Efficient Mass Spectral Analysis of Active Transporters Overexpressed in <i>Escherichia coli</i>

Structural analysis of purified active membrane proteins can be performed by mass spectrometry (MS). However, no large-scale expression systems for active eukaryotic membrane proteins are available. Moreover, because membrane proteins cannot easily be digested by trypsin and ionized, they are difficult to analyze by MS. We developed a method for mass spectral analysis of eukaryotic membrane proteins combined with an overexpression system in <i>Escherichia coli</i>. Vesicular glutamate transporter 2 (VGLUT2/SLC17A6) with a soluble α-helical protein and histidine tag on the N- and C-terminus, respectively, was overexpressed in <i>E. coli</i>, solubilized with detergent, and purified by Ni-NTA affinity chromatography. Proteoliposomes containing VGLUT2 retained glutamate transport activity. For MS analysis, the detergent was removed from purified VGLUT2 by trichloroacetic acid precipitation, and VGLUT2 was then subjected to reductive alkylation and tryptic digestion. The resulting peptides were detected with 88% coverage by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS with or without liquid chromatography. Vesicular excitatory amino acid transporter and vesicular acetylcholine transporter were also detected with similar coverage by the same method. Thus this methodology could be used to analyze purified eukaryotic active transporters. Structural analysis with chemical modifiers by MS could have applications in functional binding analysis for drug discovery

Structures suggest a mechanism for energy coupling by a family of organic anion transporters.

Members of the solute carrier 17 (SLC17) family use divergent mechanisms to concentrate organic anions. Membrane potential drives uptake of the principal excitatory neurotransmitter glutamate into synaptic vesicles, whereas closely related proteins use proton cotransport to drive efflux from the lysosome. To delineate the divergent features of ionic coupling by the SLC17 family, we determined the structure of Escherichia coli D-galactonate/H+ symporter D-galactonate transporter (DgoT) in 2 states: one open to the cytoplasmic side and the other open to the periplasmic side with substrate bound. The structures suggest a mechanism that couples H+ flux to substrate recognition. A transition in the role of H+ from flux coupling to allostery may confer regulation by trafficking to and from the plasma membrane

Structures suggest a mechanism for energy coupling by a family of organic anion transporters.

Members of the solute carrier 17 (SLC17) family use divergent mechanisms to concentrate organic anions. Membrane potential drives uptake of the principal excitatory neurotransmitter glutamate into synaptic vesicles, whereas closely related proteins use proton cotransport to drive efflux from the lysosome. To delineate the divergent features of ionic coupling by the SLC17 family, we determined the structure of Escherichia coli D-galactonate/H+ symporter D-galactonate transporter (DgoT) in 2 states: one open to the cytoplasmic side and the other open to the periplasmic side with substrate bound. The structures suggest a mechanism that couples H+ flux to substrate recognition. A transition in the role of H+ from flux coupling to allostery may confer regulation by trafficking to and from the plasma membrane

Structures suggest a mechanism for energy coupling by a family of organic anion transporters.

Members of the solute carrier 17 (SLC17) family use divergent mechanisms to concentrate organic anions. Membrane potential drives uptake of the principal excitatory neurotransmitter glutamate into synaptic vesicles, whereas closely related proteins use proton cotransport to drive efflux from the lysosome. To delineate the divergent features of ionic coupling by the SLC17 family, we determined the structure of Escherichia coli D-galactonate/H+ symporter D-galactonate transporter (DgoT) in 2 states: one open to the cytoplasmic side and the other open to the periplasmic side with substrate bound. The structures suggest a mechanism that couples H+ flux to substrate recognition. A transition in the role of H+ from flux coupling to allostery may confer regulation by trafficking to and from the plasma membrane

Vesicular Inhibitory Amino Acid Transporter Is a Cl−/γ-Aminobutyrate Co-transporter*

The vesicular inhibitory amino acid transporter (VIAAT) is a synaptic vesicle protein responsible for the vesicular storage of γ-aminobutyrate (GABA) and glycine which plays an essential role in GABAergic and glycinergic neurotransmission. The transport mechanism of VIAAT remains largely unknown. Here, we show that proteoliposomes containing purified VIAAT actively took up GABA upon formation of membrane potential (Δψ) (positive inside) but not ΔpH. VIAAT-mediated GABA uptake had an absolute requirement for Cl− and actually accompanied Cl− movement. Kinetic analysis indicated that one GABA molecule and two Cl− equivalents were transported during one transport cycle. VIAAT in which Glu213 was specifically mutated to alanine completely lost the ability to take up both GABA and Cl−. Essentially the same results were obtained with glycine, another substrate of VIAAT. These results demonstrated that VIAAT is a vesicular Cl− transporter that co-transports Cl− with GABA or glycine in a Δψ dependent manner. It is concluded that Cl− plays an essential role in vesicular storage of GABA and glycine

Outward open conformation of a Major Facilitator Superfamily multidrug/H⁺ antiporter provides insights into switching mechanism

薬剤耐性の原因「薬剤汲み出しタンパク質」の排出メカニズムを解明 --多剤排出トランスポーターMdfAの分子機構-- 京都大学プレスリリース. 2018-10-03.Multidrug resistance (MDR) poses a major challenge to medicine. A principle cause of MDR is through active efflux by MDR transporters situated in the bacterial membrane. Here we present the crystal structure of the major facilitator superfamily (MFS) drug/H⁺ antiporter MdfA from Escherichia coli in an outward open conformation. Comparison with the inward facing (drug binding) state shows that, in addition to the expected change in relative orientations of the N- and C-terminal lobes of the antiporter, the conformation of TM5 is kinked and twisted. In vitro reconstitution experiments demonstrate the importance of selected residues for transport and molecular dynamics simulations are used to gain insights into antiporter switching. With the availability of structures of alternative conformational states, we anticipate that MdfA will serve as a model system for understanding drug efflux in MFS MDR antiporters