SummaryActive transport of substrates across cytoplasmic membranes is of great physiological, medical and pharmaceutical importance. The glycerol-3-phosphate (G3P) transporter (GlpT) of the E. coli inner membrane is a secondary active antiporter from the ubiquitous major facilitator superfamily that couples the import of G3P to the efflux of inorganic phosphate (P i ) down its concentration gradient. Integrating information from a novel combination of structural, molecular dynamics simulations and biochemical studies, we identify the residues involved directly in binding of substrate to the inwardfacing conformation of GlpT, thus defining the structural basis for the substrate-specificity of this transporter. The substrate binding mechanism involves protonation of a histidine residue at the binding site. Furthermore, our data suggest that the formation and breaking of inter-and intradomain salt bridges control the conformational change of the transporter that accompanies substrate translocation across the membrane. The mechanism we propose may be a paradigm for organophosphate/phosphate antiporters.

Secondary active transport of substrate across the cell membrane is crucial to many cellular and physiological processes. The crystal structure of one member of the secondary active transporter family, the sn-glycerol-3-phosphate (G3P) transporter (GlpT) of the inner membrane of Escherichia coli, suggests a mechanism for substrate translocation across the membrane that involves a rockerswitch-type movement of the protein. This rocker-switch mechanism makes two specific predictions with respect to kinetic behavior: the transport rate increases with the temperature, whereas the binding affinity of the transporter to a substrate is temperature-independent. In this work, we directly tested these two predictions by transport kinetics and substrate-binding experiments, integrating the data on this single system into a coherent set of observations. The transport kinetics of the physiologically relevant G3P-phosphate antiport reaction were characterized at different temperatures using both E. coli whole cells and GlpT reconstituted into proteoliposomes. Substrate-binding affinity of the transporter was measured using tryptophan fluorescence quenching in detergent solution. Indeed, the substrate transport velocity of GlpT increased dramatically with temperature. In contrast, neither the apparent Michaelis constant (K m ) nor the apparent substrate-binding dissociation constant (K d ) showed temperature dependence. Moreover, GlpT-catalyzed G3P translocation exhibited a completely linear Arrhenius function with an activation energy of 35.2 kJ mol −1 for the transporter reconstituted into proteoliposomes, suggesting that the substrate-loaded transporter is delicately poised between the inward-and outward-facing conformations. When these results are taken together, they are in agreement with a rocker-switch mechanism for GlpT.Secondary active membrane transporter proteins use an electrochemical gradient generated across the membrane by primary active transport as the driving force for substrate translocation (1,2). Over 100 families of secondary active membrane transporters have been identified to date, the largest of which is the major facilitator superfamily (MFS), 1 with over 5000 known members (3-5). The MFS, members of which are ubiquitous in all three kingdoms of life, includes many medically and pharmaceutically important transporters, such as the efflux pumps that confer resistance to antibiotics in bacteria and chemotherapeutic drugs in humans (6,7). † This work was supported in part by NIH Grant . The work of P.C.M. was supported by NIH Grant GM-24195. * To whom correspondence should be addressed. Telephone: (212) 263-8951. E-mail: wang@saturn.med.nyu.edu. ‡ New York University School of Medicine. § Johns Hopkins School of Medicine.1 Abbreviations: GlpT, glycerol-3-phosphate transporter; G3P, glycerol-3-phosphate; P i , inorganic phosphate; MFS, major facilitator superfamily; E a , activation energy; DDM, N-dodecyl-β-D-maltoside. Over the years, extensive efforts have been made to understand the molecular ...

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Background Perfusing fixatives through the cerebrovascular system is the gold standard approach in animals to prepare brain tissue for spatial biomolecular profiling, circuit tracing, and ultrastructural studies such as connectomics. Translating these discoveries to humans requires examination of postmortem autopsy brain tissue. Yet banked brain tissue is routinely prepared using immersion fixation, which is a significant barrier to optimal preservation of tissue architecture. The challenges involved in adopting perfusion fixation in brain banks and the extent to which it improves histology quality are not well defined. Methodology We searched four databases to identify studies that have performed perfusion fixation in human brain tissue and screened the references of the eligible studies to identify further studies. From the included studies, we extracted data about the methods that they used, as well as any data comparing perfusion fixation to immersion fixation. The protocol was preregistered at the Open Science Framework: https://osf.io/cv3ys/ . Results We screened 4489 abstracts, 214 full-text publications, and identified 35 studies that met our inclusion criteria, which collectively reported on the perfusion fixation of 558 human brains. We identified a wide variety of approaches to perfusion fixation, including perfusion fixation of the brain in situ and ex situ, perfusion fixation through different sets of blood vessels, and perfusion fixation with different washout solutions, fixatives, perfusion pressures, and postfixation tissue processing methods. Through a qualitative synthesis of data comparing the outcomes of perfusion and immersion fixation, we found moderate confidence evidence showing that perfusion fixation results in equal or greater subjective histology quality compared to immersion fixation of relatively large volumes of brain tissue, in an equal or shorter amount of time. Conclusions This manuscript serves as a resource for investigators interested in building upon the methods and results of previous research in designing their own perfusion fixation studies in human brains or other large animal brains. We also suggest several future research directions, such as comparing the in situ and ex situ approaches to perfusion fixation, studying the efficacy of different washout solutions, and elucidating the types of brain donors in which perfusion fixation is likely to result in higher fixation quality than immersion fixation. Electronic supplementary material The online version of this article (10.1186/s40478-019-0799-y) contains supplementary material, which is available to authorized users.

Postprandial triglyceride-rich lipoproteins (TRL)exert proatherogenic effects at the arterial wall, including lipid deposition. Following consumption of a mixed meal (1,200 kcal), plasma-mediated cellular free cholesterol (FC) efflux, lecithin:cholesterol acyltransferase (LCAT), and cholesteryl ester transfer protein (CETP) activities were determined in subjects (n ‫؍‬ 12) displaying type IIB hyperlipidemia and compared with those in a normolipidemic control group (n ‫؍‬ 14). The relative capacity of plasma to induce FC efflux from Fu5AH cells via the SR-BI receptor was significantly increased 4 h postprandially ( ؉ 23%; P Ͻ 0.005) in the type IIB group, whereas it remained unchanged for postprandial plasma from normolipidemic subjects. LCAT activity was significantly elevated 2 h postprandially in both the IIB and control groups, ( ؉ 46% and ؉ 36%, respectively; P Ͻ 0.005 vs. respective baseline value). In type IIB subjects, total cholesteryl ester (CE) mass transfer from HDL to total TRL [chylomicrons (CMs) ؉ VLDL-1 ؉ VLDL-2 ؉ IDL] increased progressively from 15 ؎ 2 g CE/h/ml at baseline to 28 ؎ 2 g CE transferred/h/ml ( ؉ 87%; P ‫؍‬ 0.0004) at 4 h postprandially. CE transfer to CMs and VLDL-1 was preferentially stimulated (2.6-fold and 2.3-fold respectively) at 4 h in IIB subjects and occurred concomitantly with elevation in mass and particle number of both CMs (2.3-fold) and VLDL-1 (1.3-fold). Furthermore, in type IIB subjects, CETP-mediated total CE flux over the 8 h postprandial period from HDL to potentially atherogenic TRL was significantly enhanced, and notably to VLDL-1 (32-fold elevation; P Ͻ 0.005), relative to control subjects. Such CE transfer flux was reflected in a significant postprandial increase in CE-TG ratio in both CMs and VLDL-1 in type IIB plasmas. In conclusion, HDL-CE is preferentially targeted to VLDL-1 via the action of CETP during alimentary lipemia, thereby favoring formation and accumulation of atherogenic CE-rich remnant particles. In order to maintain cholesterol homeostasis in peripheral tissues, excess cellular cholesterol is returned to the liver for excretion via a multistep process termed "reverse cholesterol transport" (RCT) (1). A key component of this process involves the transfer of a significant portion of the cholesteryl ester (CE) pool in HDL to apoB-containing lipoproteins (VLDL, IDL, and LDL) via the action of the cholesteryl ester transfer protein (CETP) (2).Hyperlipidemia of phenotype IIB is associated with an increased risk of premature coronary artery disease and is characterized by concomitant elevation of circulating levels of atherogenic apoB-containing, triglyceride-rich (VLDL) and cholesterol-rich lipoproteins (VLDL remnants, IDL, and LDL including small, dense LDL) (3). In type IIB hyperlipidemia during the fasting state, CETP is implicated in the intravascular formation of atherogenic small, dense LDL through an indirect mechanism involving an elevated rate of CE transfer from HDL to VLDL, and more specifically, to large VLDL-1 particles (4, 5)....