Publications by authors named "Ralf B Nehring"

16 Publications

  • Page 1 of 1

Two mechanisms of chromosome fragility at replication-termination sites in bacteria.

Sci Adv 2021 Jun 18;7(25). Epub 2021 Jun 18.

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

Chromosomal fragile sites are implicated in promoting genome instability, which drives cancers and neurological diseases. Yet, the causes and mechanisms of chromosome fragility remain speculative. Here, we identify three spontaneous fragile sites in the genome and define their DNA damage and repair intermediates at high resolution. We find that all three sites, all in the region of replication termination, display recurrent four-way DNA or Holliday junctions (HJs) and recurrent DNA breaks. Homology-directed double-strand break repair generates the recurrent HJs at all of these sites; however, distinct mechanisms of DNA breakage are implicated: replication fork collapse at natural replication barriers and, unexpectedly, frequent shearing of unsegregated sister chromosomes at cell division. We propose that mechanisms such as both of these may occur ubiquitously, including in humans, and may constitute some of the earliest events that underlie somatic cell mosaicism, cancers, and other diseases of genome instability.
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http://dx.doi.org/10.1126/sciadv.abe2846DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8213236PMC
June 2021

Bacteria-to-Human Protein Networks Reveal Origins of Endogenous DNA Damage.

Cell 2019 01;176(1-2):127-143.e24

Graduate Program in Quantitative and Computational Biosciences, Baylor College of Medicine, Houston, TX 77030, USA; Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

DNA damage provokes mutations and cancer and results from external carcinogens or endogenous cellular processes. However, the intrinsic instigators of endogenous DNA damage are poorly understood. Here, we identify proteins that promote endogenous DNA damage when overproduced: the DNA "damage-up" proteins (DDPs). We discover a large network of DDPs in Escherichia coli and deconvolute them into six function clusters, demonstrating DDP mechanisms in three: reactive oxygen increase by transmembrane transporters, chromosome loss by replisome binding, and replication stalling by transcription factors. Their 284 human homologs are over-represented among known cancer drivers, and their RNAs in tumors predict heavy mutagenesis and a poor prognosis. Half of the tested human homologs promote DNA damage and mutation when overproduced in human cells, with DNA damage-elevating mechanisms like those in E. coli. Our work identifies networks of DDPs that provoke endogenous DNA damage and may reveal DNA damage-associated functions of many human known and newly implicated cancer-promoting proteins.
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http://dx.doi.org/10.1016/j.cell.2018.12.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6344048PMC
January 2019

ELKS1 localizes the synaptic vesicle priming protein bMunc13-2 to a specific subset of active zones.

J Cell Biol 2017 04 6;216(4):1143-1161. Epub 2017 Mar 6.

Department of Molecular Neurobiology, Center for Nanoscale Microscopy and Molecular Physiology of the Brain, Max Planck Institute of Experimental Medicine, 37075 Göttingen, Germany

Presynaptic active zones (AZs) are unique subcellular structures at neuronal synapses, which contain a network of specific proteins that control synaptic vesicle (SV) tethering, priming, and fusion. Munc13s are core AZ proteins with an essential function in SV priming. In hippocampal neurons, two different Munc13s-Munc13-1 and bMunc13-2-mediate opposite forms of presynaptic short-term plasticity and thus differentially affect neuronal network characteristics. We found that most presynapses of cortical and hippocampal neurons contain only Munc13-1, whereas ∼10% contain both Munc13-1 and bMunc13-2. Whereas the presynaptic recruitment and activation of Munc13-1 depends on Rab3-interacting proteins (RIMs), we demonstrate here that bMunc13-2 is recruited to synapses by the AZ protein ELKS1, but not ELKS2, and that this recruitment determines basal SV priming and short-term plasticity. Thus, synapse-specific interactions of different Munc13 isoforms with ELKS1 or RIMs are key determinants of the molecular and functional heterogeneity of presynaptic AZs.
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http://dx.doi.org/10.1083/jcb.201606086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5379939PMC
April 2017

Holliday junction trap shows how cells use recombination and a junction-guardian role of RecQ helicase.

Sci Adv 2016 Nov 18;2(11):e1601605. Epub 2016 Nov 18.

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.; Department of Biochemistry, Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.; Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA.; Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.; Graduate Program in Integrative Molecular and Biomedical Sciences, Baylor College of Medicine, Houston, TX 77030, USA.; Systems, Synthetic, and Physical Biology Program, Rice University, Houston, TX 77030, USA.

DNA repair by homologous recombination (HR) underpins cell survival and fuels genome instability, cancer, and evolution. However, the main kinds and sources of DNA damage repaired by HR in somatic cells and the roles of important HR proteins remain elusive. We present engineered proteins that trap, map, and quantify Holliday junctions (HJs), a central DNA intermediate in HR, based on catalytically deficient mutant RuvC protein of . We use RuvCDefGFP (RDG) to map genomic footprints of HR at defined DNA breaks in and demonstrate genome-scale directionality of double-strand break (DSB) repair along the chromosome. Unexpectedly, most spontaneous HR-HJ foci are instigated, not by DSBs, but rather by single-stranded DNA damage generated by replication. We show that RecQ, the ortholog of five human cancer proteins, nonredundantly promotes HR-HJ formation in single cells and, in a novel junction-guardian role, also prevents apparent non-HR-HJs promoted by RecA overproduction. We propose that one or more human RecQ orthologs may act similarly in human cancers overexpressing the RecA ortholog and find that cancer genome expression data implicate the orthologs BLM and RECQL4 in conjunction with EME1 and GEN1 as probable HJ reducers in such cancers. Our results support RecA-overproducing as a model of the many human tumors with up-regulated and provide the first glimpses of important, previously elusive reaction intermediates in DNA replication and repair in single living cells.
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http://dx.doi.org/10.1126/sciadv.1601605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5222578PMC
November 2016

An ultra-dense library resource for rapid deconvolution of mutations that cause phenotypes in Escherichia coli.

Nucleic Acids Res 2016 Mar 17;44(5):e41. Epub 2015 Nov 17.

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA The Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA

With the wide availability of whole-genome sequencing (WGS), genetic mapping has become the rate-limiting step, inhibiting unbiased forward genetics in even the most tractable model organisms. We introduce a rapid deconvolution resource and method for untagged causative mutations after mutagenesis, screens, and WGS in Escherichia coli. We created Deconvoluter-ordered libraries with selectable insertions every 50 kb in the E. coli genome. The Deconvoluter method uses these for replacement of untagged mutations in the genome using a phage-P1-based gene-replacement strategy. We validate the Deconvoluter resource by deconvolution of 17 of 17 phenotype-altering mutations from a screen of N-ethyl-N-nitrosourea-induced mutants. The Deconvoluter resource permits rapid unbiased screens and gene/function identification and will enable exploration of functions of essential genes and undiscovered genes/sites/alleles not represented in existing deletion collections. This resource for unbiased forward-genetic screens with mapping-by-sequencing ('forward genomics') demonstrates a strategy that could similarly enable rapid screens in many other microbes.
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http://dx.doi.org/10.1093/nar/gkv1131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4797258PMC
March 2016

Thymineless death is inhibited by CsrA in Escherichia coli lacking the SOS response.

DNA Repair (Amst) 2013 Nov 25;12(11):993-9. Epub 2013 Sep 25.

Departments of Molecular and Human Genetics, Biochemistry and Molecular Biology, Molecular Virology and Microbiology and the Dan L Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030-3411, United States.

Thymineless death (TLD) is the rapid loss of colony-forming ability in bacterial, yeast and human cells starved for thymine, and is the mechanism of action of common chemotherapeutic drugs. In Escherichia coli, significant loss of viability during TLD requires the SOS replication-stress/DNA-damage response, specifically its role in inducing the inhibitor of cell division, SulA. An independent RecQ- and RecJ-dependent TLD pathway accounts for a similarly large additional component of TLD, and a third SOS- and RecQ/J-independent TLD pathway has also been observed. Although two groups have implicated the SOS-response in TLD, an SOS-deficient mutant strain from an earlier study was found to be sensitive to thymine deprivation. We performed whole-genome resequencing on that SOS-deficient strain and find that, compared with the SOS-proficient control strain, it contains five mutations in addition to the SOS-blocking lexA(Ind(-)) mutation. One of the additional mutations, csrA, confers TLD sensitivity specifically in SOS-defective strains. We find that CsrA, a carbon storage regulator, reduces TLD in SOS- or SulA-defective cells, and that the increased TLD that occurs in csrA(-) SOS-defective cells is dependent on RecQ. We consider a hypothesis in which the modulation of nucleotide pools by CsrA might inhibit TLD specifically in SOS-deficient (SulA-deficient) cells.
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http://dx.doi.org/10.1016/j.dnarep.2013.08.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3898814PMC
November 2013

A role for vesicular glutamate transporter 1 in synaptic vesicle clustering and mobility.

Eur J Neurosci 2013 May 15;37(10):1631-42. Epub 2013 Apr 15.

Institute of Biology of the Ecole Normale Supérieure, Paris, France.

Synaptic vesicles (SVs) from excitatory synapses carry vesicular glutamate transporters (VGLUTs) that fill the vesicles with neurotransmitter. Although the essential function of VGLUTs as glutamate transporters has been well established, the evidence for additional cell-biological functions is more controversial. Both VGLUT1 and VGLUT2 disruptions in mice result in a reduced number of SVs away from release sites, flattening of SVs, and the appearance of tubular structures. Therefore, we analysed the morphology, biochemical composition and trafficking of SVs at synapses of VGLUT1(-/-) mice in order to test for a function of VGLUTs in the formation or clustering of SVs. Analyses with high-pressure freezing immobilisation and electron tomography pointed to a role of VGLUT1 transport function in the tonicity of excitatory SVs, explaining the aldehyde-induced flattening of SVs observed in VGLUT1(-/-) synapses. We confirmed the steep reduction in the number of SVs previously observed in VGLUT1(-/-) presynaptic terminals, but did not observe accumulation of endocytotic intermediates. Furthermore, SV proteins of adult VGLUT1(-/-) mouse brain tissue were expressed at normal levels in all subcellular fractions, suggesting that they were not displaced to another organelle. We thus assessed the mobility of the recently documented superpool of SVs. Synaptobrevin2-enhanced green fluorescent protein time lapse experiments revealed an oversized superpool of SVs in VGLUT1(-/-) neurons. Our results support the idea that, beyond glutamate loading, VGLUT1 enhances the tonicity of excitatory SVs and stabilises SVs at presynaptic terminals.
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http://dx.doi.org/10.1111/ejn.12199DOI Listing
May 2013

Identity and function of a large gene network underlying mutagenic repair of DNA breaks.

Science 2012 Dec;338(6112):1344-8

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030-3411, USA.

Mechanisms of DNA repair and mutagenesis are defined on the basis of relatively few proteins acting on DNA, yet the identities and functions of all proteins required are unknown. Here, we identify the network that underlies mutagenic repair of DNA breaks in stressed Escherichia coli and define functions for much of it. Using a comprehensive screen, we identified a network of ≥93 genes that function in mutation. Most operate upstream of activation of three required stress responses (RpoS, RpoE, and SOS, key network hubs), apparently sensing stress. The results reveal how a network integrates mutagenic repair into the biology of the cell, show specific pathways of environmental sensing, demonstrate the centrality of stress responses, and imply that these responses are attractive as potential drug targets for blocking the evolution of pathogens.
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http://dx.doi.org/10.1126/science.1226683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3782309PMC
December 2012

Interplay between VGLUT isoforms and endophilin A1 regulates neurotransmitter release and short-term plasticity.

Neuron 2011 Mar;69(6):1147-59

Departments of Neuroscience and Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

Vesicular glutamate transporters (VGLUTs) are essential for filling synaptic vesicles with glutamate and mammals express three VGLUT isoforms (VGLUT1-3) with distinct spatiotemporal expression patterns. Here, we find that neurons expressing VGLUT1 have lower release probability and less short-term depression than neurons expressing VGLUT2 or VGLUT3. Investigation of the underlying mechanism identified endophilin A1 as a positive regulator of exocytosis whose expression levels are positively correlated with release efficiency and showed that the differences in release efficiency between VGLUT1- and VGLUT2-expressing neurons are due to VGLUT1's ability to bind endophilin A1 and inhibit endophilin-induced enhancement of release probability.
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http://dx.doi.org/10.1016/j.neuron.2011.02.002DOI Listing
March 2011

Characterization of Eag1 channel lateral mobility in rat hippocampal cultures by single-particle-tracking with quantum dots.

PLoS One 2010 Jan 25;5(1):e8858. Epub 2010 Jan 25.

Department of Molecular Biology of Neuronal Signals, Max-Planck Institute of Experimental Medicine, Göttingen, Germany.

Voltage-gated ion channels are main players involved in fast synaptic events. However, only slow intracellular mechanisms have so far been described for controlling their localization as real-time visualization of endogenous voltage-gated channels at high temporal and spatial resolution has not been achieved yet. Using a specific extracellular antibody and quantum dots we reveal and characterize lateral mobility as a faster mechanism to dynamically control the number of endogenous ether-a-go-go (Eag)1 ion channels inside synapses. We visualize Eag1 entering and leaving synapses by lateral diffusion in the plasma membrane of rat hippocampal neurons. Mathematical analysis of their trajectories revealed how the motion of Eag1 gets restricted when the channels diffuse into the synapse, suggesting molecular interactions between Eag1 and synaptic components. In contrast, Eag1 channels switch to Brownian movement when they exit synapses and diffuse into extrasynaptic membranes. Furthermore, we demonstrate that the mobility of Eag1 channels is specifically regulated inside synapses by actin filaments, microtubules and electrical activity. In summary, using single-particle-tracking techniques with quantum dots nanocrystals, our study shows for the first time the lateral diffusion of an endogenous voltage-gated ion channel in neurons. The location-dependent constraints imposed by cytoskeletal elements together with the regulatory role of electrical activity strongly suggest a pivotal role for the mobility of voltage-gated ion channels in synaptic activity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0008858PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2810327PMC
January 2010

Differential abilities of SNAP-25 homologs to support neuronal function.

J Neurosci 2007 Aug;27(35):9380-91

Department of Membrane Biophysics, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany.

The SNAP receptor (SNARE) complex, consisting of synaptosome-associated protein of 25 kDa (SNAP-25), synaptobrevin-2, and syntaxin-1, is involved in synaptic vesicles exocytosis. In addition, SNAP-25 has been implicated in constitutive exocytosis processes required for neurite outgrowth. However, at least three isoforms of SNAP-25 have been reported from neurons: SNAP-23, which is also present in non-neuronal cells, and the two alternative splice variants SNAP-25a and SNAP-25b. Here, we studied the differential ability of these isoforms to support the functions previously broadly ascribed to "SNAP-25." We studied the rescue of snap-25 null neurons in culture with different SNAP-25 homologs. We find that deletion of SNAP-25 leads to strongly reduced neuron survival, and, in the few surviving cells, impaired arborization, reduced spontaneous release, and complete arrest of evoked release. Lentiviral expression of SNAP-25a, SNAP-25b, or SNAP-23 rescued neuronal survival, arborization, amplitude, and frequency of spontaneous events. Also evoked release was rescued by all isoforms, but synchronous release required SNAP-25a/b in both glutamatergic and GABAergic neurons. SNAP-23 supported asynchronous release only, reminiscent of synaptotagmin-1 null neurons. SNAP-25b was superior to SNAP-25a in vesicle priming, resembling the shift to larger releasable vesicle pools that accompanies synaptic maturation. These data demonstrate a differential ability of SNAP-25b, SNAP-25a, and SNAP-23 to support neuronal function.
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http://dx.doi.org/10.1523/JNEUROSCI.5092-06.2007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6673127PMC
August 2007

Monitoring clathrin-mediated endocytosis during synaptic activity.

J Neurosci 2004 Feb;24(8):2004-12

Department of Membrane Biophysics, Max-Planck Institute for Biophysical Chemistry, D-37077 Goettingen, Germany.

To visualize clathrin redistribution during endocytosis in hippocampal boutons, we used a fusion protein of clathrin light chain with enhanced green fluorescent protein. Both high potassium and electric field stimulation lead after a stimulus-dependent delay to a transient increase of fluorescence in synapses, but a slight and transient decrease in adjacent axonal segments. We conclude that the rise and fall of the signal in boutons, with decay kinetics remarkably similar to previous estimates of the endocytic time course, reflects coat assembly and disassembly. Thus, we could selectively measure clathrin-mediated endocytosis and separate its kinetics from other modes of membrane retrieval in CNS synapses. A long-lasting delay preceding the fluorescent transients shows that endocytosis during the first few seconds of continuing stimulation cannot be mediated by newly formed clathrin-coated pits. Therefore, a fast mode of endocytosis is either clathrin-independent or involves preassembled (easily retrievable) clathrin lattices at sites of endocytosis.
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http://dx.doi.org/10.1523/JNEUROSCI.4080-03.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6730391PMC
February 2004

Differential control of the releasable vesicle pools by SNAP-25 splice variants and SNAP-23.

Cell 2003 Jul;114(1):75-86

Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.

The SNARE complex, consisting of synaptobrevin, syntaxin, and SNAP-25, is essential for calcium-triggered exocytosis in neurosecretory cells. Little is known, however, about how developmentally regulated isoforms and other cognate SNARE components regulate vesicular fusion. To address this question, we examined neuroexocytosis from chromaffin cells of Snap25 null mice rescued by the two splice variants SNAP-25a and SNAP-25b and the ubiquitously expressed homolog SNAP-23. In the absence of SNAP-25, vesicle docking persisted, but primed vesicle pools were empty and fast calcium-triggered release abolished. Single vesicular fusion events showed normal characteristics, except for a shorter duration of the fusion pore. Overexpression of SNAP-25a, SNAP-25b, and SNAP-23 resulted in three distinct phenotypes; SNAP-25b induced larger primed vesicle pools than SNAP-25a, whereas SNAP-23 did not support a standing pool of primed vesicles. We conclude that three alternative SNARE components support exocytosis, but they differ in their ability to stabilize vesicles in the primed state.
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http://dx.doi.org/10.1016/s0092-8674(03)00477-xDOI Listing
July 2003

Protein kinase C-dependent phosphorylation of synaptosome-associated protein of 25 kDa at Ser187 potentiates vesicle recruitment.

J Neurosci 2002 Nov;22(21):9278-86

Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.

Activation of protein kinase C (PKC) constitutes a key event in the upregulation of secretory strength in neurons and neurosecretory cells during extensive stimulation, presumably by speeding up vesicle supply. However, the molecular targets and their mode of action remain elusive. We studied the only PKC-dependent phosphorylation site in the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, Ser(187), in synaptosome-associated protein of 25 kDa (SNAP-25). This phosphorylation site is located within the negatively charged C-terminal end of SNAP-25, which has been shown to be of critical importance in calcium-triggered exocytosis. We combined mutational studies that used overexpression in chromaffin cells with capacitance measurements and flash photolysis of caged calcium, allowing for high time resolution during both the stimulation and measurement of exocytosis. Overexpression of mutants simulating the phosphorylated form of Ser(187) accelerated vesicle recruitment after the emptying of the releasable vesicle pools. Overexpression of mutants simulating the nonphosphorylated form, or block of PKC, impaired the refilling of the vesicle pools to similar extents. Biochemical studies verified the phosphorylation of a subpopulation of SNAP-25 after elevation of intracellular calcium concentrations. Some of the mutations led to a moderately decreased fast exocytotic burst component, which did not seem to be associated with the phosphorylation state of SNAP-25. Thus the C terminus of SNAP-25 plays a role for both fast exocytosis triggering and vesicle recruitment, and the latter process is regulated by PKC-dependent phosphorylation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6758053PMC
November 2002

The SNARE protein SNAP-25 is linked to fast calcium triggering of exocytosis.

Proc Natl Acad Sci U S A 2002 Feb;99(3):1627-32

Max-Planck-Institut für Biophysikalische Chemie, Am Fassberg 11, 37077 Göttingen, Germany.

Synchronous neurotransmission depends on the tight coupling between Ca(2+) influx and fusion of neurotransmitter-filled vesicles with the plasma membrane. The vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein synaptobrevin 2 and the plasma membrane SNAREs syntaxin 1 and synaptosomal protein of 25 kDa (SNAP-25) are essential for calcium-triggered exocytosis. However, the link between calcium triggering and SNARE function remains elusive. Here we describe mutations in two sites on the surface of the SNARE complex formed by acidic and hydrophilic residues of SNAP-25 and synaptobrevin 2, which were found to coordinate divalent cations in the neuronal SNARE complex crystal structure. By reducing the net charge of the site in SNAP-25 we identify a mutation that interferes with calcium triggering of exocytosis when overexpressed in chromaffin cells. Exocytosis was elicited by photorelease of calcium from a calcium cage and evaluated by using patch-clamp capacitance measurements at millisecond time resolution. We present a method for monitoring the dependence of exocytotic rate upon calcium concentration at the release site and demonstrate that the mutation decreased the steepness of this relationship, indicating that the number of sequential calcium-binding steps preceding exocytosis is reduced by one. We conclude that the SNARE complex is linked directly to calcium triggering of exocytosis, most likely in a complex with auxiliary proteins.
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http://dx.doi.org/10.1073/pnas.251673298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC122241PMC
February 2002
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