here we present evidence that Neo1p localizes to endosomes and Golgi elements. In agreement with that finding, the temperature-sensitive neo1-37 and neo1-69 mutants exhibit defects in receptor-mediated endocytosis, vacuole biogenesis, and vacuolar protein sorting. Furthermore, neo1 mutants accumulate aberrantly shaped membranous structures most likely derived from vacuoles and the endosomal/Golgi system. At permissive temperatures, HA-Neo1-69p, like wild-type Neo1p, is stable and associates with endosomes. In contrast, HA-Neo1-37p is rapidly degraded and is predominantly retained within the endoplasmic reticulum (ER). Thus, the two neo1 alleles affect the stability and localization of the mutant polypeptides in different ways. A C-terminally truncated and a C-terminally epitope-tagged version of Neo1p are nonfunctional and also mislocalize to the ER. In agreement with a role within the endomembrane system, Neo1p exhibits genetic and physical interactions with Ysl2p, a potential guanine nucleotide exchange factor for Arl1p. Interestingly, deletion of ARL1 rescues the temperature sensitivity of neo1-37 and neo1-69. We demonstrate that Arl1p in its myristoylated and GTP-bound form is responsible for the inhibitory effect. Thus, Neo1p, Ysl2p, and Arl1p represent three proteins that collaborate in membrane trafficking within the endosomal/Golgi system.     

AtNHX8, a member of the monovalent cation: proton antiporter-1 family in Arabidopsis thaliana, encodes a putative Li/H antiporter

The Arabidopsis monovalent cation:proton antiporter-1 (CPA1) family includes eight members, AtNHX1-8. AtNHX1 and AtNHX7/SOS1 have been well characterized as tonoplast and plasma membrane Na+/H+ antiporters, respectively. The proteins AtNHX2-6 have been phylogenetically linked to AtNHX1, while AtNHX8 appears to be related to AtNHX7/SOS1. Here we report functional characterization of AtNHX8. AtNHX8 T-DNA insertion mutants are hypersensitive to lithium ions (Li+) relative to wild-type plants, but not to the other metal ions such as sodium (Na+), potassium (K+) and caesium (Cs+). AtNHX8 overexpression in a triple-deletion yeast mutant AXT3 that exhibits defective Na+/Li+ transport specifically suppresses sensitivity to Li+, but does not affect Na+ sensitivity. Likewise, AtNHX8 overexpression complemented sensitivity to Li+, but not Na+, in sos1-1 mutant seedlings, and increased Li+ tolerance of both the sos1-1 mutant and wild-type seedlings. Results of Li+ and K+ measurement of loss-of-function and gain-of-function mutants indicate that AtNHX8 may be responsible for Li+ extrusion, and may be able to maintain K+ acquisition and intracellular ion homeostasis. Subcellular localization of the AtNHX8-enhanced green fluorescent protein (EGFP) fusion protein suggested that AtNHX8 protein is targeted to the plasma membrane. Taken together, our findings suggest that AtNHX8 encodes a putative plasma membrane Li+/H+ antiporter that functions in Li detoxification and ion homeostasis in Arabidopsis.     

BTG1, a member of a new family of antiproliferative genes.

The BTG1 gene locus has been shown to be involved in a t(8;12)(q24;q22) chromosomal translocation in a case of B-cell chronic lymphocytic leukemia. We report here the cloning and sequencing of the human BTG1 cDNA and establish the genomic organization of this gene. The full-length cDNA isolated from a lymphoblastoid cell line contains an open reading frame of 171 amino acids. BTG1 expression is maximal in the G0/G1 phases of the cell cycle and is down-regulated when cells progress throughout G1. Furthermore, transfection experiments of NIH3T3 cells indicate that BTG1 negatively regulates cell proliferation. The BTG1 open reading frame is 60% homologous to PC3, an immediate early gene induced by nerve growth factor in rat PC12 cells. Sequence and Northern blot analyses indicate that BTG1 and PC3 are not cognate genes. We then postulate that these two genes are the first members of a new family of antiproliferative genes.     

TaABC1, a member of the activity of bc1 complex protein kinase family from common wheat, confers enhanced tolerance to abiotic stresses in Arabidopsis

Abiotic stresses such as drought, salinity, and low temperature have drastic effects on plant growth and development. However, the molecular mechanisms regulating biochemical and physiological changes in response to stresses are not well understood. Protein kinases are major signal transduction factors among the reported molecular mechanisms mediating acclimation to environmental changes. Protein kinase ABC1 (activity of bc(1) complex) is involved in regulating coenzyme Q biosynthesis in mitochondria in yeast (Saccharomyces cersvisiae), and in balancing oxidative stress in chloroplasts in Arabidopsis thaliana. In the current study, TaABC1 (Triticum aestivum L. activity of bc(1) complex) protein kinase was localized to the cell membrane, cytoplasm, and nucleus. The effects of overexpressing TaABC1 in transgenic Arabidopsis plants on responses to drought, salt, and cold stress were further investigated. Transgenic Arabidopsis overexpressing the TaABC1 protein showed lower water loss and higher osmotic potential, photochemistry efficiency, and chlorophyll content, while cell membrane stability and controlled reactive oxygen species homeostasis were maintained. In addition, overexpression of TaABC1 increased the expression of stress-responsive genes, such as DREB1A, DREB2A, RD29A, ABF3, KIN1, CBF1, LEA, and P5CS, detected by real-time PCR analysis. The results suggest that TaABC1 overexpression enhances drought, salt, and cold stress tolerance in Arabidopsis, and imply that TaABC1 may act as a regulatory factor involved in a multiple stress response pathways.     

PAS1, a yeast gene required for peroxisome biogenesis, encodes a member of a novel family of putative ATPases

PAS genes are required for peroxisome biogenesis in the yeast S. cerevisiae. Here we describe the cloning, sequencing, and characterization of the PAS1 gene. Its gene product, Pas1p, has been identified as a rather hydrophilic 117 kd polypeptide. The predicted Pas1p sequence contains two putative ATP-binding sites and reveals a structural relationship to three other groups of proteins associated with different biological processes such as vesicle-mediated protein transport (NSF and Sec18p), control of cell cycle (Cdc48p, VCP, and p97-ATPase), and modulation of gene expression of the human immunodeficiency virus (TBP-1). The proteins share a highly conserved domain of about 185 amino acids including a consensus sequence for ATP binding. We suggest that these proteins are members of a novel family of putative ATPases and may be descendants of one common ancestor.     

Identification of a novel member of the T1R family of putative taste receptors

In the gustatory system, the recognition of sugars, amino acids and bitter-tasting compounds is the function of specialized G protein-coupled receptors. Recently, two members of novel subfamily of G protein-coupled receptors were proposed to function as taste receptors based on their specific expression in taste receptor cells. Here, we report the identification of a third member, T1R3, of this family of receptors. T1R3 maps near the telomere of mouse chromosome 4 rendering it a candidate for the Sac locus, a primary determinant of sweet preference in mice. Consistent with its candidacy for the Sac locus, T1R3 displays taste receptor cell-specific expression. In addition, taster and non-taster strains of mouse harbor different alleles of T1R3.     

ROXY1, a member of the plant glutaredoxin family, is required for petal development in Arabidopsis thaliana

We isolated three alleles of an Arabidopsis thaliana gene named ROXY1, which initiates a reduced number of petal primordia and exhibits abnormalities during further petal development. The defects are restricted to the second whorl of the flower and independent of organ identity. ROXY1 belongs to a subgroup of glutaredoxins that are specific for higher plants and we present data on the first characterization of a mutant from this large Arabidopsis gene family for which information is scarce. ROXY1 is predominantly expressed in tissues that give rise to new flower primordia, including petal precursor cells and petal primordia. Occasionally, filamentous organs with stigmatic structures are formed in the second whorl of the roxy1 mutant, indicative for an ectopic function of the class C gene AGAMOUS (AG). The function of ROXY1 in the negative regulation of AG is corroborated by premature and ectopic AG expression in roxy1-3 ap1-10 double mutants, as well as by enhanced first whorl carpeloidy in double mutants of roxy1 with repressors of AG, such as ap2 or lug. Glutaredoxins are oxidoreductases that oxidize or reduce conserved cysteine-containing motifs. Mutagenesis of conserved cysteines within the ROXY1 protein demonstrates the importance of cysteine 49 for its function. Our data demonstrate that, unexpectedly, a plant glutaredoxin is involved in flower development, probably by mediating post-translational modifications of target proteins required for normal petal organ initiation and morphogenesis.     

The C1orf9 gene encodes a putative transmembrane member of a novel protein family

Here we report the characterization of a human mRNA encoding a novel protein denoted C1orf9 (chromosome 1 open reading frame 9). The cDNA sequence, derived from a testis cDNA library, contains 5700 bp which encodes an open reading frame of 1254 amino acids. The deduced protein contains a putative N-terminal signal peptide and one putative transmembrane region, indicating membrane localization. No significant homology was found with known characterized proteins. However, a 150 amino acid region has significant homology to deduced protein sequences from other organisms, including Caenorhabditis elegans (43% identity), Saccharomyces cerevisiae (47% identity), Schizosaccharomyces pombe (48% identity), and two proteins from Arabidopsis thaliana (42% and 40% identity), suggesting a novel family of conserved domains. The C1orf9 gene was assigned to chromosome 1q24. The gene spans approximately 78.7 kb and is organized into at least 24 exons. Expression analysis revealed a single C1orf9 mRNA species of approximately 6.0 kb with a predominant expression in pancreas and testis, and only low levels of expression in other tissues examined.     

IL-36 a new member of the IL-1 family cytokines

Interleukin-36 (IL-36) is a pro-inflammatory cytokine which plays an important role in innate and adaptive immunity. IL-36 activates MAPK and NF-kB pathways and is produced by many different cells. This cytokine is a family member of interleukin-1 (IL-1) and plays an important role in the pathophysiology of several diseases. Here we summarise and review the new aspects of this important pro-inflammatory cytokine.     

Interaction of auxin and ERECTA in elaborating Arabidopsis inflorescence architecture revealed by the activation tagging of a new member of the YUCCA family putative flavin monooxygenases

The aboveground body of higher plants has a modular structure of repeating units, or phytomers. As such, the position, size, and shape of the individual phytomer dictate the plant architecture. The Arabidopsis (Arabidopsis thaliana) ERECTA (ER) gene regulates the inflorescence architecture by affecting elongation of the internode and pedicels, as well as the shape of lateral organs. A large-scale activation-tagging genetic screen was conducted in Arabidopsis to identify novel genes and pathways that interact with the ER locus. A dominant mutant, super1-D, was isolated as a nearly complete suppressor of a partial loss-of-function allele er-103. We found that SUPER1 encodes YUCCA5, a novel member of the YUCCA family of flavin monooxygenases. The activation tagging of YUCCA5 conferred increased levels of free indole acetic acid, increased auxin response, and mild phenotypic characteristics of auxin overproducers, such as elongated hypocotyls, epinastic cotyledons, and narrow leaves. Both genetic and cellular analyses indicate that auxin and the ER pathway regulate cell division and cell expansion in a largely independent but overlapping manner during elaboration of inflorescence architecture.     

Zebrafish tenascin-W,a new member of the tenascin family

A cDNA clone encoding tenascin-W, a novel member of the tenascin family, was isolated from a 20- to 28-h postfertilization (hpf) zebrafish cDNA library on the basis of the conserved epidermal growth factor-like domains represented in all tenascin molecules. An open reading frame of 2796 base pairs encodes a mature protein consisting of heptad repeats, a cysteine-rich amino terminal region, 3.5 epidermal growth factor-like repeats, five fibronectin type III homologous repeats, and a domain homologous to fibrinogen. These domains are the typical modular elements of molecules of the tenascin family. Sequence comparison demonstrated that TN-W shares homologies with the members of the tenascin family but is not a species homolog of any identified tenascin. The expression pattern of tn-w was analyzed by in situ hybridization in 1-day-old embryos, in 3-day-old larvae, and in juvenile zebrafish. At 24–25 hpf, tn-w mRNA was expressed in the lateral plate mesoderm, most conspicuously in the presumptive sclerotome. Migrating cells of sclerotomal and neural crest origins also showed high levels of expression. At 3 days, expression by sclerotomal and neural crest cells continued to be observed while expression in the somitic mesoderm was decreased. In juvenile fish, tn-w was expressed weakly by cells in the myosepta and, more strongly, by presumably nonneuronal cells in the dorsal root ganglia. In these tissues and at the same developmental stages, the expression of tn-w partially overlapped with the distribution of tn-c mRNA. In addition, tn-c was expressed in the central nervous system (CNS) and in the axial mesoderm, neither of which expressed tn-w at any of the age stages examined. The expression pattern of tn-w suggests an involvement in neural crest and sclerotome cell migration and in the formation of the skeleton. Similar and possibly overlapping functions could also be performed by tn-c, which appears to have additional functions during the development of the CNS. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 1–16, 1998     

FARE, a new family of foldback transposons in Arabidopsis

A new family of transposons, FARE, has been identified in Arabidopsis. The structure of these elements is typical of foldback transposons, a distinct subset of mobile DNA elements found in both plants and animals. The ends of FARE elements are long, conserved inverted repeat sequences typically 550 bp in length. These inverted repeats are modular in organization and are predicted to confer extensive secondary structure to the elements. FARE elements are present in high copy number, are heterogeneous in size, and can be divided into two subgroups. FARE1's average 1.1 kb in length and are composed entirely of the long inverted repeats. FARE2's are larger, up to 16.7 kb in length, and contain a large internal region in addition to the inverted repeat ends. The internal region is predicted to encode three proteins, one of which bears homology to a known transposase. FARE1.1 was isolated as an insertion polymorphism between the ecotypes Columbia and Nossen. This, coupled with the presence of 9-bp target-site duplications, strongly suggests that FARE elements have transposed recently. The termini of FARE elements and other foldback transposons are imperfect palindromic sequences, a unique organization that further distinguishes these elements from other mobile DNAs.