(65)Zn(2+) transport by isolated gill epithelial cells of the American lobster, Homarus americanus

Gills are the first site of impact by metal ions in contaminated waters. Work on whole gill cells and metal uptake has not been reported before in crustaceans. In this study, gill filaments of the American lobster, Homarus americanus, were dissociated in physiological saline and separated into several cell types on a 30, 40, 50, and 80% sucrose gradient. Cells from each sucrose solution were separately resuspended in physiological saline and incubated in (65)Zn(2+) in order to assess the nature of metal uptake by each cell type. Characteristics of zinc accumulation by each kind of cell were investigated in the presence and absence of 10 mM calcium, variable NaCl concentrations and pH values, and 100 mu M verapamil, nifedipine, and the calcium ionophore A23187. (65)Zn(2+) influxes were hyperbolic functions of zinc concentration (1-1,000 mu M) and followed Michaelis-Menten kinetics. Calcium reduced both apparent zinc binding affinity (K (m)) and maximal transport velocity (J (max)) for 30% sucrose cells, but doubled the apparent maximal transport velocity for 80% sucrose cells. Results suggest that calcium, sodium, and protons enter gill epithelial cells by an endogenous broad-specificity cation channel and trans-stimulate metal uptake by a plasma membrane carrier system. Differences in zinc transport observed between gill epithelial cell types appear related to apparent affinity differences of the transporters in each kind of cell. Low affinity cells from 30% sucrose were inhibited by calcium, while high affinity cells from 80% sucrose were stimulated. (65)Zn(2+) transport was also studied by isolated, intact, gill filament tips. These intact gill fragments generally displayed the same transport properties as did cells from 80% sucrose and provided support for metal uptake processes being an apical phenomenon. A working model for zinc transport by lobster gill cells is presented.

Acid-base transport by the renal distal nephron

The functional versatility of the distal nephron is mainly due to the large cytological heterogeneity of the segment. Part of Na(+) uptake by distal tubules is dependent on Na(+)/H(+). exchanger 2 (NHE2), implicating a role of distal convoluted cells also in acid-base homeostasis. In addition, intercalated (IC) cells expressed in distal convoluted tubules, connecting tubules and collecting ducts are involved in the final regulation of acid-base excretion. IC cells regulate acid-base handling by 2 main transport proteins, a V-type H(+)-ATPase and a Cl/HCO(3)(-) exchanger, localized at different membrane domains. Type A IC cells are characterized by a luminal H(+)-ATPase in series with a basolateral Cl/HCO(3)(-) exchanger, the anion exchanger AE1. Type B IC cells mediate HCO(3)(-) secretion through the apical Cl(-)/HCO(3)(-) exchanger pendrin in series with a H(+)-ATPase at the basolateral membrane. Alternatively, H(+)/K(+)-ATPases have also been found in several distal tubule cells, particularly in type A and B IC cells. All of these mechanisms are finely regulated, and mutations of 1 or more proteins ultimately lead to expressive disorders of acid-base balance.

Comparação de efeitos dos extratos de Hypericum perforatum (Hipérico) e de Mentha crispa (Hortelã) em diferentes modelos experiemtais

Several clinic evaluations have been possible with radiobiocomplexes labeled with technetium-99m (99mTc). Some natural and synthetic drugs are capable of to interfere on the labeling of blood constituents with 99mTc, as well as on the biodistribution of radiobiocomplexes. Authors have also reported about the toxicity of several natural products. The aim of this study was to compare the effects of the Mentha crispa (hortelã) and of the Hypericum perforatum (hipérico) in different experimental models. On the labeling of red blood cells (RBC) and plasma and cellular proteins with 99mTc, both extracts were capable of to decrease the radioactivity percentage on the cellular compartment and on the fixation on plasma and cellular proteins. On the morphometry of the RBC, only the hortelã was capable to alter the shape and the perimeter/area ratio of the RBC. On the biodistribution of the radiobiocomplex sodium pertechnetate (Na99mTcO4), the hortelã increased the Na99mTcO4 distribution in the kidney, spleen, liver and thyroid, meanwhile the hipérico decreased the Na99mTcO4 distribution in the bone, stomach, lungs and thyroid, and increased the Na99mTcO4 distribution in the pancreas. On the bacterial cultures survival, the hipérico was capable of to protect the bacteria against the stannous chloride (SnCl2) effect. The hipérico did not alter the topology of plasmidial DNA and did not protect the plasmidial DNA against the SnCl2 action. Probably, the effects presented by both extracts could be due to chemical compounds of the extracts that could alter the morphology of the RBC and the plasma membrane ions transport, and/or by phytocomplexes that could be formed with different effects dependent on the biological system considered

Interaction of human neutrophils with endothelial cells regulates the expression of endogenous proteins annexin 1, galectin-1 and galectin-3

Annexin 1 (ANXA1), galectin-1 (Gal-1) and galectin-3 (Gal-3) proteins have been identified as important mediators that promote or inhibit leukocyte migration. The expression of these proteins was studied in human neutrophils and endothelial cells (ECs) during a transmigration process induced by IL-8. Upon neutrophil adhesion to EC, a significant increase in the cleaved ANXA1 (LCS3, raised against all ANXA1 isoforms) expression was detected in the plasma membrane of adhered neutrophils and ECs compared to intact ANXA1 isoform (LCPS1, against N-terminus of protein). Adherent neutrophils had elevated Gal-3 levels in the nucleus and cytoplasm, and ECs in their plasma membranes. In contrast, a decrease in the total amounts of Gal-1 was detected in migrated compared to non-migrated neutrophils. Therefore, ANXA1 and Gal-3 changed in their content and localization when neutrophils adhere to endothelia, suggesting a process of sensitive-balance between two endogenous anti- and pro-inflammatory mediators. (c) 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Papel das Yops secretadas por Yersinia sobre a resposta imune do hospedeiro

The genus Yersinia contains three species pathogenic to humans: Y. pestis, Y. enterocolitica e Y. pseudotuberculosis. The pathogenicity of Yersinia is linked to the presence of a 70-kb virulence plasmid (pYV) that is common to the three species and codifies a type III secretion system and a set of virulence proteins, including those known as Yersinia outer proteins (Yops), that are exported by this system when the bacteria encounter host cells. Two Yops translocators (YopB and YopD) are inserted into the host plasma membrane and transport six effectors (YopO, YopH, YopM, YopJ and YopT) across the membrane into the cytosol of the host cell. The Yops effectors interfere with multiple signaling pathways of the infected cell, affecting both the innate and adaptive immune responses. This article focuses on the role of Yops in the modulation of the host immune response.

Aspectos estruturais da membrana eritrocitária

This article describes the structures and functions of the erythrocyte membrane and its importance in transfusional medicine. The erythrocyte membrane is one of the best known membranes in terms of structure, function and genetic disorders. As any other plasma membrane, it mediates transport functions. It also provides the erythrocytes with their resilience and deformability. According to the International Society of Blood Transfusion (ISBT), more than 500 antigens are expressed in the erythrocyte membrane, and around 270 are involved in transfusion reaction cases and hemolytic diseases of the fetus and newborn. In the ISBT classification, the high frequency series is represented by antigens in more than 99% of population (high prevalence antigen). In transfusion, the absence of these antigens determines severe problems as for example, one woman without the P antigen suffered 6 repetitive miscarriages due to placental insufficiency, which was caused by an antibody formed against the absent P antigen. Some important erythrocyte membrane proteins are described here including Band 3, Glycophorins and spectrin. The most abundant integral membrane protein is Band 3 and its main function is to mediate exchange of chloride and bicarbonate anions across the plasma membrane. The second most abundant integral membrane protein in the human erythrocyte is sialoglycoprotein glycophorin A (GPA). With its high sialic acid content, GPA is the main contributor to the net negative cell-surface charge and is thus critical for minimizing cell-cell interactions and preventing red cell aggregation. Glycophorin C (GPC) is the receptor for PfEBP-2 (baebl, EBA-140), the newly identified erythrocyte binding ligand of Plasmodium falciparum. The ternary complex of spectrin, actin and 4.1R defines the nodes of the erythrocyte membrane skeletal network, and is inseparable from membrane stability when under mechanical stress. This erythrocyte membrane review is important for a better understanding of transfusion reactions, where the antibody formation against high prevalence antigens makes compatible transfusions difficult. The study of antigen diversity and biochemical characterization of different proteins will contribute to healthcare, as well as diagnosis, development of technology such as monoclonal antibody production and the therapeutic conduct of many diseases.

C-terminal processing of the teneurin proteins: Independent actions of a teneurin C-terminal associated peptide in hippocampal cells

Many neuropsychiatric conditions have a common set of neurological substrates associated with the integration of sensorimotor processing. The teneurins are a recently described family of proteins that play a significant role in visual and auditory development. Encoded on the terminal exon of the teneurin genes is a family of bioactive peptides, termed teneurin C-terminal associated peptides (TCAP), which regulate mood-disorder associated behaviors. Thus, the teneurin-TCAP system could represent a novel neurological system underlying the origins of a number of complex neuropsychiatric conditions. However, it is not known if TCAP-1 exerts its effects as part of a direct teneurin function, whereby TCAP represents a functional region of the larger teneurin protein, or if it has an independent role, either as a splice variant or post-translational proteolytic cleavage product of teneurin. In this study, we show that TCAP-1 can be transcribed as a smaller mRNA transcript. After translation, further processing yields a smaller 15. kDa protein containing the TCAP-1 region. In the mouse hippocampus, immunoreactive (ir) TCAP-1 is exclusively localized to the pyramidal layers of the CA1, CA2 and CA3 regions. Although the localization of TCAP and teneurin in hippocampal regions is similar, they are distinct within the cell as most ir-teneurin is found at the plasma membrane, whereas ir-TCAP-1 is predominantly found in the cytosol. Moreover, in mouse embryonic hippocampal cell culture, FITC-labeled TCAP-1 binds to the plasma membrane and is taken up into the cytosol via dynamin-dependent caveolae-mediated endocytosis. Our data provides novel evidence that TCAP-1 is structurally and functionally distinct from the larger teneurins. © 2012.

Avaliação dos constituintes proteicos do plasma seminal e da membrana plasmática se espermatozóides e sua correlação com a fertilidade de garanhões

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Relative CO2/NH3 permeabilities of human RhAG, RhBG and RhCG

Mammalian glycosylated rhesus (Rh) proteins include the erythroid RhAG and the nonerythroid RhBG and RhCG. RhBG and RhCG are expressed in multiple tissues, including hepatocytes and the collecting duct (CD) of the kidney. Here, we expressed human RhAG, RhBG and RhCG in Xenopus oocytes (vs. H2O-injected control oocytes) and used microelectrodes to monitor the maximum transient change in surface pH (ΔpHS) caused by exposing the same oocyte to 5 % CO2/33 mM HCO3 − (an increase) or 0.5 mM NH3/NH4 + (a decrease). Subtracting the respective values for day-matched, H2O-injected control oocytes yielded channel-specific values (*). (ΔpH∗S)CO2 and (−ΔpH∗S)NH3 were each significantly >0 for all channels, indicating that RhBG and RhCG—like RhAG—can carry CO2 and NH3. We also investigated the role of a conserved aspartate residue, which was reported to inhibit NH3 transport. However, surface biotinylation experiments indicate the mutants RhBGD178N and RhCGD177N have at most a very low abundance in the oocyte plasma membrane. We demonstrate for the first time that RhBG and RhCG—like RhAG—have significant CO2 permeability, and we confirm that RhAG, RhBG and RhCG all have significant NH3 permeability. However, as evidenced by (ΔpH∗S)CO2/(−ΔpH∗S)NH3 values, we could not distinguish among the CO2/NH3 permeability ratios for RhAG, RhBG and RhCG. Finally, we propose a mechanism whereby RhBG and RhCG contribute to acid secretion in the CD by enhancing the transport of not only NH3 but also CO2 across the membranes of CD cells.

Identificazione delle Lamine di tipo A come nuovi substrati della protein chinasi Akt/PKB

Akt (also called PKB) is a 63 kDa serine/threonine kinase involved in promotion of cell survival, proliferation a nd metabolic responses downstream the phosphoinositide-3-kinase (PI 3-kinase) signaling pathway. In resting cells, Akt is a predominantly cytosolic enzyme; however generation of PI 3-kinase lipid products recruits Akt to the plasma membrane, resulting in a conformational change which confers full enzymatic activity through the phosphorylation of the membrane-bound protein at two residues, Thr308, and Ser473. Activated Akt redistributes to cytoplasm and nucleus, where phosphorylation of specific substrates occurs. Both the presence and the activity of Akt in the nucleus have been described. An interesting mechanism that mediates nuclear translocation of Akt has been described in human mature T-cell leukemia: the product of TCL1 gene, Tcl1, interacts with the PH domain of phosphorylated Akt, thus driving Akt to the nucleus. In this context, Tcl1 may act as a direct transporter of Akt or may contribute to the formation of a complex that promotes the transport of active Akt to the nucleus, where it can phosphorylate nuclear substrates. A well described nuclear substrate if Foxo. IGF-1 triggers phosphorylation of Foxo by Akt inside the nucleus, where phospho-Foxo associates to 14.3.3 proteins that, in turn, promote its export to the cytoplasm where it is sequestered. Remarkably, Foxo phosphorylation by Akt has been shown to be a crucial event in Akt-dependent myogenesis. However, most Akt nuclear substrates have so far remained elusive, as well as nuclear Akt functions. This lack of information prompted us to undertake a search of substrates of Akt in the nucleus, by the combined use of 2D-separation/mass spectrometry and anti-Akt-phosphosubstrate antibody. This study presents evidence of A-type lamins as novel nuclear substrates of Akt. Lamins are type V intermediate filaments proteins found in the nucleus of higher eukaryotes where, together with lamin-binding proteins, they form the lamina at the nuclear envelope, providing mechanical stability for the nuclear membrane. By coimmunoprecipitation, it is demonstrated here that endogenous lamin A and Akt interact, and that A-type lamins are phosphorylated by Akt both in vitro and in vivo. Moreover, by phosphoaminoacid analysis and mutagenesis, it is further demonstrated that Akt phosphorylates lamin A at Ser404, and, more importantly, that while lamin A/C phosphorylation is stable throughout the cell cycle, phosphorylation of the precursor prelamin A becomes detectable as cells enter the G2 phase, picking at G2/M. This study also shows that lamin phosphorylation by Akt creates a binding site for 14.3.3 adaptors which, in turn, promote prelamin A degradation. While this mechanism is in agreement with a general role of Akt in the regulation of a subset of its substrates, opposite to what has been described, degradation is not mediated through a ubiquitination and proteasomal mechanism but through a lysosomal pathway, as indicated by the reverting action of the lysosomal inhibitor cloroquine. Phosphorylation is a key event in the mitotic breakdown of the nuclear lamina. However, the kinases and the precise sites of phosphorylation are scarcely known. Therefore, these results represent an important breakthrough in this very significant but understudied area. The phosphorylation of the precursor protein prelamin A and its subsequent degradation at G2/M, when both the nuclear envelop and the nuclear lamina disassemble, can be view as part of a mechanism to dispose off the precursor that is not needed in this precise context. The recently reported finding that patients affected by Emery-Dreifuss muscular dystrophy carry a mutation at Arg 401, in the Akt phosphorylation motif, open new perspective that warrant further investigation in this very important field.

Ruolo delle specie reattive dell'ossigeno e di caveole/raft lipidici nella biosegnalazione in linee cellulari umane

Membrane lipid rafts are detergent-resistant microdomains containing glycosphingolipids, cholesterol and glycosylphosphatidylinositol-linked proteins; they seem to be actively involved in many cellular processes including signal transduction, apoptosis, cell adhesion and migration. Lipid rafts may represent important functional platforms where redox signals are produced and transmitted in response to various agonists or stimuli. In addition, a new concept is emerging that could be used to define the interactions or amplification of both redox signalling and lipid raft-associated signalling. This concept is characterized by redox-mediated feed forward amplification in lipid platforms. It is proposed that lipid rafts are formed in response to various stimuli; for instance, NAD(P)H oxidase (Nox) subunits are aggregated or recruited in these platforms, increasing Nox activity. Superoxide and hydrogen peroxide generation could induce various regulatory activities, such as the induction of glucose transport activity and proliferation in leukaemia cells. The aim of our study is to probe: i) the involvement of lipid rafts in the modulation of the glucose transporter Glut1 in human acute leukemia cells; ii) the involvement of plasma membrane caveolae/lipid rafts in VEGF-mediated redox signaling via Nox activation in human leukemic cells; iii) the role of p66shc, an adaptor protein, in VEGF signaling and ROS production in endothelial cells (ECs); iv) the role of Sindecan-2, a transmembrane heparan sulphate proteoglycan, in VEGF signaling and physiological response in ECs and v) the antioxidant and pro-apoptotic activities of simple dietary phenolic acids, i. e. caffeic, syringic and protocatechuic acids in leukemia cells, characterized by a very high ROS content. Our results suggest that the role played by NAD(P)H oxidase-derived ROS in the regulation of glucose uptake, proliferation and migration of leukaemia and endothelial cells could likely occur through the control of lipid raft-associated signalling.

Endocytic trafficking and oligodendroglial exosome secretion in axon-glia communication and myelination

Oligodendrocytes form specialized plasma membrane extensions which spirally enwrap axons, thereby building up the myelin sheath. During myelination, oligodendrocytes produce large amounts of membrane components. Oligodendrocytes can be seen as a complex polarized cell type with two distinct membrane domains, the plasma membrane surrounding the cell body and the myelin membrane. SNARE proteins mediate the fusion of vesicular cargoes with their target membrane. We propose a model in which the major myelin protein PLP is transported by two different pathways. VAMP3 mediates the non-polarized transport of newly synthesized PLP via recycling endosomes to the plasma membrane, while transport of PLP from late endosomes/lysosomes to myelin is controlled by VAMP7. In the second part of the thesis, the role of exosome secretion in glia to axon signaling was studied. Further studies are required to clarify whether VAMP7 also controls exosome secretion. The thesis further focused on putative metabolic effects in the target neurons. Oligodendroglial exosomes showed no obvious influences on neuronal metabolic activity. Analysis of the phosphorylation levels of the neurofilament heavy subunit revealed a decrease in presence of oligodendrocytes, indicating effects of oligodendroglial exosomes on the neuronal cytoskeleton. Finally, candidates for kinases which are possibly activated upon influence of oligodendroglial exosomes and could influence neuronal survival were identified.

Cellular localization and mechanism of amyloid precursor protein (APP) homodimer formation in an oxidizing environment

The amyloid precursor protein (APP) is a type I transmembrane glycoprotein, which resembles a cell surface receptor, comprising a large ectodomain, a single spanning transmembrane part and a short C-terminal, cytoplasmic domain. It belongs to a conserved gene family, with over 17 members, including also the two mammalian APP homologues proteins APLP1 and APLP2 („amyloid precursor like proteins“). APP is encoded by 19 exons, of which exons 7, 8, and 15 can be alternatively spliced to produce three major protein isoforms APP770, APP751 and APP695, reflecting the number of amino acids. The neuronal APP695 is the only isoform that lacks a Kunitz Protease Inhibitor (KPI) domain in its extracellular portion whereas the two larger, peripheral APP isoforms, contain the 57-amino-acid KPI insert. rnRecently, research effort has suggested that APP metabolism and function is thought to be influenced by homodimerization and that the oligomerization state of APP could also play a role in the pathology of Alzheimer's disease (AD), by regulating its processing and amyloid beta production. Several independent studies have shown that APP can form homodimers within the cell, driven by motifs present in the extracellular domain, as well as in the juxtamembrane (JM) and transmembrane (TM) regions of the molecule, whereby the exact molecular mechanism and the origin of dimer formation remains elusive. Therefore, we focused in our study on the actual subcellular origin of APP homodimerization within the cell, an underlying mechanism, and a possible impact on dimerization properties of its homologue APLP1. Furthermore, we analyzed homodimerization of various APP isoforms, in particular APP695, APP751 and APP770, which differ in the presence of a Kunitz-type protease inhibitor domain (KPI) in the extracellular region. In order to assess the cellular origin of dimerization under different cellular conditions, we established a mammalian cell culture model-system in CHO-K1 (chinese hamster ovary) cells, stably overexpressing human APP, harboring dilysine based organelle sorting motifs at the very C-terminus [KKAA-Endoplasmic Reticulum (ER); KKFF-Golgi]. In this study we show that APP exists as disulfide-bound, SDS-stable dimers, when it was retained in the ER, unlike when it progressed further to the cis-Golgi, due to the KKFF ER exit determinant. These stable APP complexes were isolated from cells, and analyzed by SDS–polyacrylamide gel electrophoresis under non-reducing conditions, whereas strong denaturing and reducing conditions completely converted those dimers to monomers. Our findings suggested that APP homodimer formation starts early in the secretory pathway and that the unique oxidizing environment of the ER likely promotes intermolecular disulfide bond formation between APP molecules. We particularly visualized APP dimerization employing a variety of biochemical experiments and investigated the origin of its generation by using a Bimolecular Fluorescence Complementation (BiFC) approach with split GFP-APP chimeras. Moreover, using N-terminal deletion constructs, we demonstrate that intermolecular disulfide linkage between cysteine residues, exclusively located in the extracellular E1 domain, represents another mechanism of how an APP sub-fraction can dimerize within the cell. Additionally, mutational studies revealed that cysteines at positions 98 and 105, embedded in the conserved loop region within the E1 domain, are critical for interchain disulfide bond formation. Using a pharmacological treatment approach, we show that once generated in the oxidative environment of the ER, APP dimers remain stably associated during transport, reaching the plasma membrane. In addition, we demonstrate that APP isoforms, encompassing the KPI domain, exhibit a strongly reduced ability to form cis-directed dimers in the ER, whereas trans-directed cell aggregation of Drosophila Schneider (S2)-cells was isoform independent, mediating cell-cell contacts. Thus, suggesting that steric properties of KPI-APP might be the cause for weaker cis-interaction in the ER, compared to APP695. Finally, we provide evidence that APP/APLP1 heterointeractions are likewise initiated in the ER, suggesting a similar mechanism for heterodimerization. Therefore, dynamic alterations of APP between monomeric, homodimeric, and possibly heterodimeric status could at least partially explain some of the variety in the physiological functions of APP.rn

Identification and characterisation of Stx5 a novel interactor of VLDL-R affecting its intracellular trafficking and processing

We identified syntaxin 5 (Stx5), a protein involved in intracellular vesicle trafficking, as a novel interaction partner of the very low density lipoprotein (VLDL)-receptor (VLDL-R), a member of the LDL-receptor family. In addition, we investigated the effect of Stx5 on VLDL-R maturation, trafficking and processing. Here, we demonstrated mutual association of both proteins using several in vitro approaches. Furthermore, we detected a special maturation phenotype of VLDL-R resulting from Stx5 overexpression. We found that Stx5 prevented Golgi-maturation of VLDL-R, but did not cause accumulation of the immature protein in ER to Golgi compartments, the main expression sites of Stx5. Rather more, abundantly present Stx5 was capable of translocating ER-/N-glycosylated VLDL-R to the plasma membrane, and thus was insensitive to BFA treatment and incubation at low temperature. Based on our findings, we postulate that Stx5 can directly bind to the C-terminal domain of VLDL-R, thereby influencing the receptor’s glycosylation, trafficking and processing characteristics. Resulting from that, we further suggest that Stx5, which is highly expressed in neurons along with VLDL-R, might play a role in modulating the receptor’s physiology by participating in a novel/undetermined alternative pathway bypassing the Golgi apparatus.

Na +-coupled betaine symporters involved in osmotic stress response in prokaryotes and eukaryotes

The betaine/GABA transporter BGT1 is one of the most important osmolyte transporters in the kidney. BGT1 is a member of the neurotransmitter sodium symporter (NSS) family, facilitates Na+/Cl--coupled betaine uptake to cope with hyperosmotic stress. Betaine transport in kidney cells is upregulated under hypertonic conditions by a yet unknown mechanism when increasing amounts of intracellular BGT1 are inserted into the plasma membrane. Re-establishing isotonicity results in ensuing depletion of BGT1 from the membrane. BGT1 phosphorylation on serines and threonines might be a regulation mechanism. In the present study, four potential PKC phosphorylation sites were mutated to alanines and the responses to PKC activators, phorbol 12-myristate acetate (PMA) and dioctanoyl-sn-glycerol (DOG) were determined. GABA-sensitive currents were diminished after 30 min preincubation with these PKC activators. Staurosporine blocked the response to DOG. Three mutants evoked normal GABA-sensitive currents but currents in oocytes expressing the mutant T40A were greatly diminished. [3H]GABA uptake was also determined in HEK-293 cells expressing EGFP-tagged BGT1 with the same mutations. Three mutants showed normal upregulation of GABA uptake after hypertonic stress, and downregulation by PMA was normal compared to EGFP-BGT1. In contrast, GABA uptake by the T40A mutant showed no response to hypertonicity or PMA. Confocal microscopy of the EGFP-BGT1 mutants expressed in MDCK cells, grown on glass or filters, revealed that T40A was present in the cytoplasm after 24 h hypertonic stress while the other mutants and EGFP-BGT1 were predominantely present in the plasma membrane. All four mutants co-migrated with EGFP-BGT1 on Western blots suggesting they are full-length proteins. In conclusion, T235, S428, and S564 are not involved in downregulation of BGT1 due to phosphorylation by PKC. However, T40 near the N-terminus may be part of a hot spot important for normal trafficking or insertion of BGT1 into the plasma membrane. Additionally, a link between substrate transport regulation, insertion of BGT1 into the plasma membrane and N-glycosylation in the extracellular loop 2 (EL2) could be revealed. The functional importance of two predicted N-glycosylation sites, which are conserved in EL2 within the NSS family were investigated for trafficking, transport and regulated plasma membrane insertion by immunogold-labelling, electron microscopy, mutagenesis, two-electrode voltage clamp measurements in Xenopus laevis oocytes and uptake of radioactive-labelled substrate into MDCK cells. Trafficking and plasma membrane insertion of BGT1 was clearly promoted by proper N-glycosylation in both, oocytes and MDCK cells. De-glycosylation with PNGase F or tunicamycin led to a decrease in substrate affinity and transport rate. Mutagenesis studies revealed that in BGT1 N183 is the major N-glycosylation site responsible for full protein activity. Replacement of N183 with aspartate resulted in a mutant, which was not able to bind N-glycans suggesting that N171 is a non-glycosylated site in BGT1. N183D exhibited close to WT transport properties in oocytes. Surprisingly, in MDCK cells plasma membrane insertion of the N183D mutant was no longer regulated by osmotic stress indicating unambiguously that association with N-glycans at this position is linked to osmotic stress-induced transport regulation in BGT1. The molecular transport mechanism of BGT1 remains largely unknown in the absence of a crystal structure. Therefore investigating the structure-function relationship of BGT1 by a combination of structural biology (2D and 3D crystallization) and membrane protein biochemistry (cell culture, substrate transport by radioactive labeled GABA uptake into cells and proteoliposomes) was the aim of this work. While the functional assays are well established, structure determination of eukaryotic membrane transporters is still a challenge. Therefore, a suitable heterologous expression system could be defined, starting with cloning and overexpression of an optimized gene. The achieved expression levels in P. pastoris were high enough to proceed with isolation of BGT1. Furthermore, purification protocols could be established and resulted in pure protein, which could even be reconstituted in an active form. The quality and homogeneity of the protein allowed already 2D and 3D crystallization, in which initial crystals could be obtained. Interestingly, the striking structural similarity of BGT1 to the bacterial betaine transporter BetP, which became a paradigm for osmoregulated betaine transport, provided information on substrate coordination in BGT1. The structure of a BetP mutant that showed activity for GABA was solved to 3.2Å in complex with GABA in an inward facing open state. This structure shed some light into the molecular transport mechanisms in BGT1 and might help in future to design conformationally locked BGT1 to enforce the on-going structure determination.