scholarly journals Hexose transport in Torulaspora delbrueckii: identification of Igt1, a new dual-affinity transporter

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Andreia Pacheco ◽  
Lorena Donzella ◽  
Maria Jose Hernandez-Lopez ◽  
Maria Judite Almeida ◽  
Jose Antonio Prieto ◽  
...  
Keyword(s):  
Glucose Transporter ◽  
Functional Characterization ◽  
Sugar Transport ◽  
Sugar Metabolism ◽  
Torulaspora Delbrueckii ◽  
Sugar Transporters ◽  
Rate Limiting Step ◽  
Efficient Fermentation ◽  
Rate Limiting

ABSTRACT Torulaspora delbrueckii is a yeast species receiving increasing attention from the biotechnology industry, with particular relevance in the wine, beer and baking sectors. However, little is known about its sugar transporters and sugar transport capacity, frequently a rate-limiting step of sugar metabolism and efficient fermentation. Actually, only one glucose transporter, Lgt1, has been characterized so far. Here we report the identification and characterization of a second glucose transporter gene, IGT1, located in a cluster, upstream of LGT1 and downstream of two other putative hexose transporters. Functional characterization of IGT1 in a Saccharomyces cerevisiae hxt-null strain revealed that it encodes a transporter able to mediate uptake of glucose, fructose and mannose and established that its affinity, as measured by Km, could be modulated by glucose concentration in the medium. In fact, IGT1-transformed S. cerevisiae hxt-null cells, grown in 0.1% glucose displayed biphasic glucose uptake kinetics with an intermediate- (Km = 6.5 ± 2.0 mM) and a high-affinity (Km = 0.10 ± 0.01 mM) component, whereas cells grown in 2% glucose displayed monophasic kinetics with an intermediate-affinity (Km of 11.5 ± 1.5 mM). This work contributes to a better characterization of glucose transport in T. delbrueckii, with relevant implications for its exploitation in the food industry.

scholarly journals Imaging specific cellular glycan structures using glycosyltransferases via click chemistry

2017 ◽  
Author(s):  
Zhengliang L Wu ◽  
Anthony Person ◽  
Matthew Anderson ◽  
Barbara Burroughs ◽  
Timothy Tatge ◽  
...  
Keyword(s):  
Umbilical Vein ◽  
Functional Characterization ◽  
Tn Antigen ◽  
Stem Cell Line ◽  
Rate Limiting Step ◽  
Architectural Features ◽  
Huvec Cells ◽  
Umbilical Vein Endothelial Cells ◽  
Rate Limiting

AbstractHeparan sulfate (HS) is a polysaccharide fundamentally important for biologically activities. T/Tn antigens are universal carbohydrate cancer markers. Here, we report the specific imaging of these carbohydrates using a mesenchymal stem cell line and human umbilical vein endothelial cells (HUVEC). The staining specificities were demonstrated by comparing imaging of different glycans and validated by either removal of target glycans, which results in loss of signal, or installation of target glycans, which results in gain of signal. As controls, representative key glycans including O-GlcNAc, lactosaminyl glycans and hyaluronan were also imaged. HS staining revealed novel architectural features of the extracellular matrix (ECM) of HUVEC cells. Results from T/Tn antigen staining suggest that O-GalNAcylation is a rate-limiting step for O-glycan synthesis. Overall, these highly specific approaches for HS and T/Tn antigen imaging should greatly facilitate the detection and functional characterization of these biologically important glycans.

Functional characterization of mouse sodium/glucose transporter type 3b

2010 ◽  
Vol 299 (1) ◽  
pp. C58-C65 ◽  
Author(s):  
Oscar Aljure ◽  
Ana Díez-Sampedro
Keyword(s):  
Steady State ◽  
Human Genome ◽  
Conformational Changes ◽  
Glucose Transporter ◽  
Glucose Sensor ◽  
Functional Characterization ◽  
Sugar Transport ◽  
Tightly Coupled ◽  
Type 3

Despite belonging to a family of sugar cotransporters, human sodium/glucose transporter type 3 (hSGLT3) does not transport sugar, but it depolarizes the cell in the presence of extracellular sugar, and thus it has been suggested to work as a sugar sensor. In the human genome there is one SGLT3 gene, yet in mouse there are two. In this study we cloned one of them, mouse SGLT3b (mSGLT3b) and characterized the protein. We found that mSGLT3b has low affinity for sugars, as does hSGLT3, but surprisingly, mSGLT3b transports sugar, although the sugar transport is not as tightly coupled to cations as in SGLT1. Moreover, the sugar specificity of mSGLT3b has characteristics reminiscent of both SGLT1 and hSGLT3: mSGLT3b does not respond to galactose, similar to hSGLT3, but neither does it respond to 1-deoxynojirimycin, unlike hSGLT3 but similar to SGLT1. mSGLT3b has low apparent affinities for sugar and Na+ and, furthermore, displays pre-steady-state currents, which in SGLT1 report on conformational changes in the protein. Finally, phlorizin, the typical inhibitor of SGLT proteins, also inhibits mSGLT3b. In summary, although mSGLT3b has some characteristics that resemble SGLT1 and others that are similar to hSGLT3, its low sugar affinity and uncoupled sugar transport lead us to conclude that mSGLT3b likely functions as a physiological glucose sensor similar to hSGLT3.

Identification of a novel glucose transporter-like protein—GLUT-12

2002 ◽  
Vol 282 (3) ◽  
pp. E733-E738 ◽  
Author(s):  
Suzanne Rogers ◽  
Maria L. Macheda ◽  
Susan E. Docherty ◽  
Maynard D. Carty ◽  
Michael A. Henderson ◽  
...  
Keyword(s):  
Breast Cancer Cells ◽  
Glucose Transporter ◽  
Sugar Transport ◽  
Amino Acid Identity ◽  
Specific Expression ◽  
Glut 4 ◽  
Glucose Transport System ◽  
Metabolic Properties ◽  
Mcf 7

Facilitative glucose transporters exhibit variable hexose affinity and tissue-specific expression. These characteristics contribute to specialized metabolic properties of cells. Here we describe the characterization of a novel glucose transporter-like molecule, GLUT-12. GLUT-12 was identified in MCF-7 breast cancer cells by homology to the insulin-regulatable glucose transporter GLUT-4. The GLUT-12 cDNA encodes 617 amino acids, which possess features essential for sugar transport. Di-leucine motifs are present in NH2 and COOH termini at positions similar to the GLUT-4 FQQI and LL targeting motifs. GLUT-12 exhibits 29% amino acid identity with GLUT-4 and 40% to the recently described GLUT-10. Like GLUT-10, a large extracellular domain is predicted between transmembrane domains 9 and 10. Genomic organization of GLUT-12 is highly conserved with GLUT-10 but distinct from GLUTs 1–5. Immunofluorescence showed that, in the absence of insulin, GLUT-12 is localized to the perinuclear region in MCF-7 cells. Immunoblotting demonstrated GLUT-12 expression in skeletal muscle, adipose tissue, and small intestine. Thus GLUT-12 is potentially part of a second insulin-responsive glucose transport system.

Molecular and functional characterization and tissue localization of 2 glucose transporter homologues (TGTP1 and TGTP2) from the tapeworm Taenia solium

Parasitology ◽  
1998 ◽  
Vol 117 (6) ◽  
pp. 579-588 ◽  
Author(s):  
D. RODRÍGUEZ-CONTRERAS ◽  
P. J. SKELLY ◽  
A. LANDA ◽  
C. B. SHOEMAKER ◽  
J. P. LACLETTE
Keyword(s):  
Glucose Transporter ◽  
Cytochalasin B ◽  
Functional Characterization ◽  
Taenia Solium ◽  
Cdna Clones ◽  
Facilitated Diffusion ◽  
Porcine Cysticercosis ◽  
Isolation And Characterization ◽  
Hexose Uptake

Tapeworms absorb and consume large quantities of glucose through their syncytial tegument, storing the excess as glycogen. Although some studies on the metabolism of glucose in several tapeworms are available, the proteins that mediate its uptake and distribution in their tissue have not been identified. We describe the isolation and characterization of cDNA clones encoding 2 facilitated diffusion glucose transporters (TGTP1 and TGTP2) from Taenia solium, the causal agent of human and porcine cysticercosis. Radio-isotope labelled hexose uptake mediated by TGTP1 expressed in Xenopus oocytes is inhibited by the natural stereoisomers d-glucose and d-mannose but not by l-glucose. Transport by TGTP1 is sensitive to classical inhibitors of facilitated diffusion such as phloretin and cytochalasin B, and insensitive to ouabain. TGTP2 did not function in Xenopus oocytes. Localization studies using specific anti-TGTP1 and anti-TGTP2 antibodies show that TGTP1 is abundant in a number of structures underlying the tegument in adult parasites and larvae, whereas TGTP2 appears to be localized only on the tegumentary surface of the larvae and is not detected in adults.

Saturable and nonsaturable hexose uptake in cultured human skin fibroblasts

1978 ◽  
Vol 56 (2) ◽  
pp. 80-88 ◽  
Author(s):  
Ralph J. Germinario ◽  
Maureen Oliveira ◽  
Hyman Leung
Keyword(s):  
Human Skin ◽  
Cytochalasin B ◽  
Competitive Inhibition ◽  
Sugar Transport ◽  
Skin Fibroblasts ◽  
Sugar Uptake ◽  
Human Skin Fibroblasts ◽  
Rate Limiting Step ◽  
Hexose Uptake ◽  
Rate Limiting

The saturable transport of 2-deoxy-D-glucose across the cell membrane of cultured human skin fibroblasts was measured in sparse and confluent cultures. The contribution of nonsaturable sugar uptake to total sugar uptake was monitored by determining L-glucose uptake. The uptake of 2-deoxy-D-glucose was studied as a function of time and substrate concentration. Greater than 70% of transported 2-deoxy-D-glucose was phosphorylated after incubation for 2 min or less at all substrate concentrations employed (0.1 to 3.0 mM), and phosphorylation paralleled sugar uptake at these time intervals. Experiments with cytochalasin B demonstrated that an inhibition of transport was always paralleled by an equal inhibition of sugar phosphorylation.The kinetic constants for the uptake and phosphorylation of 2-deoxy-D-glucose and the inhibition of transport by competing sugars and cytochalasin B were calculated from Line-weaver-Burk plots. The Km and Vmax for saturable sugar uptake were calculated for sparse and confluent cultures after subtracting the contribution of nonsaturable sugar uptake. The resulting Km values for sugar uptake in the sparse and confluent cultures were 1.21 ± 0.04 and 0.88 ± 0.2 mM respectively. The corresponding Vmax values were 15.5 ± 1 nmol/mg protein∙min−1 for the sparse cultures and 10.1 ± 1 nmol/mg protein∙min−1 for the confluent cultures. In both sparse and confluent cultures, the Ki values for the competitive inhibition of sugar transport by D-glucose and 3-O-methyl-D-glucose were 0.8 and 2.7 mM respectively; the Ki value for the noncompetitive inhibition of sugar transport by cytochalasin B was 0.5 μM. The Km values for sugar phosphorylation by cell-free homogenates of sparse and confluent cultures were 0.57 ± 0.1 and 0.6 ± 0.1 mM respectively, while their respective Vmax values were 160 ± 53 and 139 ± 43 nmol/mg protein∙min−1.The data are in agreement with the concept that in cultured human skin fibroblasts sugar transport is the rate-limiting step in 2-deoxy-D-glucose metabolism and that phosphorylation is distinct from transport.

scholarly journals Mth1 regulates the interaction between the Rgt1 repressor and the Ssn6-Tup1 corepressor complex by modulating PKA-dependent phosphorylation of Rgt1

2013 ◽  
Vol 24 (9) ◽  
pp. 1493-1503 ◽  
Author(s):  
Adhiraj Roy ◽  
Yong Jae Shin ◽  
Kyu Hong Cho ◽  
Jeong-Ho Kim
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Glucose Transporter ◽  
Binding Activity ◽  
Supporting Evidence ◽  
Dna Binding Activity ◽  
Rate Limiting Step ◽  
Corepressor Complex ◽  
Necessary And Sufficient ◽  
Rate Limiting

Glucose uptake, the first, rate-limiting step of its utilization, is facilitated by glucose transporters. Expression of several glucose transporter (HXT) genes in yeast is repressed by the Rgt1 repressor, which recruits the glucose-responsive transcription factor Mth1 and the general corepressor complex Ssn6-Tup1 in the absence of glucose; however, it is derepressed when Mth1 is inactivated by glucose. Here we show that Ssn6-Tup1 interferes with the DNA-binding ability of Rgt1 in the absence of Mth1 and that the Rgt1 function abrogated by Ssn6 overexpression is restored by co-overexpression of Mth1. Thus Mth1 likely regulates Rgt1 function not by modulating its DNA-binding activity directly but by functionally antagonizing Ssn6-Tup1. Mth1 does so by acting as a scaffold-like protein to recruit Ssn6-Tup1 to Rgt1. Supporting evidence shows that Mth1 blocks the protein kinase A–dependent phosphorylation of Rgt1 that impairs the ability of Rgt1 to interact with Ssn6-Tup1. Of note, Rgt1 can bind DNA in the absence of Ssn6-Tup1 but does not inhibit transcription, suggesting that dissociation of Rgt1 from Ssn6-Tup1, but not from DNA, is necessary and sufficient for the expression of its target genes. Taken together, these findings show that Mth1 is a transcriptional corepressor that facilitates the recruitment of Ssn6-Tup1 by Rgt1.

Wavelet Based Structural Analysis of Electroless Deposits in the Diffusion Limited Regime

1994 ◽  
Vol 367 ◽  
Author(s):  
A. Arneodo ◽  
F. Argoul ◽  
A. Kuhn ◽  
J.F. Muzy
Keyword(s):  
Electroless Deposition ◽  
Side Reactions ◽  
Statistical Features ◽  
Diffusion Limited ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Fractal Patterns ◽  
Rate Limiting ◽  
Comparative Structural Characterization

AbstractWe discuss the actual relevance of thin gap geometry electrodeposition to generate fractal patterns that mimic the morphology of Witten and Sander's diffusion-limited aggregates (DLA). Eliminating migration and convection, as well as electrochemical side reactions, we show that electroless deposition is a good candidate to meet the requirements for diffusion to be the rate limiting step of the growth process. We use the wavelet transform microscope to achieve a comparative structural characterization of both experimental electroless deposits and numerical DLA clusters. The fact that five-fold symmetry and Fibonacci hierarchical ordering are found as common predominant statistical features is, to our knowledge, the first demonstration, relying on an appropriate structural fractal analysis, of the existence of DLA morphologies in an experimental context.

Sign in / Sign up

Export Citation Format

Share Document