Expression and lysosomal targeting of CLN7, a major facilitator superfamily transporter associated with variant late-infantile neuronal ceroid lipofuscinosis.

Neuronal ceroid lipofuscinoses (NCLs) constitute a group of progressive neurodegenerative disorders resulting from mutations in at least eight different genes. Mutations in the most recently identified NCL gene, MFSD8/CLN7, underlie a variant of late-infantile NCL (vLINCL). The MFSD8/CLN7 gene encodes a polytopic protein with unknown function, which shares homology with ion-coupled membrane transporters. In this study, we confirmed the lysosomal localization of the native CLN7 protein. This localization of CLN7 is not impaired by the presence of pathogenic missense mutations or after genetic ablation of the N-glycans. Expression of chimeric and full-length constructs showed that lysosomal targeting of CLN7 is mainly determined by an N-terminal dileucine motif, which specifically binds to the heterotetrameric adaptor AP-1 in vitro. We also show that CLN7 mRNA is more abundant in neurons than astrocytes and microglia, and that it is expressed throughout rat brain, with increased levels in the granular layer of cerebellum and hippocampal pyramidal cells. Interestingly, this cellular and regional distribution is in good agreement with the autofluorescent lysosomal storage and cell loss patterns found in brains from CLN7-defective patients. Overall, these data highlight lysosomes as the primary site of action for CLN7, and suggest that the pathophysiology underpinning CLN7-associated vLINCL is a cell-autonomous process.

Molecular physiology and pathophysiology of lysosomal membrane transporters.

In contrast to lysosomal hydrolytic enzymes, the lysosomal membrane remains poorly characterized. In particular, although the genetic study of cystinosis and sialic acid storage disorders led to the identification of two lysosomal transporters for cystine and sialic acids, respectively, ten years ago, most transporters responsible for exporting lysosomal hydrolysis products to the cytosol are still unknown at the molecular level. However, two lines of investigation recently started to fill this gap in the knowledge of lysosomal biology. First, novel proteomic approaches are now able to provide a reliable inventory of lysosomal membrane proteins. On the other hand, a novel functional approach based on intracellular trafficking mechanisms allows direct transport measurement in whole cells by redirecting recombinant lysosomal transporters to the cell surface. After surveying the current state of knowledge in this field, the review focuses on the sialic acid transporter sialin and shows how recent functional data using the above whole-cell approach shed new light on the pathogenesis of sialic acid storage disorders by revealing the existence of a residual transport activity associated with Salla disease.

The existence of a second vesicular glutamate transporter specifies subpopulations of glutamatergic neurons.

Before their exocytotic release during stimulation of nerve terminals, nonpeptide neurotransmitters are loaded into synaptic vesicles by specific transporters. Recently, a protein initially identified as brain-specific Na(+)-dependent inorganic phosphate transporter I (BNPI) has been shown to represent a vesicular glutamate transporter (VGLUT1). In this study, we investigated whether a highly homologous "differentiation-associated Na(+)-dependent inorganic phosphate transporter" (DNPI) is involved in glutamatergic transmission. Vesicles isolated from BON cells expressing recombinant DNPI accumulated l-glutamate with bioenergetical and pharmacological characteristics identical to those displayed by VGLUT1 and by brain synaptic vesicles. Moreover, DNPI localized to synaptic vesicles, at synapses exhibiting classical excitatory features. DNPI thus represents a novel vesicular glutamate transporter (VGLUT2). The distributions of each VGLUT transcript in brain were highly complementary, with only a partial regional and cellular overlap. At the protein level, we could only detect either VGLUT1- or VGLUT2-expressing presynaptic boutons. The existence of two VGLUTs thus defines distinct subsets of glutamatergic neurons.

Cystinosin, the protein defective in cystinosis, is a H(+)-driven lysosomal cystine transporter.

Cystinosis is an inherited lysosomal storage disease characterized by defective transport of cystine out of lysosomes. However, the causative gene, CTNS, encodes a seven transmembrane domain lysosomal protein, cystinosin, unrelated to known transporters. To investigate the molecular function of cystinosin, the protein was redirected from lysosomes to the plasma membrane by deletion of its C-terminal GYDQL sorting motif (cystinosin-DeltaGYDQL), thereby exposing the intralysosomal side of cystinosin to the extracellular medium. COS cells expressing cystinosin-DeltaGYDQL selectively take up L-cystine from the extracellular medium at acidic pH. Disruption of the transmembrane pH gradient or incubation of the cells at neutral pH strongly inhibits the uptake. Cystinosin-DeltaGYDQL is directly involved in the observed cystine transport, since this activity is highly reduced when the GYDQL motif is restored and is abolished upon introduction of a point mutation inducing early-onset cystinosis. We conclude that cystinosin represents a novel H(+)-driven transporter that is responsible for cystine export from lysosomes, and propose that cystinosin homologues, such as mammalian SL15/Lec35 and Saccharomyces cerevisiae ERS1, may perform similar transport processes at other cellular membranes.

Identification and characterization of a lysosomal transporter for small neutral amino acids.

In eukaryotic cells, lysosomes represent a major site for macromolecule degradation. Hydrolysis products are eventually exported from this acidic organelle into the cytosol through specific transporters. Impairment of this process at either the hydrolysis or the efflux step is responsible of several lysosomal storage diseases. However, most lysosomal transporters, although biochemically characterized, remain unknown at the molecular level. In this study, we report the molecular and functional characterization of a lysosomal amino acid transporter (LYAAT-1), remotely related to a family of H+-coupled plasma membrane and synaptic vesicle amino acid transporters. LYAAT-1 is expressed in most rat tissues, with highest levels in the brain where it is present in neurons. Upon overexpression in COS-7 cells, the recombinant protein mediates the accumulation of neutral amino acids, such as gamma-aminobutyric acid, l-alanine, and l-proline, through an H+/amino acid symport. Confocal microscopy on brain sections revealed that this transporter colocalizes with cathepsin D, an established lysosomal marker. LYAAT-1 thus appears as a lysosomal transporter that actively exports neutral amino acids from lysosomes by chemiosmotic coupling to the H+-ATPase of these organelles. Homology searching in eukaryotic genomes suggests that LYAAT-1 defines a subgroup of lysosomal transporters in the amino acid/auxin permease family.

Gephyrin-independent clustering of postsynaptic GABA(A) receptor subtypes.

Gephyrin has been shown to be essential for the synaptic localization of the inhibitory glycine receptor and major GABA(A) receptor (GABA(A)R) subtypes. However, in retina certain GABA(A)R subunits are found at synaptic sites in the absence of gephyrin. Here, we quantitatively analyzed GABA(A)R alpha1, alpha2, alpha3, alpha5, beta2/3, and gamma2 subunit immunoreactivities in spinal cord sections derived from wild-type and gephyrin-deficient (geph -/-) mice. The punctate staining of GABA(A)R alpha1 and alpha5 subunits was unaltered in geph -/- mice, whereas the numbers of alpha2-, alpha3-, beta2/3-, and gamma2-subunit-immunoreactive synaptic sites were significantly or even strikingly reduced in the mutant animals. Immunostaining with an antibody specific for the vesicular inhibitory amino acid transporter revealed that the number of inhibitory presynaptic terminals is unaltered upon gephyrin deficiency. These data show that in addition to gephyrin other clustering proteins must exist that mediate the synaptic localization of selected GABA(A)R subtypes.

Formation of mixed glycine and GABAergic synapses in cultured spinal cord neurons.

In the spinal cord, GABA and glycine mediate inhibition at separate or mixed synapses containing glycine and/or GABA(A) receptors (GlyR and GABA(A)R, respectively). We have analysed here the sequence of events leading to inhibitory synapse formation during synaptogenesis of embryonic spinal cord neurons between 1 and 11 days in vitro (DIV). We used immunocytochemical methods to detect simultaneously an antigen specific to inhibitory terminals, the vesicular inhibitory amino acid transporter (VIAAT), and one of the following postsynaptic elements: GlyR, GABA(A)R or gephyrin, the anchoring protein of GlyR, which is also associated with GABA(A)R. Quantitative analysis revealed that until 5 DIV most gephyrin clusters were not adjacent to VIAAT-positive profiles, but became associated with them at later stages. In contrast, GlyR and GABAAR clustered predominantly in front of VIAAT-containing terminals at all stages. However, about 10% of receptor aggregates were detected at nonsynaptic loci. The two receptors colocalized in 66.2+/-2.5% of the inhibitory postsynaptic domains after 11 DIV, while 30.3+/-2.6% and 3.4+/-0.8% of them contained only GlyR and GABA(A)R, respectively. Interestingly, at 3 DIV GABA(A)R clustered at a postsynaptic location prior to gephyrin and GlyR; GABA(A)R could thus be the initiating element in the construction of mixed glycine and GABAergic synapses. The late colocalization of gephyrin with GABA(A)R, and the demonstration by other groups that, in the absence of gephyrin, postsynaptic GABA(A)R is not detected, suggest that gephyrin is involved in the stabilization of GABA(A)R rather than in its initial accumulation at synaptic sites.

Constitutive phosphorylation of the vesicular inhibitory amino acid transporter in rat central nervous system.

gamma-Aminobutyric acid (GABA) and glycine are stored into synaptic vesicles by a recently identified vesicular inhibitory amino acid transporter [VIAAT, also called vesicular GABA transporter (VGAT)]. Immunoblotting analysis revealed that rat brain VIAAT migrated as a doublet during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a predominant slower band in all areas examined except olfactory bulb and retina. The slower band corresponded to a phosphorylated form of VIAAT as it was converted to the faster one by treating brain homogenates with alkaline phosphatase or with an endogenous phosphatase identified as type 2A protein-serine/threonine phosphatase using okadaic acid. In contrast, the recombinant protein expressed in COS-7 or PC12 cells co-migrated with the faster band of the brain doublet and was insensitive to alkaline phosphatase. To investigate the influence of VIAAT phosphorylation on vesicular neurotransmitter loading, purified synaptic vesicles were treated with alkaline phosphatase and assayed for amino acid uptake. However, neither GABA nor glycine uptake was affected by VIAAT phosphorylation. These results indicate that VIAAT is constitutively phosphorylated on cytosolic serine or threonine residues in most, but not all, regions of the rat brain. This phosphorylation does not regulate the vesicular loading of GABA or glycine, suggesting that it is involved at other stages of the synaptic vesicle life cycle.

The loading of neurotransmitters into synaptic vesicles.

Classical (non-peptide) transmitters are stored into secretory vesicles by a secondary active transporter driven by a V-type H(+)-ATPase. Five vesicular neurotransmitter uptake activities have been characterized in vitro and, for three of them, the transporters involved have been identified at the molecular level using cDNA cloning and/or Caenorhabditis elegans genetics. These transporters belong to two protein families, which are both unrelated to the Na(+)-coupled neurotransmitter transporters operating at the plasma membrane. The two isoforms of the mammalian vesicular monoamine transporter, VMAT1 and VMAT2, are related to the vesicular acetylcholine transporter (VACHT), while a novel, unrelated vesicular inhibitory amino acid transporter (VIAAT), also designated vesicular GABA transporter (VGAT), is responsible for the storage of GABA, glycine or, at some synapses, both amino acids into synaptic vesicles. The observed effects of experimentally altered levels of VACHT or VMAT2 on synaptic transmission and behavior, as well as the recent awareness that GABAergic or glutamatergic receptors are not always saturated at central synapses, suggest a potential role of vesicular loading in synaptic plasticity.

Molecular dissection of regulated secretory pathways in human gastric enterochromaffin-like cells: an immunohistochemical analysis.

Enterochromaffin-like (ECL) cells regulate gastric acid secretion through vesicular release of histamine. Until now, the molecular machinery of human ECL cells involved in the formation and release of vesicles is largely unknown. We analyzed tissue samples obtained from normal human gastric mucosa (n=4) and ECLomas (n=5) immunohistochemically using the APAAP method or double immunofluorescence confocal laser microscopy. Human pheochromocytomas (n=5) were investigated in parallel and compared to ECL cells. Secretory pathways were characterized using antibodies specific for marker proteins of large dense-core vesicles (LDCVs; islet cell antigen 512, chromogranin A, pancreastatin, and vesicular monoamine transporter 2) and small synaptic vesicle (SSV) analogues (synaptophysin). Tissues were also analyzed for expression of the peptide hormone processing enzymes, carboxypeptidase E and prohormone convertase 1, as well as the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, 25-kDa synaptosome-associated protein (SNAP25), syntaxin, and synaptobrevin. Immunoreactivity for markers of LDCVs and SSV analogues were detected in normal ECL cells and ECLomas. Both tissues also showed expression of carboxypeptidase E and prohormone convertase 1. Analysis of vesicular SNARE (v-SNARE) and target membrane SNARE (t-SNARE) proteins revealed the presence of SNAP25, syntaxin, and synaptobrevin in normal and neoplastic ECL cells. Our data suggest that ECL cells possess the two vesicle types of regulated neuroendocrine secretory pathways, LDCVs and SSV analogues. Since ECL cells also contain typical SNARE proteins, the molecular machinery underlying secretory processes in this cell type appears to be identical to the secretory apparatus of neuroendocrine cells and neurons. In addition, our findings suggest that the secretory apparatus of ECL cells is maintained during neoplastic transformation.

Presence of the vesicular inhibitory amino acid transporter in GABAergic and glycinergic synaptic terminal boutons.

The characterization of the Caenorhabditis elegans unc-47 gene recently allowed the identification of a mammalian (gamma)-amino butyric acid (GABA) transporter, presumed to be located in the synaptic vesicle membrane. In situ hybridization data in rat brain suggested that it might also take up glycine and thus represent a general Vesicular Inhibitory Amino Acid Transporter (VIAAT). In the present study, we have investigated the localization of VIAAT in neurons by using a polyclonal antibody raised against the hydrophilic N-terminal domain of the protein. Light microscopy and immunocytochemistry in primary cultures or tissue sections of the rat spinal cord revealed that VIAAT was localized in a subset (63-65%) of synaptophysin-immunoreactive terminal boutons; among the VIAAT-positive terminals around motoneuronal somata, 32.9% of them were also immunoreactive for GAD65, a marker of GABAergic presynaptic endings. Labelling was also found apposed to clusters positive for the glycine receptor or for its associated protein gephyrin. At the ultrastructural level, VIAAT immunoreactivity was restricted to presynaptic boutons exhibiting classical inhibitory features and, within the boutons, concentrated over synaptic vesicle clusters. Pre-embedding detection of VIAAT followed by post-embedding detection of GABA or glycine on serial sections of the spinal cord or cerebellar cortex indicated that VIAAT was present in glycine-, GABA- or GABA- and glycine-containing boutons. Taken together, these data further support the view of a common vesicular transporter for these two inhibitory transmitters, which would be responsible for their costorage in the same synaptic vesicle and subsequent corelease at mixed GABA-and-glycine synapses.

The vesicular monoamine transporter: from chromaffin granule to brain.

All characterized monoaminergic cells utilize the same transport system for the vesicular accumulation of monoamines prior to their release. This system operates in neuronal (catecholaminergic, serotoninergic or histaminergic) as well as in endocrine or neuroendocrine cells. For several decades, chromaffin granules from bovine adrenal medulla have been used as a model system, allowing progress in the understanding of the biophysics, the biochemistry and the pharmacology of the monoamine vesicular transporter. The transporters from rat, bovine and man have been cloned. Surprisingly, two genes encode different isoforms of the protein which are differentially expressed in monoaminergic systems. The conjunction of recombinant DNA techniques and expression in secretory or non-secretory cells with the large body of data obtained on the chromaffin granule transporter has allowed rapid progress in the study of the protein. But interestingly enough, this progress has open new possibilities in the study of biological problems, especially in the brain. The transporter is useful for the determination of the relationship between small and large dense core vesicles, for the understanding of the mechanism of the drugs such as 1-methyl-4-phenylpyridinium (MPP+), tetrabenazine or amphetamines, and as a marker in brain development. The possibility of regulations at the vesicular transporter level and of their effect on the quantum size has to be investigated. The vesicular monoamine transporter is also an important target for brain imaging.

Cloning of a functional vesicular GABA and glycine transporter by screening of genome databases.

The unc-47 locus of Caenorhabditis elegans has been suggested to encode a synaptic vesicle GABA transporter. Here we used hydropathy plot analysis to identify a candidate vesicular GABA transporter in genomic sequences derived from a region of the physical map comprising unc-47. A mouse homologue was identified and cloned from EST database information. In situ hybridization in rat brain revealed codistribution with both GABAergic and glycinergic neuronal markers. Moreover, expression in COS-7 and PC12 cells induced an intracellular, glycine-sensitive GABA uptake activity. These observations, consistent with previous data on GABA and glycine uptake by synaptic vesicles, demonstrate that the mouse clone encodes a vesicular inhibitory amino acid transporter.

The photoactivatable inhibitor 7-azido-8-iodoketanserin labels the N terminus of the vesicular monoamine transporter from bovine chromaffin granules.

In monoaminergic cells, the hormone or neurotransmitter is concentrated into secretory vesicles by a tetrabenazine- and reserpine-sensitive vesicular monoamine transporter (VMAT), catalyzing a H+/monoamine antiport. Ketanserin is another powerful inhibitor of VMAT that binds to the tetrabenazine binding site. A photoactivatable derivative, 7-azido-8-iodoketanserin (AZIK), labels covalently the transporter from bovine chromaffin granules, VMAT-2. Digestion with endoproteinases V8 or Lys-C, which cleave peptide bonds at acidic or lysine residues, respectively, revealed that the AZIK label is located in a 7 kDa segment of the VMAT-2 polypeptide. The photolabeled chromaffin granule transporter was purified by DEAE and WGA chromatography followed by selective aggregation and size-exclusion HPLC. After treatment by V8 or Lys-C, digestion products were separated by electrophoresis in SDS and sequenced. For both enzymes, the material comigrating with the labeled peptide produced a sequence matching the N terminus of VMAT-2. A K55E mutant of the bovine VMAT-2 cDNA was constructed and expressed in COS-7 cells. The mutant protein exhibited a full VMAT activity and could be labeled by AZIK. However, the formation of the 7 kDa labeled peptide upon Lys-C proteolysis was prevented in the mutant, with a redistribution of the label in higher-molecular mass digestion products. The localization of the label upstream of lysine 55 was confirmed by an immunological and enzymatic analysis. We conclude that the segment 2-55 of bovine VMAT-2, which encompasses the cytosolic N terminus and the first transmembrane segment in the current topological model of the transporter, contains residues involved in the binding of ketanserin and, possibly, tetrabenazine.

SDS-resistant aggregation of membrane proteins: application to the purification of the vesicular monoamine transporter.

The vesicular monoamine transporter, which catalyses a H+/ monoamine antiport in monoaminergic vesicle membrane, is a very hydrophobic intrinsic membrane protein. After solubilization, this protein was found to have a high tendency to aggregate, as shown by SDS/PAGE, especially when samples were boiled in the classical Laemmli buffer before electrophoresis. This behavior was analysed in some detail. The aggregation was promoted by high temperatures, organic solvents and acidic pH, suggesting that it resulted from the unfolding of structure remaining in SDS. The aggregates were very stable and could be dissociated only by suspension in anhydrous trifluoroacetic acid. This SDS-resistant aggregation behaviour was shared by very few intrinsic proteins of the chromaffin granule membrane. Consequently, a purification procedure was based on this property. A detergent extract of chromaffin granule membranes enriched in monoamine transporter was heated and the aggregates were isolated by size-exclusion HPLC in SDS. The aggregates, containing the transporter, were dissociated in the presence of trifluoroacetic acid and analysed on the same HPLC column. This strategy might be of general interest for the purification of membrane proteins that exhibit SDS-resistant aggregation.

Biochemistry and molecular biology of the vesicular monoamine transporter from chromaffin granules.

Prior to secretion, monoamines (catecholamines, serotonin, histamine) are concentrated from the cytoplasm into vesicles by vesicular monoamine transporters (VMAT). These transporters also carry non-physiological compounds, e.g. the neurotoxin methyl-4-phenylpyridinium. VMAT acts as an electrogenic antiporter (exchanger) of protons and monoamines, using a proton electrochemical gradient. Vesicular transport is inhibited by specific ligands, including tetrabenazine, ketanserin and reserpine. The mechanism of transport and the biochemistry of VMAT have been analyzed with the help of these tools, using mainly the chromaffin granules from bovine adrenal glands as a source of transporter. Although biochemical studies did not suggest a multiplicity of VMATs, two homologous but distinct VMAT genes have recently been cloned from rat, bovine and human adrenal glands. The VMAT proteins are predicted to possess 12 transmembrane segments, with both extremities lying on the cytoplasmic side. They possess N-glycosylation sites in a putative luminal loop and phosphorylation sites in cytoplasmic domains. In rat, VMAT1 is expressed in the adrenal gland whereas VMAT2 is expressed in the brain. In contrast, we found that the bovine adrenal gland expressed both VMAT1 and VMAT2. VMAT2 corresponds to the major transporter of chromaffin granules, as shown by partial peptidic sequences of the purified protein and by a pharmacological analysis of the transport obtained in transfected COS cells (COS cells are monkey kidney cells possessing the ability to replicate SV-40-origin-containing plasmids). We discuss the possibility that VMAT1 may be specifically addressed to large secretory granules vesicles, whereas VMAT2 may also be addressed to small synaptic vesicles; species differences would then reflect the distinct physiological roles of the small synaptic vesicles in the adrenal gland.