Identification of residues critical for proton-coupled glutathione translocation in the yeast glutathione transporter, Hgt1p

2017 ◽  
Vol 474 (11) ◽  
pp. 1807-1821
Author(s):  
Mohammad Zulkifli ◽  
Anand Kumar Bachhawat
Keyword(s):  
Proton Transport ◽  
Driving Force ◽  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Random Mutagenesis ◽  
Substrate Transport ◽  
Transporter Family ◽  
Proton Relay ◽  
Proton Symport ◽  
Glutathione Transport

The proton gradient acts as the driving force for the transport of many metabolites across fungal and plant plasma membranes. Identifying the mechanism of proton relay is critical for understanding the mechanism of transport mediated by these transporters. We investigated two strategies for identifying residues critical for proton-dependent substrate transport in the yeast glutathione transporter, Hgt1p, a member of the poorly understood oligopeptide transporter family of transporters. In the first strategy, we tried to identify the pH-independent mutants that could grow at higher pH when dependant on glutathione transport. Screening a library of 269 alanine mutants of the transmembrane domains (TMDs) along with a random mutagenesis strategy yielded two residues (E135K on the cusp of TMD2 and N710S on TMD12) that permitted growth on glutathione at pH 8.0. Further analysis revealed that these residues were not involved in proton symport even though they conferred better transport at a higher pH. The second strategy involved a knowledge-driven approach, targeting 31 potential residues based on charge, conservation and location. Mutation of these residues followed by functional and biochemical characterization revealed E177A, Y193A, D335A, Y374A, H445A and R554A as being defective in proton transport. Further analysis enabled possible roles of these residues to be assigned in proton relay. The implications of these findings in relation to Hgt1p and the suitability of these strategic approaches for identifying such residues are discussed.

scholarly journals Immunocytochemical localization and biochemical characterization of a novel plasma membrane-associated, neutral pH optimum α-l-fucosidase from rat testis and epididymal spermatozoa

1996 ◽  
Vol 318 (3) ◽  
pp. 821-831 ◽  
Author(s):  
Manuel AVILÉS ◽  
Irene ABASCAL ◽  
José Angel MARTÍNEZ-MENÁRGUEZ ◽  
María Teresa CASTELLS ◽  
Sheri R. SKALABAN ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Gradient Centrifugation ◽  
Light And Electron Microscopy ◽  
Enzymic Activity ◽  
Epididymal Sperm ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum ◽  
Neutral Ph

1. Immunocytochemical and biochemical techniques have been used to localize and characterize a novel plasma membrane-associated, neutral-pH-optimum α-l-fucosidase from rat spermatozoa. Light and electron microscopy specifically localized the fucosidase on the plasma membrane of the convex region of the principal segment of testicular and cauda epididymal sperm heads. Immunoreactivity for α-l-fucosidase was also detected in the Golgi apparatus of spermatocytes and spermatids but no immunoreactivity was observed in the acrosome. 2. Fractionation of epididymal sperm homogenates indicated that over 90% of the α-l-fucosidase activity was associated with the 48000 g pellet. This pellet-associated activity could be solubilized with 0.5 M NaCl but not with 0.5% Triton X-100, suggesting that fucosidase is peripherally associated with membranes. Sucrose-density-gradient centrifugation of sperm homogenates indicated that fucosidase was enriched in the plasma membrane-enriched fraction. Analysis of α-l-fucosidase on intact epididymal sperm indicated that the enzyme was active, displayed linear kinetics and had a pH–activity curve (with an optimum near 7) which was comparable to that of fucosidase from epididymal sperm extracts. These results further suggest that fucosidase is associated with plasma membranes, and that its active site is accessible to fucoconjugates. Evidence that most of the fucosidase is associated with the exterior of the plasma membrane came from studies in which intact sperm had fucosidase activity comparable to that of sperm sonicates, and from studies in which approx. 90% of the fucosidase activity on intact sperm could be released from the sperm by gentle shaking with 0.5 M NaCl. Isoelectric focusing indicated that the NaCl-solubilized epididymal sperm fucosidase appears to have one major and one minor isoform with pIs near 7.2 and 5.2, respectively. SDS/PAGE and Western blotting indicated that the NaCl-solubilized extract of epididymal sperm contains two protein bands of 54 and 50 kDa which were highly immunoreactive with the IgG fraction of anti-fucosidase antibodies. Although the function of the novel sperm fucosidase is not known, its specific localization to the plasma membrane of the region of the rat sperm head involved in sperm–egg binding and its high enzymic activity at neutral pH on intact sperm suggest that this enzyme may have a role in sperm–egg interactions.

Affinity labeling the bovine gallbladder cholecystokinin receptor using a battery of probes

1988 ◽  
Vol 255 (5) ◽  
pp. G579-G586
Author(s):  
B. Schjoldager ◽  
S. P. Powers ◽  
L. J. Miller
Keyword(s):  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Peptide Hormone ◽  
Molecular Probes ◽  
Affinity Labeling ◽  
Molecular Heterogeneity ◽  
Cck Receptors ◽  
Amino Terminal ◽  
Binding Subunit

Although the gallbladder was the first recognized target of the peptide hormone cholecystokinin (CCK) and is a physiologically important target, only one preliminary report of the biochemical characterization of this receptor exists. Recently, a series of molecular probes for the affinity labeling of different domains of the pancreatic CCK receptor have been developed. In this work we report the application of several of those probes toward the biochemical characterization of the bovine gallbladder muscularis receptor. These include "long" (125I-Bolton-Hunter-CCK-33) and "short" (125I-D-Tyr-Gly-[Nle28,31)CCK-(26-33)]) probes chemically cross-linkable through their amino-terminal amino groups and monofunctional probes with their photolabile moieties at their amino terminus (2-diazo-3,3,3-trifluoropropionyl-125I-D-Tyr-Gly-[(Nle28,31) CCK-(26-33)]) and carboxyl terminus (125I-D-Tyr-Gly-[(Nle28,31,pNO2-Phe33)CCK-(26-33)]), that span the receptor-binding region. Each of these bound specifically and saturably to a preparation enriched in plasma membranes from bovine gallbladder muscularis (mean inhibitor constants: 5.2, 1.1, 0.8, and 1.8 nM, respectively). A major relative molecular weight (Mr) 70,000-85,000 band was specifically and reproducibly labeled with the appropriate apparent affinity by each of the probes, whereas labeling of minor bands of Mr 40,000-50,000, Mr 92,000, Mr 120,000, and Mr 200,000 was dependent on cross-linker type or concentration. These observations support the identification of the Mr 70,000-85,000 protein as the bovine gallbladder CCK-binding subunit and, since this is a different size from the pancreatic CCK-binding subunit, provide biochemical evidence for molecular heterogeneity of peripheral CCK receptors.

scholarly journals Biochemical Characterization of the C4-Dicarboxylate Transporter DctA from Bacillus subtilis

2010 ◽  
Vol 192 (11) ◽  
pp. 2900-2907 ◽  
Author(s):  
Maarten Groeneveld ◽  
Ruud G. J. Detert Oude Weme ◽  
Ria H. Duurkens ◽  
Dirk Jan Slotboom
Keyword(s):  
Bacillus Subtilis ◽  
Biochemical Characterization ◽  
Krebs Cycle ◽  
Membrane Vesicles ◽  
Positive Bacterium ◽  
Proton Symport ◽  
Negative Membrane Potential ◽  
Isolated Membrane Vesicles

ABSTRACT Bacterial secondary transporters of the DctA family mediate ion-coupled uptake of C4-dicarboxylates. Here, we have expressed the DctA homologue from Bacillus subtilis in the Gram-positive bacterium Lactococcus lactis. Transport of dicarboxylates in vitro in isolated membrane vesicles was assayed. We determined the substrate specificity, the type of cotransported ions, the electrogenic nature of transport, and the pH and temperature dependence patterns. DctA was found to catalyze proton-coupled symport of the four C4-dicarboxylates from the Krebs cycle (succinate, fumurate, malate, and oxaloacetate) but not of other mono- and dicarboxylates. Because (i) succinate-proton symport was electrogenic (stimulated by an internal negative membrane potential) and (ii) the divalent anionic form of succinate was recognized by DctA, at least three protons must be cotransported with succinate. The results were interpreted in the light of the crystal structure of the homologous aspartate transporter GltPh from Pyrococcus horikoshii.

Enrichment and biochemical characterization of Toxoplasma gondii tachyzoite plasmalemma

Parasitology ◽  
1997 ◽  
Vol 114 (5) ◽  
pp. 421-426 ◽  
Author(s):  
A. RABJEAU ◽  
F. FOUSSARD ◽  
G. MAURAS ◽  
J. F. DUBREMETZ
Keyword(s):  
Toxoplasma Gondii ◽  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Surface Membrane ◽  
Surface Protein ◽  
Protozoan Parasite ◽  
Membrane Complex ◽  
Inner Membrane Complex ◽  
Partial Dissociation ◽  
Major Surface

The protozoan parasite Toxoplasma gondii possesses a triple surface membrane called the pellicle. This is made of an outer plasmalemma and an inner membrane complex lying under the plasmalemma. Using a high salt glycerol treatment followed by sonication, we have obtained a partial dissociation of the pellicle. A plasmalemma-enriched fraction was isolated on 0·7M sucrose. It was identified by immunodetection of the tachyzoite major surface antigens. Protein content, resolved by SDS–PAGE, revealed that the surface protein SAG1 is the major component of the plasmalemma. The plasmalemma fraction is made of small vesicles (20–100 nm) which possess a low density (1·085–1·090 g/cm3 in sucrose) contrasting with other eukaryotic plasma membranes (1·12–1·16 g/cm3).

L-Malate transport and proton symport in vesicles prepared from Pseudomonas putida

1986 ◽  
Vol 64 (11) ◽  
pp. 1190-1194 ◽  
Author(s):  
F. R. Agbanyo ◽  
G. Moses ◽  
N. F. Taylor
Keyword(s):  
Membrane Potential ◽  
Pseudomonas Putida ◽  
Kinetic Analysis ◽  
Transport System ◽  
Electron Donor ◽  
Proton Transport ◽  
Proton Motive Force ◽  
Alkaline Ph ◽  
Proton Symport ◽  
Malate Transport

In vesicles from glucose-grown Pseudomonas putida, L-malate is transported by nonspecific physical diffusion. L-Malate also acts as an electron donor and generates a proton motive force (Δp) of 129 mV which is composed of a membrane potential (Δψ) of 60 mV and a ΔpH of 69 mV. In contrast, vesicles from succinate-grown cells (a) transport L-malate by a carrier-mediated system with a Km value of 14.3 mM and a Vmax of 313 nmol∙mg protein−1∙min−1, (b) generate no Δψ, ΔpH, or Δp when L-malate is the electron donor, and (c) produce an extravesicular alkaline pH during the transport of L-malate. A kinetic analysis of this L-malate-induced proton transport gives a Km value of 16 mM and a Vmax of 667 nmol H+∙mg protein−1∙min−1. This corresponds to a H+/L-malate ratio of 2.1. The failure to generate a Δp in these vesicles is considered, therefore, to be consistent with the induction in succinate-grown cells of an electrogenic proton symport L-malate transport system.

scholarly journals Regular structures in unit membranes. II. Morphological and biochemical characterization of two water-soluble membrane proteins isolated from the suckling rat ileum.

1976 ◽  
Vol 70 (1) ◽  
pp. 97-111 ◽  
Author(s):  
E R Jakoi ◽  
G Zampighi ◽  
J D Robertson
Keyword(s):  
High Speed ◽  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Membrane Surface ◽  
Gel Filtration ◽  
Water Soluble ◽  
Regular Structures ◽  
Membrane Surfaces ◽  
Negative Stain ◽  
Ordered Array

Specialized plasma membranes from the endocytic complex of ileal epithelial cells of suckling rats were isolated by differential flotation. Thin-section and negative-stain electron microscopy showed the luminal surfaces of these membranes to be covered by an ordered array of particles 14.5-nm separations in long rows. This particulate coating was released from the membrane surfaces by 10 mM CaCl2 and recovered free of membranes after dialysis against 0.5 mM EGTA and high-speed centrifugation. Two proteins were resolved by gel filtration to be in supernate: n-acetyl-beta-glucosaminidase and a filamentous protein which attaches n-acetylglucosaminidase to the membrane surface thereby providing bidirectionality to the array of enzyme. We believe that the filamentous protein has not been previously described. Therefore we have called it ligatin from the latin ligare, which translates "to bind together". Furthermore, we suggest that the membranes of the endocytic complex contain sites for the extracellular digestion of carbohydrate moieties in the maternal milk.

scholarly journals Effects of cholesterol content on activity of P-glycoproteins and membrane physical state, and consequences for anthelmintic resistance in the nematode Haemonchus contortus

Parasite ◽  
2020 ◽  
Vol 27 ◽  
pp. 3 ◽  
Author(s):  
Mickaël Riou ◽  
Fabrice Guégnard ◽  
Yves Le Vern ◽  
Isabelle Grasseau ◽  
Christine Koch ◽  
...  
Keyword(s):  
Haemonchus Contortus ◽  
Plasma Membranes ◽  
Anthelmintic Resistance ◽  
Active Role ◽  
Cholesterol Content ◽  
Cellular Mechanisms ◽  
Chemical Attack ◽  
Transporter Family ◽  
Anthelminthic Resistance

Eukaryote plasma membranes protect cells from chemical attack. Xenobiotics, taken up through passive diffusion, accumulate in the membranes, where they are captured by transporters, among which P-glycoproteins (Pgps). In nematodes such as Haemonchus contortus, eggshells and cuticles provide additional protective barriers against xenobiotics. Little is known about the role of these structures in the transport of chemical molecules. Pgps, members of the ABC transporter family, are present in eggshells and cuticles. Changes in the activity of these proteins have also been correlated with alterations in lipids, such as cholesterol content, in eggshells. However, the cellular mechanisms underlying these effects remain unclear. We show here that an experimental decrease in the cholesterol content of eggshells of Haemonchus contortus, with Methyl-beta-CycloDextrin (MβCD), results in an increase in membrane fluidity, favouring Pgp activity and leading to an increase in resistance to anthelmintics. This effect is modulated by the initial degree of anthelminthic resistance of the eggs. These results suggest that eggshell fluidity plays a major role in the modulation of Pgp activity. They confirm that Pgp activity is highly influenced by the local microenvironment, in particular sterols, as observed in some vertebrate models. Thus, eggshell barriers could play an active role in the transport of xenobiotics.

scholarly journals Differential targeting of glucose transporter isoforms heterologously expressed in Xenopus oocytes

1993 ◽  
Vol 290 (3) ◽  
pp. 707-715 ◽  
Author(s):  
H M Thomas ◽  
J Takeda ◽  
G W Gould
Keyword(s):  
Glucose Transport ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Subcellular Fractionation ◽  
Transport Activity ◽  
Glut 4 ◽  
Transporter Family ◽  
Native Cell

We have examined the subcellular distribution of three members of the human glucose transporter family expressed in oocytes from Xenopus laevis. Following injection of in vitro-transcribed mRNA encoding the transporter isoform to be studied, we have determined the subcellular localization of the expressed protein by immunofluorescence and by subcellular fractionation coupled with immunoblotting using specific anti-peptide antibodies. We have shown that both the liver-type (GLUT 2) and brain-type (GLUT 3) glucose transporters are expressed predominantly in the plasma membranes of oocytes, and in both cases high levels of glucose transport activity are exhibited. In contrast, the insulin-regulatable glucose transporter (GLUT 4) is localized predominantly to an intracellular membrane pool, and the levels of transport activity recorded in oocytes expressing GLUT 4 are correspondingly lower. The localization of the different transporter isoforms to distinct subcellular fractions mirrors the situation observed in their native cell type and thus demonstrates that oocytes may prove to be a useful system with which to study the targeting signals for this important class of membrane proteins. In addition, the determination of the amounts of the transporters expressed per oocyte together with a knowledge of their Km values has allowed us to estimate the turnover numbers of these transporters. Insulin was without effect on glucose transport in oocytes expressing any of these transporter isoforms. Microinjection of guanosine 5′-[gamma-thio]triphosphate into oocytes expressing GLUT 4 was also without effect on the transport rate.

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