'Active' sugar transport in eukaryotes.

1994 ◽  
Vol 196 (1) ◽  
pp. 197-212 ◽  
Author(s):  
E M Wright ◽  
D D Loo ◽  
M Panayotova-Heiermann ◽  
M P Lostao ◽  
B H Hirayama ◽  
...  
Keyword(s):  
Gene Family ◽  
Conformational Changes ◽  
Sugar Transport ◽  
Large Family ◽  
Site Directed Mutagenesis ◽  
Xenopus Laevis Oocytes ◽  
Alpha Helices ◽  
Sugar Transporters ◽  
Ultrastructural Studies ◽  
Kinetics Of

Sugar transporters in prokaryotes and eukaryotes belong to a large family of membrane proteins containing 12 transmembrane alpha-helices. They are divided into two classes: one facilitative (uniporters) and the other concentrative (cotransporters or symporters). The concentrative transporters are energised by either H+ or Na+ gradients, which are generated and maintained by ion pumps. The facilitative and H(+)-driven sugar transporters belong to a gene family with a distinctive secondary structure profile. The Na(+)-driven transporters belong to a separate, small gene family with no homology at either the primary or secondary structural levels. It is likely that the Na(+)- and H(+)-driven sugar cotransporters share common transport mechanisms. To explore these mechanisms, we have expressed cloned eukaryote Na+/sugar cotransporters (SGLT) in Xenopus laevis oocytes and measured the kinetics of sugar transport using two-electrode voltage-clamp techniques. For SGLT1, we have developed a six-state ordered model that accounts for the experimental data. To test the model we have carried out the following experiments. (i) We measured pre-steady-state kinetics of SGLT1 using voltage-jump techniques. In the absence of sugar, SGLT1 exhibits transient carrier currents that reflect voltage-dependent conformational changes of the protein. Time constants for the carrier currents give estimates of rate constants for the conformational changes, and the charge movements, integrals of the transient currents, give estimates of the number and valence of SGLT1 proteins in the plasma membrane. Ultrastructural studies have confirmed these estimates of SGLT1 density. (ii) We have perturbed the kinetics of the cotransporter by site-directed mutagenesis of selected residues.(ABSTRACT TRUNCATED AT 250 WORDS)

The pre-steady-state kinetics of conformational changes in sugar transporters

1994 ◽  
Vol 22 (3) ◽  
pp. 650-654 ◽  
Author(s):  
Adrian R. Walmsley ◽  
Giles E. M. Martin ◽  
Terence P. McDonald ◽  
Peter J. F. Henderson
Keyword(s):  
Steady State ◽  
Conformational Changes ◽  
Sugar Transporters ◽  
Steady State Kinetics ◽  
Kinetics Of

Molecular Basis of Glucose Transport Mechanism in Plants

2019 ◽  
Author(s):  
Balaji Selvam ◽  
Ya-Chi Yu ◽  
Liqing Chen ◽  
Diwakar Shukla
Keyword(s):  
Molecular Dynamics ◽  
Free Energy ◽  
Molecular Dynamics Simulations ◽  
Conformational Changes ◽  
Transport Mechanism ◽  
Conformational Dynamics ◽  
Site Directed Mutagenesis ◽  
Free Energy Barrier ◽  
Transport Cycle ◽  
Dynamics Simulations

<p>The SWEET family belongs to a class of transporters in plants that undergoes large conformational changes to facilitate transport of sugar molecules across the cell membrane. However, the structures of their functionally relevant conformational states in the transport cycle have not been reported. In this study, we have characterized the conformational dynamics and complete transport cycle of glucose in OsSWEET2b transporter using extensive molecular dynamics simulations. Using Markov state models, we estimated the free energy barrier associated with different states as well as 1 for the glucose the transport mechanism. SWEETs undergoes structural transition to outward-facing (OF), Occluded (OC) and inward-facing (IF) and strongly support alternate access transport mechanism. The glucose diffuses freely from outside to inside the cell without causing major conformational changes which means that the conformations of glucose unbound and bound snapshots are exactly same for OF, OC and IF states. We identified a network of hydrophobic core residues at the center of the transporter that restricts the glucose entry to the cytoplasmic side and act as an intracellular hydrophobic gate. The mechanistic predictions from molecular dynamics simulations are validated using site-directed mutagenesis experiments. Our simulation also revealed hourglass like intermediate states making the pore radius narrower at the center. This work provides new fundamental insights into how substrate-transporter interactions actively change the free energy landscape of the transport cycle to facilitate enhanced transport activity.</p>

Kinetic analysis of mechanism of intestinal Na+-dependent sugar transport

1985 ◽  
Vol 248 (5) ◽  
pp. C498-C509 ◽  
Author(s):  
D. Restrepo ◽  
G. A. Kimmich
Keyword(s):  
Experimental Data ◽  
Steady State ◽  
Sugar Transport ◽  
Enzyme Kinetic ◽  
Steady State Distribution ◽  
State Distribution ◽  
Least Squares Fit ◽  
Coupling Stoichiometry ◽  
Rate Limiting ◽  
Kinetics Of

Zero-trans kinetics of Na+-sugar cotransport were investigated. Sugar influx was measured at various sodium and sugar concentrations in K+-loaded cells treated with rotenone and valinomycin. Sugar influx follows Michaelis-Menten kinetics as a function of sugar concentration but not as a function of Na+ concentration. Nine models with 1:1 or 2:1 sodium:sugar stoichiometry were considered. The flux equations for these models were solved assuming steady-state distribution of carrier forms and that translocation across the membrane is rate limiting. Classical enzyme kinetic methods and a least-squares fit of flux equations to the experimental data were used to assess the fit of the different models. Four models can be discarded on this basis. Of the remaining models, we discard two on the basis of the trans sodium dependence and the coupling stoichiometry [G. A. Kimmich and J. Randles, Am. J. Physiol. 247 (Cell Physiol. 16): C74-C82, 1984]. The remaining models are terter ordered mechanisms with sodium debinding first at the trans side. If transfer across the membrane is rate limiting, the binding order can be determined to be sodium:sugar:sodium.

scholarly journals The Molecular Physiology of Sodium- and Proton-Coupled Solute Transporters

Physiology ◽  
1998 ◽  
Vol 13 (3) ◽  
pp. 123-131 ◽  
Author(s):  
Angela Steel ◽  
Matthias A. Hediger
Keyword(s):  
Xenopus Laevis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Xenopus Laevis Oocytes ◽  
Missense Mutations ◽  
Molecular Physiology ◽  
Inherited Diseases ◽  
Biophysical Analysis ◽  
Solute Transporters

The expression of cloned Na+- and H+-coupled solute transporters in Xenopus laevis oocytes has permitted detailed molecular and biophysical analysis and illuminated unique mechanistic features. The identification of missense mutations in inherited diseases and site-directed mutagenesis studies have enhanced our understanding of their roles in physiological and pathological processes.

scholarly journals Genome- Wide Identification, Structure Characterization and Expression Pattern Profiling of the Aquaporin Gene Family in Betula pendula

2021 ◽  
Vol 22 (14) ◽  
pp. 7269
Author(s):  
Jean-Stéphane Venisse ◽  
Eele Õunapuu-Pikas ◽  
Maxime Dupont ◽  
Aurélie Gousset-Dupont ◽  
Mouadh Saadaoui ◽  
...  
Keyword(s):  
Cold Stress ◽  
Gene Family ◽  
Betula Pendula ◽  
Large Family ◽  
Biochemical Properties ◽  
Silver Birch ◽  
Structure Characterization ◽  
Transcriptional Expression ◽  
Genome Wide ◽  
A Genome

Aquaporin water channels (AQPs) constitute a large family of transmembrane proteins present throughout all kingdoms of life. They play key roles in the flux of water and many solutes across the membranes. The AQP diversity, protein features, and biological functions of silver birch are still unknown. A genome analysis of Betula pendula identified 33 putative genes encoding full-length AQP sequences (BpeAQPs). They are grouped into five subfamilies, representing ten plasma membrane intrinsic proteins (PIPs), eight tonoplast intrinsic proteins (TIPs), eight NOD26-like intrinsic proteins (NIPs), four X intrinsic proteins (XIPs), and three small basic intrinsic proteins (SIPs). The BpeAQP gene structure is conserved within each subfamily, with exon numbers ranging from one to five. The predictions of the aromatic/arginine selectivity filter (ar/R), Froger’s positions, specificity-determining positions, and 2D and 3D biochemical properties indicate noticeable transport specificities to various non-aqueous substrates between members and/or subfamilies. Nevertheless, overall, the BpePIPs display mostly hydrophilic ar/R selective filter and lining-pore residues, whereas the BpeTIP, BpeNIP, BpeSIP, and BpeXIP subfamilies mostly contain hydrophobic permeation signatures. Transcriptional expression analyses indicate that 23 BpeAQP genes are transcribed, including five organ-related expressions. Surprisingly, no significant transcriptional expression is monitored in leaves in response to cold stress (6 °C), although interesting trends can be distinguished and will be discussed, notably in relation to the plasticity of this pioneer species, B. pendula. The current study presents the first detailed genome-wide analysis of the AQP gene family in a Betulaceae species, and our results lay a foundation for a better understanding of the specific functions of the BpeAQP genes in the responses of the silver birch trees to cold stress.

scholarly journals Multimerization of Hepatitis Delta Antigen Is a Critical Determinant of RNA Binding Specificity

2009 ◽  
Vol 84 (3) ◽  
pp. 1406-1413 ◽  
Author(s):  
Brian C. Lin ◽  
Dawn A. Defenbaugh ◽  
John L. Casey
Keyword(s):  
Conformational Changes ◽  
Rna Binding ◽  
Site Directed Mutagenesis ◽  
Minimum Length ◽  
Critical Determinant ◽  
Hepatitis Delta ◽  
Ribonucleoprotein Complex ◽  
Delta Antigen ◽  
Hepatitis Delta Antigen

ABSTRACT Hepatitis delta virus (HDV) RNA forms an unbranched rod structure that is associated with hepatitis delta antigen (HDAg) in cells replicating HDV. Previous in vitro binding experiments using bacterially expressed HDAg showed that the formation of a minimal ribonucleoprotein complex requires an HDV unbranched rod RNA of at least about 300 nucleotides (nt) and suggested that HDAg binds the RNA as a multimer of fixed size. The present study specifically examines the role of HDAg multimerization in the formation of the HDV ribonucleoprotein complex (RNP). Disruption of HDAg multimerization by site-directed mutagenesis was found to profoundly alter the nature of RNP formation. Mutant HDAg proteins defective for multimerization exhibited neither the 300-nt RNA size requirement for binding nor specificity for the unbranched rod structure. The results unambiguously demonstrate that HDAg binds HDV RNA as a multimer and that the HDAg multimer is formed prior to binding the RNA. RNP formation was found to be temperature dependent, which is consistent with conformational changes occurring on binding. Finally, analysis of RNPs constructed with unbranched rod RNAs successively longer than the minimum length indicated that multimeric binding is not limited to the first HDAg bound and that a minimum RNA length of between 604 and 714 nt is required for binding of a second multimer. The results confirm the previous proposal that HDAg binds as a large multimer and demonstrate that the multimer is a critical determinant of the structure of the HDV RNP.

scholarly journals Structures ofDrosophila melanogasterRab2 and Rab3 bound to GMPPNP

2015 ◽  
Vol 71 (1) ◽  
pp. 34-40 ◽  
Author(s):  
Jennifer A. Lardong ◽  
Jan H. Driller ◽  
Harald Depner ◽  
Christoph Weise ◽  
Astrid Petzoldt ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Intracellular Trafficking ◽  
Large Family ◽  
Cysteine Residue ◽  
Effector Proteins ◽  
Rab Proteins ◽  
Rab Gtpases ◽  
Ras Proteins ◽  
Transport Vesicles ◽  
Switch Regions

Rab GTPases belong to the large family of Ras proteins. They act as key regulators of membrane organization and intracellular trafficking. Functionally, they act as switches. In the active GTP-bound form they can bind to effector proteins to facilitate the delivery of transport vesicles. Upon stimulation, the GTP is hydrolyzed and the Rab proteins undergo conformational changes in their switch regions. This study focuses on Rab2 and Rab3 fromDrosophila melanogaster. Whereas Rab2 is involved in vesicle transport between the Golgi and the endoplasmatic reticulum, Rab3 is a key player in exocytosis, and in the synapse it is involved in the assembly of the presynaptic active zone. Here, high-resolution crystal structures of Rab2 and Rab3 in complex with GMPPNP and Mg2+are presented. In the structure of Rab3 a modified cysteine residue is observed with an enigmatic electron density attached to its thiol function.

scholarly journals Structure–Function Relations of the First and Fourth Predicted Extracellular Linkers of the Type IIa Na+/Pi Cotransporter

2004 ◽  
Vol 124 (5) ◽  
pp. 475-488 ◽  
Author(s):  
Colin Ehnes ◽  
Ian C. Forster ◽  
Katja Kohler ◽  
Andrea Bacconi ◽  
Gerti Stange ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Reaction Rates ◽  
Transport Pathway ◽  
Extracellular Medium ◽  
Voltage Range ◽  
Substituted Cysteine Accessibility ◽  
Voltage Dependent ◽  
Opposite Behavior ◽  
Rate Limiting ◽  
Kinetics Of

The putative first intracellular and third extracellular linkers are known to play important roles in defining the transport properties of the type IIa Na+-coupled phosphate cotransporter (Kohler, K., I.C. Forster, G. Stange, J. Biber, and H. Murer. 2002b. J. Gen. Physiol. 120:693–705). To investigate whether other stretches that link predicted transmembrane domains are also involved, the substituted cysteine accessibility method (SCAM) was applied to sites in the predicted first and fourth extracellular linkers (ECL-1 and ECL-4). Mutants based on the wild-type (WT) backbone, with substituted novel cysteines, were expressed in Xenopus oocytes, and their function was assayed by isotope uptake and electrophysiology. Functionally important sites were identified in both linkers by exposing cells to membrane permeant and impermeant methanethiosulfonate (MTS) reagents. The cysteine modification reaction rates for sites in ECL-1 were faster than those in ECL-4, which suggested that the latter were less accessible from the extracellular medium. Generally, a finite cotransport activity remained at the end of the modification reaction. The change in activity was due to altered voltage-dependent kinetics of the Pi-dependent current. For example, cys substitution at Gly-134 in ECL-1 resulted in rate-limiting, voltage-independent cotransport activity for V ≤ −80 mV, whereas the WT exhibited a linear voltage dependency. After cys modification, this mutant displayed a supralinear voltage dependency in the same voltage range. The opposite behavior was documented for cys substitution at Met-533 in ECL-4. Modification of cysteines at two other sites in ECL-1 (Ile-136 and Phe-137) also resulted in supralinear voltage dependencies for hyperpolarizing potentials. Taken together, these findings suggest that ECL-1 and ECL-4 may not directly form part of the transport pathway, but specific sites in these linkers can interact directly or indirectly with parts of NaPi-IIa that undergo voltage-dependent conformational changes and thereby influence the voltage dependency of cotransport.

Human organic anion transporter 1B1 and 1B3 function as bidirectional carriers and do not mediate GSH-bile acid cotransport

2007 ◽  
Vol 293 (1) ◽  
pp. G271-G278 ◽  
Author(s):  
Chitrawina Mahagita ◽  
Steven M. Grassl ◽  
Pawinee Piyachaturawat ◽  
Nazzareno Ballatori
Keyword(s):  
Bile Acid ◽  
Transport Mechanism ◽  
Organic Anion ◽  
Large Family ◽  
Organic Anion Transporter ◽  
Xenopus Laevis Oocytes ◽  
Facilitated Diffusion ◽  
Transport Activity ◽  
Estrone Sulfate ◽  
Anion Transporter

Organic anion transporting polypeptides (OATP/ SLCO) are generally believed to function as electroneutral anion exchangers, but direct evidence for this contention has only been provided for one member of this large family of genes, rat Oatp1a1/Oatp1 ( Slco1a1). In contrast, a recent study has indicated that human OATP1B3/OATP-8 ( SLCO1B3) functions as a GSH-bile acid cotransporter. The present study examined the transport mechanism and possible GSH requirement of the two members of this protein family that are expressed in relatively high levels in the human liver, OATP1B3/OATP-8 and OATP1B1/OATP-C ( SLCO1B1). Uptake of taurocholate in Xenopus laevis oocytes expressing either OATP1B1/OATP-C, OATP1B3/OATP-8, or polymorphic forms of OATP1B3/OATP-8 (namely, S112A and/or M233I) was cis-inhibited by taurocholate and estrone sulfate but was unaffected by GSH. Likewise, taurocholate and estrone sulfate transport were trans-stimulated by estrone sulfate and taurocholate but were unaffected by GSH. OATP1B3/OATP-8 also did not mediate GSH efflux or GSH-taurocholate cotransport out of cells, indicating that GSH is not required for transport activity. In addition, estrone sulfate uptake in oocytes microinjected with OATP1B3/OATP-8 or OATP1B1/OATP-C cRNA was unaffected by depolarization of the membrane potential or by changes in pH, suggesting an electroneutral transport mechanism. Overall, these results indicate that OATP1B3/OATP-8 and OATP1B1/OATP-C most likely function as bidirectional facilitated diffusion transporters and that GSH is not a substrate or activator of their transport activity.

Overlap of IGF- and heparin-binding sites in rat IGF-binding protein-5

2000 ◽  
Vol 24 (1) ◽  
pp. 43-51 ◽  
Author(s):  
H Song ◽  
J Beattie ◽  
IW Campbell ◽  
GJ Allan
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Binding Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Heparin Binding ◽  
Igf I ◽  
Heparin Binding Domain ◽  
Igf Binding

Using site-directed mutagenesis, we have undertaken a study of a potential IGF-binding site in the C-terminal domain of rat IGFBP-5, lying close to or within a previously described heparin-binding domain (residues 201-218) in this protein. After analysis of binding activity using three different methods - ligand blotting, solution phase equilibrium binding and biosensor measurement of real-time on- and off-rates - we report that the mutation of two highly conserved residues within this region (glycine 203 and glutamine 209) reduces the affinity of the binding protein for both IGF-I and IGF-II, while having no effect on heparin binding. In addition, we confirm that mutation of basic residues within the heparin-binding domain (R201L, K202E, K206Q and R214A) results in a protein that has attenuated heparin binding but shows only a small reduction in affinity for IGF-I and -II. Previous findings have described the reduction in affinity of IGFBP-5 for IGFs that occurs after complexation of the binding protein with heparin or other components of the extracellular matrix (ECM) and have postulated that such an interaction may result in conformational changes in protein structure, affecting subsequent IGF interaction. Our data suggesting potential overlap of heparin- and IGF-binding domains argue for a more direct effect of ECM modulation of the affinity of IGFBP-5 for ligand by partial occlusion of the IGF-binding site after interaction with ECM.

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