All Title Author
Keywords Abstract

PLOS ONE  2006 

‘Fractional Recovery’ Analysis of a Presynaptic Synaptotagmin 1-Anchored Endocytic Protein Complex

DOI: 10.1371/journal.pone.0000067

Full-Text   Cite this paper   Add to My Lib

Abstract:

Background The integral synaptic vesicle protein and putative calcium sensor, synaptotagmin 1 (STG), has also been implicated in synaptic vesicle (SV) recovery. However, proteins with which STG interacts during SV endocytosis remain poorly understood. We have isolated an STG-associated endocytic complex (SAE) from presynaptic nerve terminals and have used a novel fractional recovery (FR) assay based on electrostatic dissociation to identify SAE components and map the complex structure. The location of SAE in the presynaptic terminal was determined by high-resolution quantitative immunocytochemistry at the chick ciliary ganglion giant calyx-type synapse. Methodology/Principle Findings The first step in FR analysis was to immunoprecipitate (IP) the complex with an antibody against one protein component (the IP-protein). The immobilized complex was then exposed to a high salt (1150 mM) stress-test that caused shedding of co-immunoprecipitated proteins (co-IP-proteins). A Fractional Recovery ratio (FR: recovery after high salt/recovery with control salt as assayed by Western blot) was calculated for each co-IP-protein. These FR values reflect complex structure since an easily dissociated protein, with a low FR value, cannot be intermediary between the IP-protein and a salt-resistant protein. The structure of the complex was mapped and a blueprint generated with a pair of FR analyses generated using two different IP-proteins. The blueprint of SAE contains an AP180/X/STG/stonin 2/intersectin/epsin core (X is unknown and epsin is hypothesized), and an AP2 adaptor, H-/L-clathrin coat and dynamin scission protein perimeter. Quantitative immunocytochemistry (ICA/ICQ method) at an isolated calyx-type presynaptic terminal indicates that this complex is associated with STG at the presynaptic transmitter release face but not with STG on intracellular synaptic vesicles. Conclusions/Significance We hypothesize that the SAE serves as a recognition site and also as a seed complex for clathrin-mediated synaptic vesicle recovery. The combination of FR analysis with quantitative immunocytochemistry provides a novel and effective strategy for the identification and characterization of biologically-relevant multi-molecular complexes.

References

[1]  Gentile L, Stanley EF (2005) A unified model of presynaptic release site gating by calcium channel domains. Eur J Neurosci 21: 278–282.
[2]  Murthy VN, De Camilli P (2003) Cell biology of the presynaptic terminal. Annu Rev Neurosci 26: 701–728.
[3]  Augustine GJ, Morgan JR, Villalba-Galea CA, Jin S, Prasad K, et al. (2006) Clathrin and synaptic vesicle endocytosis: studies at the squid giant synapse. Biochem Soc Trans 34: 68–72.
[4]  Mousavi SA, Malerod L, Berg T, Kjeken R (2004) Clathrin-dependent endocytosis. Biochem J 377: 1–16.
[5]  Lafer EM (2002) Clathrin-protein interactions. Traffic 3: 513–520.
[6]  Slepnev VI, De Camilli P (2000) Accessory factors in clathrin-dependent synaptic vesicle endocytosis. Nat Rev Neurosci 1: 161–172.
[7]  Xiong X, Zhou KM, Wu ZX, Xu T (2006) Silence of synaptotagmin I in INS-1 cells inhibits fast exocytosis and fast endocytosis. Biochem Biophys Res Commun 347: 76–82.
[8]  Jorgensen EM, Hartwieg E, Schuske K, Nonet ML, Jin YS, et al. (1995) Defective recycling of synaptic vesicles in synaptotagmin mutants of Caenorhabditis elegans. Nature(Lond ) 378: 196–199.
[9]  Littleton JT, Bai J, Vyas B, Desai R, Baltus AE, et al. (2001) synaptotagmin mutants reveal essential functions for the C2B domain in Ca2+-triggered fusion and recycling of synaptic vesicles in vivo. J Neurosci 21: 1421–1433.
[10]  Nicholson-Tomishima K, Ryan TA (2004) Kinetic efficiency of endocytosis at mammalian CNS synapses requires synaptotagmin I. Proc Natl Acad Sci U S A 101: 16648–16652.
[11]  Poskanzer KE, Fetter RD, Davis GW (2006) Discrete residues in the c(2)b domain of synaptotagmin I independently specify endocytic rate and synaptic vesicle size. Neuron 50: 49–62.
[12]  Willig KI, Rizzoli SO, Westphal V, Jahn R, Hell SW (2006) STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature(Lond ) 440: 935–939.
[13]  Li Q, Lau A, Morris TJ, Guo L, Stanley EF (2004) A syntaxin 1, Galpha(o), and N-type calcium channel complex at a presynaptic nerve terminal: analysis by quantitative immunocolocalization. J Neurosci 24: 4070–4081.
[14]  Yamabhai M, Hoffman NG, Hardison NL, McPherson PS, Castagnoli L, et al. (1998) Intersectin, a novel adaptor protein with two Eps15 homology and five Src homology 3 domains. J Biol Chem 273: 31401–31407.
[15]  Kelly LE, Phillips AM (2005) Molecular and genetic characterization of the interactions between the Drosophila stoned-B protein and DAP-160 (intersectin). Biochem J 388: 195–204.
[16]  Martina JA, Bonangelino CJ, Aguilar RC, Bonifacino JS (2001) Stonin 2: an adaptor-like protein that interacts with components of the endocytic machinery. J Cell Biol 153: 1111–1120.
[17]  Montesinos ML, Castellano-Munoz M, Garcia-Junco-Clemente P, Fernandez-Chacon R (2005) Recycling and EH domain proteins at the synapse. Brain Res Brain Res Rev 49: 416–428.
[18]  Drake MT, Downs MA, Traub LM (2000) Epsin binds to clathrin by associating directly with the clathrin-terminal domain. Evidence for cooperative binding through two discrete sites. J Biol Chem 275: 6479–6489.
[19]  Diril MK, Wienisch M, Jung N, Klingauf J, Haucke V (2006) Stonin 2 is an AP-2-dependent endocytic sorting adaptor for synaptotagmin internalization and recycling. Dev Cell 10: 233–244.
[20]  Fernandez-Alfonso T, Kwan R, Ryan TA (2006) Synaptic Vesicles Interchange Their Membrane Proteins with a Large Surface Reservoir during Recycling. Neuron 51: 179–186.
[21]  Wienisch M, Klingauf J (2006) Vesicular proteins exocytosed and subsequently retrieved by compensatory endocytosis are nonidentical. Nat Neurosci 9: 1019–1027.
[22]  Khanna R, Li Q, Sun L, Collins TJ, Stanley EF (2006) N type Ca(2+) channels and RIM scaffold protein covary at the presynaptic transmitter release face but are components of independent protein complexes. Neuroscience.
[23]  Mirotznik RR, Zheng X, Stanley EF (2000) G-Protein types involved in calcium channel inhibition at a presynaptic nerve terminal. J Neurosci 20: 7614–7621.
[24]  Stanley EF, Mirotznik RR (1997) Cleavage of syntaxin prevents G-protein regulation of presynaptic calcium channels. Nature(Lond ) 385: 340–343.
[25]  Walther K, Diril MK, Jung N, Haucke V (2004) Functional dissection of the interactions of stonin 2 with the adaptor complex AP-2 and synaptotagmin. Proc Natl Acad Sci U S A 101: 964–969.
[26]  Tebar F, Sorkina T, Sorkin A, Ericsson M, Kirchhausen T (1996) Eps15 is a component of clathrin-coated pits and vesicles and is located at the rim of coated pits. J Biol Chem 271: 28727–28730.
[27]  Okamoto M, Schoch S, Sudhof TC (1999) EHSH1/intersectin, a protein that contains EH and SH3 domains and binds to dynamin and SNAP-25. A protein connection between exocytosis and endocytosis? J Biol Chem 274: 18446–18454.
[28]  Keen JH, Chestnut MH, Beck KA (1987) The clathrin coat assembly polypeptide complex. Autophosphorylation and assembly activities. J Biol Chem 262: 3864–3871.
[29]  Roos J, Kelly RB (1998) Dap160, a neural-specific Eps15 homology and multiple SH3 domain-containing protein that interacts with Drosophila dynamin. J Biol Chem 273: 19108–19119.
[30]  Marie B, Sweeney ST, Poskanzer KE, Roos J, Kelly RB, et al. (2004) Dap160/intersectin scaffolds the periactive zone to achieve high-fidelity endocytosis and normal synaptic growth. Neuron 43: 207–219.
[31]  Hussain NK, Yamabhai M, Ramjaun AR, Guy AM, Baranes D, et al. (1999) Splice variants of intersectin are components of the endocytic machinery in neurons and nonneuronal cells. J Biol Chem 274: 15671–15677.
[32]  Blondeau F, Ritter B, Allaire PD, Wasiak S, Girard M, et al. (2004) Tandem MS analysis of brain clathrin-coated vesicles reveals their critical involvement in synaptic vesicle recycling. Proc Natl Acad Sci U S A 101: 3833–3838.
[33]  Koh TW, Verstreken P, Bellen HJ (2004) Dap160/intersectin acts as a stabilizing scaffold required for synaptic development and vesicle endocytosis. Neuron 43: 193–205.
[34]  Walther K, Krauss M, Diril MK, Lemke S, Ricotta D, et al. (2001) Human stoned B interacts with AP-2 and synaptotagmin and facilitates clathrin-coated vesicle uncoating. EMBO Rep 2: 634–640.
[35]  Yao PJ, Petralia RS, Bushlin I, Wang Y, Furukawa K (2005) Synaptic distribution of the endocytic accessory proteins AP180 and CALM. J Comp Neurol 481: 58–69.
[36]  Zhang JZ, Davletov BA, Südhof TC, Anderson RGW (1994) Synaptotagmin I is a high affinity receptor for clathrin AP-2: Implications for membrane recycling. Cell 78: 751–760.
[37]  von Poser C, Zhang JZ, Mineo C, Ding W, Ying Y, et al. (2000) Synaptotagmin regulation of coated pit assembly. J Biol Chem 275: 30916–30924.
[38]  Maycox PR, Link E, Reetz A, Morris SA, Jahn R (1992) Clathrin-coated vesicles in nervous tissue are involved primarily in synaptic vesicle recycling. J Cell Biol 118: 1379–1388.
[39]  Stanley EF (1991) Single calcium channels on a cholinergic presynaptic nerve terminal. Neuron 7: 585–591.
[40]  Stanley EF, Goping G (1991) Characterization of a calcium current in a vertebrate cholinergic presynaptic nerve terminal. J Neurosci 11: 985–993.

Full-Text

comments powered by Disqus