Frog Oocytes to Unveil the Structure and Supramolecular Organization of Human Transport Proteins (original) (raw)
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Structures and Models of Transporter Proteins
Journal of Pharmacology and Experimental Therapeutics, 2004
Transporter proteins in biological membranes may be divided into channels and carriers. Channels function as selective pores that open in response to a chemical or electrophysiological stimulus, allowing movement of a solute down an electrochemical gradient. Active carrier proteins use an energy producing process to translocate a substrate against a concentration gradient. Secondary active transporters use the movement of a solute down a concentration gradient to drive the translocation of another substrate across a membrane. ATP-binding cassette (ABC) transporters couple hydrolysis of adenosine triphosphate (ATP) to the translocation of various substrates across cell membranes. High-resolution three-dimensional structures have now been reported from X-ray crystallographic studies of six different transporters, including two ATP-binding cassette (ABC) transporters. These structures have explained the results
A facile approach for the in vitro assembly of multimeric membrane transport proteins
eLife, 2018
Membrane proteins such as ion channels and transporters are frequently homomeric. The homomeric nature raises important questions regarding coupling between subunits and complicates the application of techniques such as FRET or DEER spectroscopy. These challenges can be overcome if the subunits of a homomeric protein can be independently modified for functional or spectroscopic studies. Here, we describe a general approach for in vitro assembly that can be used for the generation of heteromeric variants of homomeric membrane proteins. We establish the approach using Glt, a glutamate transporter homolog that is trimeric in the native state. We use heteromeric Glt transporters to directly demonstrate the lack of coupling in substrate binding and demonstrate how heteromeric transporters considerably simplify the application of DEER spectroscopy. Further, we demonstrate the general applicability of this approach by carrying out the in vitro assembly of VcINDY, a Na-coupled succinate tra...
Structural Determinants of Water Permeation through the Sodium-Galactose Transporter vSGLT
Biophysical Journal, 2014
Human red cell AQP1 is the ®rst functionally de®ned member of the aquaporin family of membrane water channels. Here we describe an atomic model of AQP1 at 3.8 A Ê resolution from electron crystallographic data. Multiple highly conserved amino-acid residues stabilize the novel fold of AQP1. The aqueous pathway is lined with conserved hydrophobic residues that permit rapid water transport, whereas the water selectivity is due to a constriction of the pore diameter to about 3 A Ê over a span of one residue. The atomic model provides a possible molecular explanation to a longstanding puzzle in physiologyÐhow membranes can be freely permeable to water but impermeable to protons.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1983
The human red blood cell anion transport protein, band 3, was isolated and reconstituted into lipid vesicles. The main feature of the new reconstitution is the replacement of native lipids and of solubilizing detergent by externally added lipids, while band 3 protein is immobilized on a gel matrix. The vesicles formed upon detergent removal and sonication are unilameilar and sealed, and band 3 protein is the major polypeptide detectable in them. The method consists of: (a) solubilization of alkali-treated red blood cell membranes by Triton X-100; (b) binding of glycophorin and band 3 protein to diethylaminoethyl (DEAE)-ceUulose in Triton X-100 solution, followed by high ionic strength elution; (c) band 3 protein complexation to organomercurial Sepharose; (d) exchange of the Triton X-100 with the dialyzable detergent octyiglucopyranoside, while band 3 protein is complexed to the column; (e) elution of band 3 by cysteine (5 mM) in the presence of octylglucopyranoside; (f) addition of lipids (asolectin or egg phosphatidylcholine) to the protein-detergent suspension; and (g) dialysis of the mixture against 1% bovine serum albumin to remove the detergent completely. The vesicles were assayed for anion transport capacity by a novel procedure which is based on the fluorescent substrate N-(2-aminoethyisuifonate)7-nitrobenz-2-oxa-l,3-diazole (NBD-taurine) and on anti-NBD-antibodies as quenchers of extravesicular NBD-tanrine fluorescence. Efflux of NBD-tanrine from vesicles was monitored in a continuous mode as a decrease in intravesicular fluorescence. The band 3-mediated flux was approx. 50% inhibitable by externally added disulfonic stilbenes, indicating the random distribution of band 3 protein in reconstituted vesicles. Both the specific transfer rate (i.e., nmoi substrate/mg protein per min) of band 3 and its energy of activation (Ea) in the artificial lipid milieu were similar to those obtained with the native system. Glycophorin incorporation into this milieu had no significant effect on the associated anion transport properties.
Membrane transport proteins: implications of sequence comparisons
Current Opinion in Cell Biology, 1992
Analyses of the sequences and structures of many transport proteins that differ in substrate specificity, direction of transport and mechanism of transport suggest that they form a family of related proteins. Their sequence similarities imply a common mechanism of action. This hypothesis provides an objective basis for examining their mechanisms of action and relationships to other transporters.
The Journal of Physiology, 2000
System L is the major Na¤-independent amino acid transporter of mammalian cells (Christensen, 1990). It is constituted of the type II membrane protein 4F2hc (CD98) which is covalently linked to the polytopic membrane protein LAT1 via a disulfide bridge (Br oer et al. 1995, 1997a; Mastroberardino et al. 1998; Kanai et al. 1998). The transport pore of the transporter is most probably constituted by LAT1 (Pfeiffer et al. 1998), whereas 4F2hc is necessary for the translocation of the complex into the plasma membrane (Mastroberardino et al. 1998; Kanai et al. 1998; Nakamura et al. 1999). Western blotting and immunofluorescence studies show that 4F2hc is located in the plasma membrane in the absence of LAT1, whereas LAT1, when expressed alone, does not reach the surface of the oocyte (Mastroberardino et al. 1998; Kanai et al. 1998; Nakamura et al. 1999). The role of the 4F2hc protein in plasma membrane trafficking is further supported by its interaction with a family of amino acid transporters. In the absence of LAT1, expression in oocytes of 4F2hc alone results in the plasma membrane insertion of a number of endogenous amino acid transporters, e.g. the 2-amino-[2,2,1]heptane-2-carboxylic acid (BCH)-inhibitable system b 0,+ , system y¤L and some as yet unidentified transporters (Chillaron et al. 1996; Br oer et al. 1998a; Estevez et al. 1998). Subsequent to the cloning of the LAT1 cDNA, additional light chains have been identified which also interact with the 4F2hc protein to form other amino acid transporters with different substrate specificities, namely LAT2, y¤LAT1, y¤LAT2 and xCT (Torrents et al. 1998;
Nucleic Acids Research, 2006
The Transporter Classification Database (TCDB) is a web accessible, curated, relational database containing sequence, classification, structural, functional and evolutionary information about transport systems from a variety of living organisms. TCDB is a curated repository for factual information compiled from .10 000 references, encompassing 3000 representative transporters and putative transporters, classified into .400 families. The transporter classification (TC) system is an International Union of Biochemistry and Molecular Biology approved system of nomenclature for transport protein classification. TCDB is freely accessible at http://www.tcdb.org. The web interface provides several different methods for accessing the data, including step-by-step access to hierarchical classification, direct search by sequence or TC number and full-text searching. The functional ontology that underlies the database structure facilitates powerful query searches that yield valuable data in a quick and easy way. The TCDB website also offers several tools specifically designed for analyzing the unique characteristics of transport proteins. TCDB not only provides curated information and a tool for classifying newly identified membrane proteins, but also serves as a genome transporterannotation tool.