A SLC35C2 Transporter-Targeting Fluorescent Probe for the Selective Detection of B Lymphocytes Identified by SLC-CRISPRi and Unbiased Fluorescence Library Screening.

T and B lymphocytes are two major adaptive immune cells in the human defense system. To real-time monitor their diverse functions, a live-cell-selective probe for only one cell type is need to investigate the complex interaction of the immune cells. Herein, a small-molecule probe CDyB for live B cells is developed by an unbiased fluorescence library screening. The cell selectivity was confirmed by multiparametric single-cell analysis using CyTOF. Through a systematic SLC-CRISPRi library screening, the molecular target of CDyB was identified as SLC35C2 transporter based on a gating-oriented live-cell distinction (GOLD) mechanism. The gene expression analysis and knock-out experiments validated that the SLC35C2 transporter was the target for CDyB distinction. Interestingly, when CDyB was applied to study B cell development, the CDyB fluorescence and SLC35C2 expression were positively correlated with the B cell maturation process, and not involved in the T cell development.

The promiscuous binding pocket of SLC35A1 ensures redundant transport of CDP-ribitol to the Golgi.

The glycoprotein α-dystroglycan helps to link the intracellular cytoskeleton to the extracellular matrix. A unique glycan structure attached to this protein is required for its interaction with extracellular matrix proteins such as laminin. Up to now, this is the only mammalian glycan known to contain ribitol phosphate groups. Enzymes in the Golgi apparatus use CDP-ribitol to incorporate ribitol phosphate into the glycan chain of α-dystroglycan. Since CDP-ribitol is synthesized in the cytoplasm, we hypothesized that an unknown transporter must be required for its import into the Golgi apparatus. We discovered that CDP-ribitol transport relies on the CMP-sialic acid transporter SLC35A1 and the transporter SLC35A4 in a redundant manner. These two transporters are closely related, but bulky residues in the predicted binding pocket of SLC35A4 limit its size. We hypothesized that the large binding pocket SLC35A1 might accommodate the bulky CMP-sialic acid and the smaller CDP-ribitol, whereas SLC35A4 might only accept CDP-ribitol. To test this, we expressed SLC35A1 with mutations in its binding pocket in SLC35A1 KO cell lines. When we restricted the binding site of SLC35A1 by introducing the bulky residues present in SLC35A4, the mutant transporter was unable to support sialylation of proteins in cells but still supported ribitol phosphorylation. This demonstrates that the size of the binding pocket determines the substrate specificity of SLC35A1, allowing a variety of cytosine nucleotide conjugates to be transported. The redundancy with SLC35A4 also explains why patients with SLC35A1 mutations do not show symptoms of α-dystroglycan deficiency.

Structural basis for the delivery of activated sialic acid into Golgi for sialyation.

The decoration of secretory glycoproteins and glycolipids with sialic acid is critical to many physiological and pathological processes. Sialyation is dependent on a continuous supply of sialic acid into Golgi organelles in the form of CMP-sialic acid. Translocation of CMP-sialic acid into Golgi is carried out by the CMP-sialic acid transporter (CST). Mutations in human CST are linked to glycosylation disorders, and CST is important for glycopathway engineering, as it is critical for sialyation efficiency of therapeutic glycoproteins. The mechanism of how CMP-sialic acid is recognized and translocated across Golgi membranes in exchange for CMP is poorly understood. Here we have determined the crystal structure of a Zea mays CST in complex with CMP. We conclude that the specificity of CST for CMP-sialic acid is established by the recognition of the nucleotide CMP to such an extent that they are mechanistically capable of both passive and coupled antiporter activity.

Function of essential chloride and arginine residue in nucleotide binding to vesicular nucleotide transporter.

Vesicular nucleotide transporter (VNUT) plays a key role in purinergic signalling through its ability to transport nucleotides. VNUT belongs to the SLC17 family, which includes vesicular glutamate transporters (VGLUTs) and Type I Na+/phosphate cotransporters. All of these transporters exhibit membrane potential and Cl--dependent organic anion transport activity and have essential arginine in the transmembrane region. Previously, we reported that ketoacids inhibit these transporters through modulation of Cl- activation. Although this regulation is important to control signal transmission, the mechanisms underlying Cl--dependent regulation are unclear. Here, we examined the functional roles of Cl- and essential arginine residue on ATP binding to VNUT using the fluorescent ATP analogue trinitrophenyl-ATP (TNP-ATP). The fluorescence of TNP-ATP was enhanced by VNUT, whereas no enhancement was observed by VGLUT. Concentration-dependence curves showed that TNP-ATP was a high-affinity fluorescent probe for VNUT, with a Kd of 4.8 µM. TNP-ATP binding was competitive to ATP and showed similar specificity to transport activity. Addition of Cl- and ketoacids did not affect the apparent affinity for TNP-ATP. The Arg119 to Ala mutant retained TNP-ATP binding ability with slightly reduced affinity. Overall, these results indicated that Cl- and essential arginine were not important for ATP binding.

SLC35A5 Protein-A Golgi Complex Member with Putative Nucleotide Sugar Transport Activity.

Solute carrier family 35 member A5 (SLC35A5) is a member of the SLC35A protein subfamily comprising nucleotide sugar transporters. However, the function of SLC35A5 is yet to be experimentally determined. In this study, we inactivated the SLC35A5 gene in the HepG2 cell line to study a potential role of this protein in glycosylation. Introduced modification affected neither N- nor O-glycans. There was also no influence of the gene knock-out on glycolipid synthesis. However, inactivation of the SLC35A5 gene caused a slight increase in the level of chondroitin sulfate proteoglycans. Moreover, inactivation of the SLC35A5 gene resulted in the decrease of the uridine diphosphate (UDP)-glucuronic acid, UDP-N-acetylglucosamine, and UDP-N-acetylgalactosamine Golgi uptake, with no influence on the UDP-galactose transport activity. Further studies demonstrated that SLC35A5 localized exclusively to the Golgi apparatus. Careful insight into the protein sequence revealed that the C-terminus of this protein is extremely acidic and contains distinctive motifs, namely DXEE, DXD, and DXXD. Our studies show that the C-terminus is directed toward the cytosol. We also demonstrated that SLC35A5 formed homomers, as well as heteromers with other members of the SLC35A protein subfamily. In conclusion, the SLC35A5 protein might be a Golgi-resident multiprotein complex member engaged in nucleotide sugar transport.

Lysine at position 329 within a C-terminal dilysine motif is crucial for the ER localization of human SLC35B4.

SLC35B4 belongs to the solute carrier 35 (SLC35) family whose best-characterized members display a nucleotide sugar transporting activity. Using an experimental model of HepG2 cells and indirect immunofluorescent staining, we verified that SLC35B4 was localized to the endoplasmic reticulum (ER). We demonstrated that dilysine motif, especially lysine at position 329, is crucial for the ER localization of this protein in human cells and therefore one should use protein C-tagging with caution. To verify the importance of the protein in glycoconjugates synthesis, we generated SLC35B4-deficient HepG2 cell line using CRISPR-Cas9 approach. Our data showed that knock-out of the SLC35B4 gene does not affect major UDP-Xyl- and UDP-GlcNAc-dependent glycosylation pathways.

A novel family of mammalian transmembrane proteins involved in cholesterol transport.

Cholesterol is an essential compound in mammalian cells because it is involved in a wide range of functions, including as a key component of membranes, precursor of important molecules such as hormones, bile acids and vitamin D. The cholesterol transport across the circulatory system is a well-known process in contrast to the intracellular cholesterol transport, which is poorly understood. Recently in our laboratory, we identified a novel protein in C. elegans involved in dietary cholesterol uptake, which we have named ChUP-1. Insillicoanalysis identified two putative orthologue candidate proteins in mammals. The proteins SIDT1 and SIDT2 share identity and conserved cholesterol binding (CRAC) domains with C. elegans ChUP-1. Both mammalian proteins are annotated as RNA transporters in databases. In the present study, we show evidence indicating that SIDT1 and SIDT2 not only do not transport RNA, but they are involved in cholesterol transport. Furthermore, we show that single point mutations directed to disrupt the CRAC domains of both proteins prevent FRET between SIDT1 and SIDT2 and the cholesterol analogue dehydroergosterol (DHE) and alter cholesterol transport.

An insight into the orphan nucleotide sugar transporter SLC35A4.

SLC35A4 has been classified in the SLC35A subfamily based on amino acid sequence homology. Most of the proteins belonging to the SLC35 family act as transporters of nucleotide sugars. In this study, the subcellular localization of endogenous SLC35A4 was determined via immunofluorescence staining, and it was demonstrated that SLC35A4 localizes mainly to the Golgi apparatus. In silico topology prediction suggests that SLC35A4 has an uneven number of transmembrane domains and its N-terminus is directed towards the Golgi lumen. However, an experimental assay refuted this prediction: SLC35A4 has an even number of transmembrane regions with both termini facing the cytosol. In vivo interaction analysis using the FLIM-FRET approach revealed that SLC35A4 neither forms homomers nor associates with other members of the SLC35A subfamily except SLC35A5. Additional assays demonstrated that endogenous SLC35A4 is 10 to 40nm proximal to SLC35A2 and SLC35A3. To determine SLC35A4 function SLC35A4 knock-out cells were generated with the CRISPR-Cas9 approach. Although no significant changes in glycosylation were observed, the introduced mutation influenced the subcellular distribution of the SLC35A2/SLC35A3 complexes. Additional FLIM-FRET experiments revealed that overexpression of SLC35A4-BFP together with SLC35A3 and the SLC35A2-Golgi splice variant negatively affects the interaction between the two latter proteins. The results presented here strongly indicate a modulatory role for SLC35A4 in intracellular trafficking of SLC35A2/SLC35A3 complexes.

The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange.

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA.

The human SLC25A33 and SLC25A36 genes of solute carrier family 25 encode two mitochondrial pyrimidine nucleotide transporters.

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport inorganic anions, amino acids, carboxylates, nucleotides, and coenzymes across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. Here two members of this family, SLC25A33 and SLC25A36, have been thoroughly characterized biochemically. These proteins were overexpressed in bacteria and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that SLC25A33 transports uracil, thymine, and cytosine (deoxy)nucleoside di- and triphosphates by an antiport mechanism and SLC25A36 cytosine and uracil (deoxy)nucleoside mono-, di-, and triphosphates by uniport and antiport. Both carriers also transported guanine but not adenine (deoxy)nucleotides. Transport catalyzed by both carriers was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. In confirmation of their identity (i) SLC25A33 and SLC25A36 were found to be targeted to mitochondria and (ii) the phenotypes of Saccharomyces cerevisiae cells lacking RIM2, the gene encoding the well characterized yeast mitochondrial pyrimidine nucleotide carrier, were overcome by expressing SLC25A33 or SLC25A36 in these cells. The main physiological role of SLC25A33 and SLC25A36 is to import/export pyrimidine nucleotides into and from mitochondria, i.e. to accomplish transport steps essential for mitochondrial DNA and RNA synthesis and breakdown.

Incorporation of adenine nucleotide transporter, Ant1p, into proteoliposomes facilitates ATP translocation and activation of encapsulated luciferase.

We prepared functional luciferase and membrane-integrated form of adenine nucleotide transporter (Ant1p) with a wheat germ cell-free system. The reconstituted Ant1p showed transport activity of ATP/AMP exchange across the membrane. Here we demonstrate that activity of the luciferase entrapped in the Ant1p-proteoliposomes is controllable by the external supply of ATP.

Quality control and substrate-dependent downregulation of the nutrient transporter Fur4.

Upon exposure to stress conditions, unfolded cell-surface nutrient transporters are rapidly internalized and degraded via the multivesicular body (MVB) pathway. Similarly, high concentrations of nutrients result in the downregulation of the corresponding transporters. Our studies using the yeast transporter Fur4 revealed that substrate-induced downregulation and quality control utilize a common mechanism. This mechanism is based on a conformation-sensing domain, termed LID (loop interaction domain), that regulates site-specific ubiquitination (also known as degron). Conformational alterations in the transporter induced by unfolding or substrate binding are transmitted to the LID, rendering the degron accessible for ubiquitination by Rsp5. As a consequence, the transporter is rapidly degraded. We propose that the LID-degron system is a conserved, chaperone-independent mechanism responsible for conformation-induced downregulation of many cell-surface transporters under physiological and pathological conditions.

Amino acid residues important for CMP-sialic acid recognition by the CMP-sialic acid transporter: analysis of the substrate specificity of UDP-galactose/CMP-sialic acid transporter chimeras.

In our previous studies, we demonstrated that chimeric molecules of the CMP-sialic acid (CMP-Sia) transporter (CST) and the UDP-galactose (Gal) transporter (UGT) in which the seventh transmembrane helix-containing segment was derived from the CST could transport both CMP-Sia and UDP-Gal and that the CST-derived seventh transmembrane helix segment was sufficient for the chimera to recognize CMP-Sia in the otherwise UGT context. In this study, we continued to more precisely define the submolecular region that is necessary for CMP-Sia recognition, and we demonstrated that the N-terminal half of the seventh transmembrane helix of CST is essential for the CMP-Sia transport mediated by the chimeric transporters. We further showed that Tyr214Gly and Ser216Phe mutations of a chimeric transporter that was capable of transporting both CMP-Sia and UDP-Gal led to the selective loss of CMP-Sia transport activity without affecting UDP-Gal transport activity. Conversely, when a residue in a chimeric transporter that was active for UDP-Gal transport but not CMP-Sia transport was replaced by Tyr, so that Tyr occupied the same position as in the CMP-Sia transporter, the resulting mutant chimera acquired the ability to transport CMP-Sia. These results demonstrated that Tyr214 and Ser216, located in the seventh transmembrane helix of the human CST, are critically important for the recognition of CMP-Sia as a transport substrate. Identification of determinants critical for the discrimination between relevant and irrelevant substrates will advance our understanding of the mechanisms of substrate recognition by nucleotide sugar transporters.

Expression of a chloroplast ATP/ADP transporter in E. coli membranes: behind the Mistic strategy.

Eukaryotic membrane protein expression is still a major bottleneck for structural studies. Production in E. coli often leads to low expression level and/or aggregated proteins. In the last decade, strategies relying on new fusion protein expression revealed promising results. Fusion with the amphipatic Mistic protein has been described to favor expression in E. coli membranes. Although, this approach has already been reported for a few membrane proteins, little is known about the activity of the fused proteins. We used this strategy and obtained high expression levels of a chloroplast ATP/ADP transporter from A. thaliana (NTT1) and characterized its transport properties. NTT1 fused to Mistic has a very low transport activity which can be recovered after in vivo Mistic fusion cleavage. Moreover, detailed molecular characterization of purified NTT1 mature form, NTT1 fused to Mistic or NTT1 cleaved-off from this fusion highlights the correct fold of the latter one. Therefore, considering the higher quantity of purified NTT1 mature form obtained via the Mistic fusion approach, this is a valuable strategy for obtaining quantities of pure and active proteins that are adequate for structural studies.

Folding proteome of Yarrowia lipolytica targeting with uracil permease mutants.

The acquisition of the correct folding of membrane proteins is a crucial process that involves several steps from the recognition of nascent protein, its targeting to the endoplasmic reticulum membrane, its insertion, and its sorting to its final destination. Yarrowia lipolytica is a hemiascomycetous dimorphic yeast and an alternative eukaryotic yeast model with an efficient secretion pathway. To better understand the quality control of membrane proteins, we constructed a model system based on the uracil permease. Mutated forms of the permease were stabilized and retained in the cell and made the strains resistant to the 5-fluorouracil drug. To identify proteins involved in the quality control, we separated proteins extracted in nondenaturing conditions on blue native gels to keep proteins associated in complexes. Some gel fragments where the model protein was immunodetected were subjected to mass spectrometry analysis. The proteins identified gave a picture of the folding proteome, from the translocation across the endoplasmic reticulum membrane, the folding of the proteins, to the vesicle transport to Golgi or the degradation via the proteasome. For example, EMC complex, Gsf2p or Yet3p, chaperone membrane proteins of the endoplasmic reticulum were identified in the Y. lipolytica native proteome.

Involvement of SLC17A9-dependent vesicular exocytosis in the mechanism of ATP release during T cell activation.

Recent reports have shown that T cell receptor (TCR)-dependent ATP release from T cells is involved in production of interleukin-2 (IL-2) through activation of P2 receptors. Stimulation of TCR induces ATP release from T cells through gap junction hemichannels and maxianion channels, at least in part. However, the mechanisms of ATP release from activated T cells are not fully understood. Here, we studied the mechanisms of ATP release during TCR-dependent T cell activation by investigating the effects of various inhibitors on TCR-dependent ATP release from murine T cells. We found that not only anion channel and gap junction hemichannel inhibitors, but also exocytosis inhibitors suppressed the ATP release. These results suggest that ATP release from murine T cells is regulated by various mechanisms, including exocytosis. An inhibitor of exocytosis, bafilomycin A, significantly blocked TCR signaling, such as Ca(2+) elevation and IL-2 production. Furthermore, bafilomycin A, ectonucleotidase, and P2Y(6) receptor antagonist significantly inhibited production of pro-inflammatory cytokines from external antigen-restimulated splenocytes, indicating that vesicular exocytosis-mediated purinergic signaling has a significant role in TCR-dependent cytokine production. We also detected vesicular ATP in murine T cells and human T lymphoma Jurkat cells, both of which also expressed mRNA of SLC17A9, a vesicular nucleotide transporter. Knockdown of SLC17A9 in Jurkat cells markedly reduced ATP release and cytosolic Ca(2+) elevation after TCR stimulation, suggesting involvement of SLC17A9-dependent vesicular exocytosis in ATP release and T cell activation. In conclusion, vesicular exocytosis of ATP appears to play a role in T cell activation and immune responses.

Identification of essential amino acid residues in the hydrophilic loop regions of the CMP-sialic acid transporter and UDP-galactose transporter.

The Golgi CMP-sialic acid transporter (CST) is a type III transmembrane protein with 10 transmembrane domains that are linked by eight hydrophilic loops. To investigate the function of these hydrophilic loops, the green fluorescent protein (GFP) was inserted into each loop of the transporter. Expression and localization of the resulting CST-GFP fusion proteins were confirmed by analyzing the fluorescence of GFP. The transport activity of the CST-GFP proteins was analyzed by a previously described erythropoietin/isoelectric focusing assay in CST-deficient MAR-11 cells. Interruption of the second and fourth lumenal loops and the fourth cytosolic loop of CST with GFP resulted in complete or partial loss of transport activity. Regions in these loops that play crucial roles in CST's activity were identified by Gly substitutions. Single amino acid substitution experiments revealed that Lys(272) of the fourth loop on the cytosolic side of CST is essential for transport activity. Mutation of the conserved Lys residue (Lys(297)) in the UDP-galactose transporter (UGT) also resulted in a complete loss of its activity. Point mutations of highly conserved amino acid residues in the loop regions identified Leu(136) of CST as essential for its activity. However, mutation of the conserved Leu residue in UGT (Leu(160)) did not affect the transport activity of UGT. Finally, mutation of Leu(224) in UGT completely inactivated the activity of UGT, although mutation of its conserved counterpart in CST, Leu(199), did not have any effect on CST. This study provides a structure-function analysis of the loop regions in CST and UGT.

The nucleotide sugar transporters AtUTr1 and AtUTr3 are required for the incorporation of UDP-glucose into the endoplasmic reticulum, are essential for pollen development and are needed for embryo sac progress in Arabidopsis thaliana.

Uridine 5'-diphosphate (UDP)-glucose is transported into the lumen of the endoplasmic reticulum (ER), and the Arabidopsis nucleotide sugar transporter AtUTr1 has been proposed to play a role in this process; however, different lines of evidence suggest that another transporter(s) may also be involved. Here we show that AtUTr3 is involved in the transport of UDP-glucose and is located at the ER but also at the Golgi. Insertional mutants in AtUTr3 showed no obvious phenotype. Biochemical analysis in both AtUTr1 and AtUTr3 mutants indicates that uptake of UDP-glucose into the ER is mostly driven by these two transporters. Interestingly, the expression of AtUTr3 is induced by stimuli that trigger the unfolded protein response (UPR), a phenomenon also observed for AtUTr1, suggesting that both AtUTr1 and AtUTr3 are involved in supplying UDP-glucose into the ER lumen when misfolded proteins are accumulated. Disruption of both AtUTr1 and AtUTr3 causes lethality. Genetic analysis showed that the atutr1 atutr3 combination was not transmitted by pollen and was poorly transmitted by the ovules. Cell biology analysis indicates that knocking out both genes leads to abnormalities in both male and female germ line development. These results show that the nucleotide sugar transporters AtUTr1 and AtUTr3 are required for the incorporation of UDP-glucose into the ER, are essential for pollen development and are needed for embryo sac progress in Arabidopsis thaliana.

Tyrosine phosphorylation by Src within the cavity of the adenine nucleotide translocase 1 regulates ADP/ATP exchange in mitochondria.

Phosphorylation of adenine nucleotide translocator 1 (ANT1) at residue Y194, which is part of the aromatic ladder located within the lumen of the carrier, critically regulates mitochondrial metabolism. Recent data support the concept that members of the Src family of nonreceptor tyrosine kinases are constitutively present in mitochondria and key to regulation of mitochondrial function. Herein, we demonstrate that site mutations of ANT1 (Y190-->F190, Y194-->F194) mimicking dephosphorylation of the aromatic ladder resulted in loss of oxidative growth and ADP/ATP exchange activity in respiration-incompetent yeast expressing mutant chimeric yN-hANT1. ANT1 is phosphorylated at Y194 by the Src family kinase members Src and Lck, and increased phosphorylation is tightly linked to reduced cell injury in preconditioned protected vs. unprotected cardiac mitochondria. Molecular dynamics simulations find the overall structure of the phosphorylated ANT1 stable, but with an increased steric flexibility in the region of the aromatic ladder, matrix loop m2, and four helix-linking regions. Combined with an analysis of the putative cytosolic salt bridge network, we reason that the effect of phosphorylation on transport is likely due to an accelerated transition between the main two conformational states (c<-->m) of the carrier during the transport cycle. Since "aromatic signatures" are typical for other mitochondrial carrier proteins with important biological functions, our results may be more general and applicable to these carriers.