Identification and functional characterization of EhClC-A, an Entamoeba histolytica ClC chloride channel located at plasma membrane

ABSTRACT Pre-mRNA splicing is essential to ensure accurate expression of many genes in eukaryotic organisms. In Entamoeba histolytica, a deep-branching eukaryote, approximately 30% of the annotated genes are predicted to contain introns; however, the accuracy of these predictions has not been tested. In this study, we mined an expressed sequence tag (EST) library representing 7% of amoebic genes and found evidence supporting splicing of 60% of the testable intron predictions, the majority of which contain a GUUUGU 5′ splice site and a UAG 3′ splice site. Additionally, we identified several splice site misannotations, evidence for the existence of 30 novel introns in previously annotated genes, and identified novel genes through uncovering their spliced ESTs. Finally, we provided molecular evidence for the E. histolytica U2, U4, and U5 snRNAs. These data lay the foundation for further dissection of the role of RNA processing in E. histolytica gene expression.

Download Full-text

Expression, purification, and initial structural characterization of YadQ, a bacterial homolog of mammalian ClC chloride channel proteins

FEBS Letters ◽

10.1016/s0014-5793(99)01740-8 ◽

2000 ◽

Vol 466 (1) ◽

pp. 26-28 ◽

Cited By ~ 15

Author(s):

Michael D. Purdy ◽

Michael C. Wiener

Keyword(s):

Structural Characterization ◽

Chloride Channel ◽

Channel Proteins ◽

Bacterial Homolog ◽

Clc Chloride Channel

Download Full-text

Functional characterization of a novel plasma membrane intrinsic protein2 in barley

Plant Signaling & Behavior ◽

10.4161/psb.22294 ◽

2012 ◽

Vol 7 (12) ◽

pp. 1648-1652 ◽

Cited By ~ 7

Author(s):

Mineo Shibasaka ◽

Sizuka Sasano ◽

Sigeko Utsugi ◽

Maki Katsuhara

Keyword(s):

Plasma Membrane ◽

Functional Characterization

Download Full-text

Functional characterization of a plasma membrane Na+/H+ antiporter from alkali grass (Puccinellia tenuiflora)

Molecular Biology Reports ◽

10.1007/s11033-010-0624-y ◽

2010 ◽

Vol 38 (7) ◽

pp. 4813-4822 ◽

Cited By ~ 28

Author(s):

Xin Wang ◽

Ru Yang ◽

Baichen Wang ◽

Guifeng Liu ◽

Chuanping Yang ◽

...

Keyword(s):

Plasma Membrane ◽

Functional Characterization ◽

Puccinellia Tenuiflora

Download Full-text

Detection of the Endosomal Sorting Complex Required for Transport inEntamoeba histolyticaand Characterization of the EhVps4 Protein

Journal of Biomedicine and Biotechnology ◽

10.1155/2010/890674 ◽

2010 ◽

Vol 2010 ◽

pp. 1-15 ◽

Cited By ~ 12

Author(s):

Israel López-Reyes ◽

Guillermina García-Rivera ◽

Cecilia Bañuelos ◽

Silvia Herranz ◽

Olivier Vincent ◽

...

Keyword(s):

Plasma Membrane ◽

Entamoeba Histolytica ◽

Hepatic Damage ◽

Multivesicular Bodies ◽

Protein Overexpression ◽

Specific Antibodies ◽

Endosomal Sorting ◽

Biochemical Studies

Eukaryotic endocytosis involves multivesicular bodies formation, which is driven by endosomal sorting complexes required for transport (ESCRT). Here, we showed the presence and expression of homologous ESCRT genes inEntamoeba histolytica. We cloned and expressed theEhvps4gene, an ESCRT member, to obtain the recombinant EhVps4 and generate specific antibodies, which immunodetected EhVps4 in cytoplasm of trophozoites. Bioinformatics and biochemical studies evidenced that rEhVps4 is an ATPase, whose activity depends on the conserved E211 residue. Next, we generated trophozoites overexpressing EhVps4 and mutant EhVps4-E211Q FLAG-tagged proteins. The EhVps4-FLAG was located in cytosol and at plasma membrane, whereas the EhVps4-E211Q-FLAG was detected as abundant cytoplasmic dots in trophozoites. Erythrophagocytosis, cytopathic activity, and hepatic damage in hamsters were not improved in trophozoites overexpressing EhVps4-FLAG. In contrast, EhVps4-E211Q-FLAG protein overexpression impaired these properties. The localization of EhVps4-FLAG around ingested erythrocytes, together with our previous results, strengthens the role for EhVps4 inE. histolyticaphagocytosis and virulence.

Download Full-text

RsSOS1 Responding to Salt Stress Might Be Involved in Regulating Salt Tolerance by Maintaining Na+ Homeostasis in Radish (Raphanus sativus L.)

Horticulturae ◽

10.3390/horticulturae7110458 ◽

2021 ◽

Vol 7 (11) ◽

pp. 458

Author(s):

Wanting Zhang ◽

Jingxue Li ◽

Junhui Dong ◽

Yan Wang ◽

Liang Xu ◽

...

Keyword(s):

Plasma Membrane ◽

Salt Stress ◽

Salt Tolerance ◽

Functional Characterization ◽

Vital Role ◽

Salt Stress Response ◽

Reading Frame ◽

Yield And Quality

Radish is a kind of moderately salt-sensitive vegetable. Salt stress seriously decreases the yield and quality of radish. The plasma membrane Na+/H+ antiporter protein Salt Overly Sensitive 1 (SOS1) plays a crucial role in protecting plant cells against salt stress, but the biological function of the RsSOS1 gene in radish remains to be elucidated. In this study, the RsSOS1 gene was isolated from radish genotype ‘NAU-TR17’, and contains an open reading frame of 3414 bp encoding 1137 amino acids. Phylogenetic analysis showed that RsSOS1 had a high homology with BnSOS1, and clustered together with Arabidopsis plasma membrane Na+/H+ antiporter (AtNHX7). The result of subcellular localization indicated that the RsSOS1 was localized in the plasma membrane. Furthermore, RsSOS1 was strongly induced in roots of radish under 150 mmol/L NaCl treatment, and its expression level in salt-tolerant genotypes was significantly higher than that in salt-sensitive ones. In addition, overexpression of RsSOS1 in Arabidopsis could significantly improve the salt tolerance of transgenic plants. Meanwhile, the transformation of RsSOS1△999 could rescue Na+ efflux function of AXT3 yeast. In summary, the plasma membrane Na+/H+ antiporter RsSOS1 plays a vital role in regulating salt-tolerance of radish by controlling Na+ homeostasis. These results provided useful information for further functional characterization of RsSOS1 and facilitate clarifying the molecular mechanism underlying salt stress response in radish.

Download Full-text

Structural and Functional Characterization of Insect Genes Encoding Ligand-Gated Chloride-Channel Subunits

ACS Symposium Series - Molecular Action of Insecticides on Ion Channels ◽

10.1021/bk-1995-0591.ch013 ◽

1995 ◽

pp. 205-215 ◽

Cited By ~ 3

Author(s):

Douglas C. Knipple ◽

Joseph E. Henderson ◽

David M. Soderlund

Keyword(s):

Chloride Channel ◽

Functional Characterization ◽

Genes Encoding

Download Full-text

Two Novel Classes of Enzymes Are Required for the Biosynthesis of Aurofusarin in Fusarium graminearum

Journal of Biological Chemistry ◽

10.1074/jbc.m110.179853 ◽

2011 ◽

Vol 286 (12) ◽

pp. 10419-10428 ◽

Cited By ~ 56

Author(s):

Rasmus J. N. Frandsen ◽

Claes Schütt ◽

Birgitte W. Lund ◽

Dan Staerk ◽

John Nielsen ◽

...

Keyword(s):

Plasma Membrane ◽

Fusarium Graminearum ◽

Polyketide Synthase ◽

Functional Characterization ◽

Extracellular Enzyme ◽

Gene Replacement ◽

Orphan Genes ◽

Pigment Biosynthesis ◽

Targeted Gene Replacement

Previous studies have reported the functional characterization of 9 out of 11 genes found in the gene cluster responsible for biosynthesis of the polyketide pigment aurofusarin in Fusarium graminearum. Here we reanalyze the function of a putative aurofusarin pump (AurT) and the two remaining orphan genes, aurZ and aurS. Targeted gene replacement of aurZ resulted in the discovery that the compound YWA1, rather than nor-rubrofusarin, is the primary product of F. graminearum polyketide synthase 12 (FgPKS12). AurZ is the first representative of a novel class of dehydratases that act on hydroxylated γ-pyrones. Replacement of the aurS gene resulted in accumulation of rubrofusarin, an intermediate that also accumulates when the GIP1, aurF, or aurO genes in the aurofusarin cluster are deleted. Based on the shared phenotype and predicted subcellular localization, we propose that AurS is a member of an extracellular enzyme complex (GIP1-AurF-AurO-AurS) responsible for converting rubrofusarin into aurofusarin. This implies that rubrofusarin, rather than aurofusarin, is pumped across the plasma membrane. Replacement of the putative aurofusarin pump aurT increased the rubrofusarin-to- aurofusarin ratio, supporting that rubrofusarin is normally pumped across the plasma membrane. These results provide functional information on two novel classes of proteins and their contribution to polyketide pigment biosynthesis.

Download Full-text