Mads Grønborg

Molecular Anatomy of a Trafficking Organelle

Membrane traffic in eukaryotic cells involves transport of vesicles that bud from a donor compartment and fuse with an acceptor compartment. Common principles of budding and fusion have emerged, and many of the proteins involved in these events are now known. However, a detailed picture of an entire trafficking organelle is not yet available. Using synaptic vesicles as a model, we have now determined the protein and lipid composition; measured vesicle size, density, and mass; calculated the average protein and lipid mass per vesicle; and determined the copy number of more than a dozen major constituents. A model has been constructed that integrates all quantitative data and includes structural models of abundant proteins. Synaptic vesicles are dominated by proteins, possess a surprising diversity of trafficking proteins, and, with the exception of the V-ATPase that is present in only one to two copies, contain numerous copies of proteins essential for membrane traffic and neurotransmitter uptake.

Quantitative comparison of glutamatergic and GABAergic synaptic vesicles unveils selectivity for few proteins including MAL2, a novel synaptic vesicle protein.

Synaptic vesicles (SVs) store neurotransmitters and release them by exocytosis. The vesicular neurotransmitter transporters discriminate which transmitter will be sequestered and stored by the vesicles. However, it is unclear whether the neurotransmitter phenotype of SVs is solely defined by the transporters or whether it is associated with additional proteins. Here we have compared the protein composition of SVs enriched in vesicular glutamate (VGLUT-1) and GABA transporters (VGAT), respectively, using quantitative proteomics. Of >450 quantified proteins, ∼50 were differentially distributed between the populations, with only few of them being specific for SVs. Of these, the most striking differences were observed for the zinc transporter ZnT3 and the vesicle proteins SV2B and SV31 that are associated preferentially with VGLUT-1 vesicles, and for SV2C that is associated mainly with VGAT vesicles. Several additional proteins displayed a preference for VGLUT-1 vesicles including, surprisingly, synaptophysin, synaptotagmins, and syntaxin 1a. Moreover, MAL2, a membrane protein of unknown function distantly related to synaptophysins and SCAMPs, cofractionated with VGLUT-1 vesicles. Both subcellular fractionation and immunolocalization at the light and electron microscopic level revealed that MAL2 is a bona-fide membrane constituent of SVs that is preferentially associated with VGLUT-1-containing nerve terminals. We conclude that SVs specific for different neurotransmitters share the majority of their protein constituents, with only few vesicle proteins showing preferences that, however, are nonexclusive, thus confirming that the vesicular transporters are the only components essential for defining the neurotransmitter phenotype of a SV.

Role of cdk2 in the sequential phosphorylation/activation of C/EBPβ during adipocyte differentiation

Upon induction of differentiation, growth-arrested (G1 phase) 3T3-L1 preadipocytes express CCAAT/enhancer binding protein-β (C/EBPβ), initiating a transcriptional cascade. C/EBPβ immediately undergoes a priming phosphorylation (on Thr188) by MAPK/ERK. However, the acquisition of DNA binding and transactivation capacity of C/EBPβ is delayed until further phosphorylation (on Ser184 or Thr179) by GSK3β occurs. Phosphorylation by glycogen synthase kinase-3β (GSK3β) induces S phase entry and thereby mitotic clonal expansion (MCE), a requirement for terminal differentiation. Because MAPK activity is down-regulated before S phase is completed, we sought to identify the kinase that maintains C/EBPβ in the primed phosphorylated state throughout S phase and MCE. We show here that cdk2/cyclinA, whose expression is activated at the onset of S phase, functions in this capacity. Ex vivo and in vitro experiments show that cdk2/cyclinA catalyzes this delayed priming phosphorylation. Mass spectrometric analysis revealed that cdk2/cyclinA phosphorylates C/EBPβ on Thr188 and is required for phosphorylation (on Ser184 or Thr179) of C/EBPβ by GSK3β and maintenance of DNA binding activity. Suppression of cdk2 activity by RNA interference or pharmacologic inhibitor disrupts subsequent events in the differentiation program. Thus, MAPK and cdk2/cyclinA act sequentially to maintain Thr188 of C/EBPβ in the primed phosphorylated state during MCE and thereby progression of terminal differentiation.

Molecular Profiling of Synaptic Vesicle Docking Sites Reveals Novel Proteins but Few Differences between Glutamatergic and GABAergic Synapses

Neurotransmission involves calcium-triggered fusion of docked synaptic vesicles at specialized presynaptic release sites. While many of the participating proteins have been identified, the molecular composition of these sites has not been characterized comprehensively. Here, we report a procedure to biochemically isolate fractions highly enriched in docked synaptic vesicles. The fraction is largely free of postsynaptic proteins and most other organelles while containing most known synaptic vesicle and active zone proteins. Numerous presynaptic transmembrane proteins were also identified, together with over 30 uncharacterized proteins, many of which are evolutionarily conserved. Quantitative proteomic comparison of glutamate- and GABA-specific docking complexes revealed that, except of neurotransmitter-specific enzymes and transporters, only few proteins were selectively enriched in either fraction. We conclude that the core machinery involved in vesicle docking and exocytosis does not show compositional differences between the two types of synapses.

Quantitative analysis of synaptic vesicle Rabs uncovers distinct yet overlapping roles for Rab3a and Rab27b in Ca2+-triggered exocytosis.

Rab GTPases are molecular switches that orchestrate protein complexes before membrane fusion reactions. In synapses, Rab3 and Rab5 proteins have been implicated in the exo-endocytic cycling of synaptic vesicles (SVs), but an involvement of additional Rabs cannot be excluded. Here, combining high-resolution mass spectrometry and chemical labeling (iTRAQ) together with quantitative immunoblotting and fluorescence microscopy, we have determined the exocytotic (Rab3a, Rab3b, Rab3c, and Rab27b) and endocytic (Rab4b, Rab5a/b, Rab10, Rab11b, and Rab14) Rab machinery of SVs. Analysis of two closely related proteins, Rab3a and Rab27b, revealed colocalization in synaptic nerve terminals, where they reside on distinct but overlapping SV pools. Moreover, whereas Rab3a readily dissociates from SVs during Ca2+-triggered exocytosis, and is susceptible to membrane extraction by Rab-GDI, Rab27b persists on SV membranes upon stimulation and is resistant to GDI-coupled Rab retrieval. Finally, we demonstrate that selective modulation of the GTP/GDP switch mechanism of Rab27b impairs SV recycling, suggesting that Rab27b, probably in concert with Rab3s, is involved in SV exocytosis.

Unique Luminal Localization of VGAT-C Terminus Allows for Selective Labeling of Active Cortical GABAergic Synapses

Neurotransmitter uptake into synaptic vesicles is mediated by vesicular neurotransmitter transporters. Although these transporters belong to different families, they all are thought to share a common overall topology with an even number of transmembrane domains. Using epitope-specific antibodies and mass spectrometry we show that the vesicular GABA transporter (VGAT) possesses an uneven number of transmembrane domains, with the N terminus facing the cytoplasm and the C terminus residing in the synaptic vesicle lumen. Antibodies recognizing the C terminus of VGAT (anti-VGAT-C) selectively label GABAergic nerve terminals of live cultured hippocampal and striatal neurons as confirmed by immunocytochemistry and patch-clamp electrophysiology. Injection of fluorochromated anti-VGAT-C into the hippocampus of mice results in specific labeling of GABAergic synapses in vivo. Overall, our data open the possibility of studying novel GABA release sites, characterizing inhibitory vesicle trafficking, and establishing their contribution to inhibitory neurotransmission at identified GABAergic synapses.

Late motor decline after accomplished remyelination: Impact for progressive multiple sclerosis

To investigate the impact of single or repeated episodes of reversible demyelination on long‐term locomotor performance and neuroaxonal integrity, and to analyze the myelin proteome after remyelination and during aging.

Casein Kinase 2 Binds to the C Terminus of Na+/H+ exchanger 3 (NHE3) and Stimulates NHE3 Basal Activity by Phosphorylating a Separate Site in NHE3

Na(+)/H(+) exchanger 3 (NHE3) is the epithelial-brush border isoform responsible for most intestinal and renal Na(+) absorption. Its activity is both up- and down-regulated under normal physiological conditions, and it is inhibited in most diarrheal diseases. NHE3 is phosphorylated under basal conditions and Ser/Thr phosphatase inhibitors stimulate basal exchange activity; however, the kinases involved are unknown. To identify kinases that regulate NHE3 under basal conditions, NHE3 was immunoprecipitated; LC-MS/MS of trypsinized NHE3 identified a novel phosphorylation site at S(719) of the C terminus, which was predicted to be a casein kinase 2 (CK2) phosphorylation site. This was confirmed by an in vitro kinase assay. The NHE3-S719A mutant but not NHE3-S719D had reduced NHE3 activity due to less plasma membrane NHE3. This was due to reduced exocytosis plus decreased plasma membrane delivery of newly synthesized NHE3. Also, NHE3 activity was inhibited by the CK2 inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole DMAT when wild-type NHE3 was expressed in fibroblasts and Caco-2 cells, but the NHE3-S(719) mutant was fully resistant to DMAT. CK2 bound to the NHE3 C-terminal domain, between amino acids 590 and 667, a site different from the site it phosphorylates. CK2 binds to the NHE3 C terminus and stimulates basal NHE3 activity by phosphorylating a separate single site on the NHE3 C terminus (S(719)), which affects NHE3 trafficking.

Proteomic screening of glutamatergic mouse brain synaptosomes isolated by fluorescence activated sorting.

For decades, neuroscientists have used enriched preparations of synaptic particles called synaptosomes to study synapse function. However, the interpretation of corresponding data is problematic as synaptosome preparations contain multiple types of synapses and non‐synaptic neuronal and glial contaminants. We established a novel Fluorescence Activated Synaptosome Sorting (FASS) method that substantially improves conventional synaptosome enrichment protocols and enables high‐resolution biochemical analyses of specific synapse subpopulations. Employing knock‐in mice with fluorescent glutamatergic synapses, we show that FASS isolates intact ultrapure synaptosomes composed of a resealed presynaptic terminal and a postsynaptic density as assessed by light and electron microscopy. FASS synaptosomes contain bona fide glutamatergic synapse proteins but are almost devoid of other synapse types and extrasynaptic or glial contaminants. We identified 163 enriched proteins in FASS samples, of which FXYD6 and Tpd52 were validated as new synaptic proteins. FASS purification thus enables high‐resolution biochemical analyses of specific synapse subpopulations in health and disease.

Structure of Tripeptidyl-peptidase I Provides Insight into the Molecular Basis of Late Infantile Neuronal Ceroid Lipofuscinosis

Late infantile neuronal ceroid lipofuscinosis, a fatal neurodegenerative disease of childhood, is caused by mutations in the TPP1 gene that encodes tripeptidyl-peptidase I. We show that purified TPP1 requires at least partial glycosylation for in vitro autoprocessing and proteolytic activity. We crystallized the fully glycosylated TPP1 precursor under conditions that implied partial autocatalytic cleavage between the prosegment and the catalytic domain. X-ray crystallographic analysis at 2.35 Å resolution reveals a globular structure with a subtilisin-like fold, a Ser475-Glu272-Asp360 catalytic triad, and an octahedrally coordinated Ca2+-binding site that are characteristic features of the S53 sedolisin family of peptidases. In contrast to other S53 peptidases, the TPP1 structure revealed steric constraints on the P4 substrate pocket explaining its preferential cleavage of tripeptides from the unsubstituted N terminus of proteins. Two alternative conformations of the catalytic Asp276 are associated with the activation status of TPP1. 28 disease-causing missense mutations are analyzed in the light of the TPP1 structure providing insight into the molecular basis of late infantile neuronal ceroid lipofuscinosis.