The alternative translation of synaptotagmin 1 mediates the non-classical release of FGF1

doi:10.1016/j.bbrc.2003.09.119

Biochemical and Biophysical Research Communications

Volume 310, Issue 4, 31 October 2003, Pages 1041-1047

https://doi.org/10.1016/j.bbrc.2003.09.119 Get rights and content

Abstract

Although the extravesicular p40 domain of the transmembrane protein, p65 synaptotagmin (Syt) 1, is essential for the non-classical export of the signal peptide-less structure, FGF1, it was not possible to identify a specific intracellular protease responsible for the processing of p65 Syt1. Surprisingly, analysis of the p65 Syt1 coding sequence revealed the presence of two potential alternative ATG codons corresponding to Met103 and Met113 both of which were flanked by Kozak sequences. Indeed, in vitro translation of a Met103Ile but not a Met113Ile p65 Syt1 point mutant exhibited reduced expression of p40 Syt1 and the double p65 Syt1 Met103Ile and Met113Ile point mutant was unable to translate the p40 Syt1 isoform. Since the expression of the p65 Syt1 double point mutant inhibited the stress-induced release of FGF1, it is likely that the alternative translation of the p65 Syt1 transcript at Met103 may be involved in the generation of intracellular p40 Syt1, a critical component of the FGF1 release pathway.

Section snippets

Results and discussion

Because the conversion of p65 Syt1 to p40 Syt1 may involve the function of an intracellular protease and we initially characterized p40 Syt1 as an ovine brain heparin-binding protein, we investigated the possibility that Capn I may be able to proteolytically modify p65 Sytl to p40 Syt1 using cell-free methods. The p65 Syt1 transcript was transcribed in vitro using the T7 RNA polymerase, translated in a wheat germ extract, and treated with Capn I. Syt1 immunoblot analysis (Fig. 1A) revealed that

Materials and methods

Preparation of p65 Syt1, calpain I incubation assay, and immunoblot analysis. A plasmid containing the p65 form of rat Synaptotagmin I was cloned into the pcDNA3.1/myc-His(−)A vector (Invitrogen), the plasmid was transcribed in vitro using T7 RNA polymerase, and the p65 Syt1 transcript was translated in either a wheat germ or reticulocyte lysate according to instructions of the manufacturer (Promega). The Syt1 cleavage assay was performed by incubating 20% (v/v) of the total product of the

Acknowledgements

The authors thank J. Zhang (Indiana Univ. Sch. Of Med., Indianapolis, IN) for the pRF dicistronic vector, T. Sudhof (Southwestern Univ., Dallas, TX) for the p65 Syt1:EYFP chimera, P.A. Greer (Queen’s Univ., Kingston, Ont.) for the gift of the Capn I null cells, and M. Landriscina (Catholic Univ., Sch. of Med., Rome, Italy) and S. Bellum (Maine Med. Ctr. Res. Inst.) for advice and critical commentary. We also acknowledge the administrative assistance of N. Albrecht in the preparation of the

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Citation Excerpt :
The latter dependence makes Syt1 a calcium sensor which in its presence facilitates the docking of synaptic vesicles to the plasma membrane followed by fusion of lipid bilayers [37]. A 40-kDa form of Syt1, generated by alternative translation initiation and comprising the cytoplasmic part only was co-purified with FGF1 from conditioned media after a heat shock [38]. Despite the absence of a signal sequence, 40-kDa Syt1 and S100A13 are released constitutively by NIH3T3 cells as opposed to FGF1, whose secretion is triggered by cellular stress [25,39].
Fibroblast growth factors 1 and 2 (FGF1 and FGF2) are mainly considered as ligands of surface receptors through which they regulate a broad spectrum of biological processes. They are secreted in non-canonical way and, unlike other growth factors, they are able to translocate from the endosome to the cell interior. These unique features, as well as the role of the intracellular pool of FGF1 and FGF2, are far from being fully understood. An increasing number of reports address this problem, focusing on the intracellular interactions of FGF1 and 2. Here, we summarize the current state of knowledge of the FGF1 and FGF2 binding partners inside the cell and the possible role of these interactions. The partner proteins are grouped according to their function, including proteins involved in secretion, cell signaling, nucleocytoplasmic transport, binding and processing of nucleic acids, ATP binding, and cytoskeleton assembly. An in-depth analysis of the network of these binding partners could indicate novel, non-classical functions of FGF1 and FGF2 and uncover an additional level of a fine control of the well-known FGF-regulated cellular processes.
Cloning and expression of hepatic synaptotagmin 1 in mouse
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Mouse hepatic synaptotagmin 1 (SYT1) cDNA was cloned, characterized and compared to the brain one. The hepatic transcript was 1807 bp in length, smaller than the brain, and only encoded by 9 of 11 gene exons. In this regard, 5′-and 3′-untranslated regions were 66 and 476 bp, respectively; the open reading frame of 1266 bp codified for a protein of 421 amino acids, identical to the brain, with a predicted molecular mass of 47.4 kDa and highly conserved across different species. Immunoblotting of protein showed two isoforms of higher molecular masses than the theoretical prediction based on amino acid sequence suggesting posttranslational modifications. Subcellular distribution of protein isoforms corresponded to plasma membrane, lysosomes and microsomes and was identical between the brain and liver. Nonetheless, the highest molecular weight isoform was smaller in the liver, irrespective of subcellular location. Quantitative mRNA tissue distribution showed that it was widely expressed and that the highest values corresponded to the brain, followed by the liver, spleen, abdominal fat, intestine and skeletal muscle. These findings indicate tissue-specific splicing of the gene and posttranslational modification and the variation in expression in the different tissues might suggest a different requirement of SYT1 for the specific function in each organ.
Copper binding affinity of the C2B domain of synaptotagmin-1 and its potential role in the nonclassical secretion of acidic fibroblast growth factor
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This form represents a product of the alternative in-frame initiation of Syt1 mRNA translation [35]. Interestingly, unlike p65 Syt1, p40 Syt1 only contains the extravesicular portion and conspicuously lacks the intravesicular and transmembrane domains [13,35]. p40 Syt 1, but not p65 Syt1, is a constituent of the FGF1 MRC whose formation is critical for the release of FGF1 through the nonclassical pathway [35–37].
Fibroblast growth factor 1 (FGF1) is a heparin-binding proangiogenic protein. FGF1 lacks the conventional N-terminal signal peptide required for secretion through the endoplasmic reticulum (ER)–Golgi secretory pathway. FGF1 is released through a Cu²⁺-mediated nonclassical secretion pathway. The secretion of FGF1 involves the formation of a Cu²⁺-mediated multiprotein release complex (MRC) including FGF1, S100A13 (a calcium-binding protein) and p40 synaptotagmin (Syt1). It is believed that the binding of Cu²⁺ to the C2B domain is important for the release of FGF1 into the extracellular medium. In this study, using a variety of biophysical studies, Cu²⁺ and lipid interactions of the C2B domain of Syt1 were characterized. Isothermal titration calorimetry (ITC) experiments reveal that the C2B domain binds to Cu²⁺ in a biphasic manner involving an initial endothermic and a subsequent exothermic phase. Fluorescence energy transfer experiments using Tb³⁺ show that there are two Cu²⁺-binding pockets on the C2B domain, and one of these is also a Ca²⁺-binding site. Lipid-binding studies using ITC demonstrate that the C2B domain preferentially binds to small unilamellar vesicles of phosphatidyl serine (PS). Results of the differential scanning calorimetry and limited trypsin digestion experiments suggest that the C2B domain is marginally destabilized upon binding to PS vesicles. These results, for the first time, suggest that the main role of the C2B domain of Syt1 is to serve as an anchor for the FGF1 MRC on the membrane bilayer. In addition, the binding of the C2B domain to the lipid bilayer is shown to significantly decrease the binding affinity of the protein to Cu²⁺. The study provides valuable insights on the sequence of structural events that occur in the nonclassical secretion of FGF1.
NMR characterization of copper and lipid interactions of the C2B domain of synaptotagmin I-relevance to the non-classical secretion of the human acidic fibroblast growth factor (hFGF-1)
2010, Biochimica et Biophysica Acta - Biomembranes
Citation Excerpt :
Both C2A and C2B domains bind to calcium (Ca2+) and partially penetrate into the lipid bilayer, and the interactions between C2A, C2B and Ca2+ are believed to be critical for the membrane fusion activity of Syt-1. The 40-kDa form of synaptotagmin (p40 Syt1) represents a product of the alternative in-frame initiation of synaptotagmin mRNA translation [27–29]. It lacks the intravesicular and transmembrane domains and corresponds to the extravesicular domain of p65 Syt1.
Human fibroblast growth factor (hFGF-1) is a ∼ 17 kDa heparin binding cytokine. It lacks the conventional hydrophobic N-terminal signal sequence and is secreted through non-classical secretion routes. Under stress, hFGF-1 is released as a multiprotein complex consisting of hFGF-1, S100A13 (a calcium binding protein), and p40 synaptotagmin (Syt1). Copper (Cu²⁺) is shown to be required for the formation of the multiprotein hFGF-1 release complex (Landriscina et al. ,2001; Di Serio et al., 2008). Syt1, containing the lipid binding C2B domain, is believed to play an important role in the eventual export of the hFGF-1 across the lipid bilayer. In this study, we characterize Cu²⁺ and lipid interactions of the C2B domain of Syt1 using multidimensional NMR spectroscopy. The results highlight how Cu²⁺ appears to stabilize the protein bound to pS vesicles. Cu²⁺ and lipid binding interface mapped using 2D ¹H–¹⁵N heteronuclear single quantum coherence experiments reveal that residues in β-strand I contributes to the unique Cu²⁺ binding site in the C2B domain. In the absence of metal ions, residues located in Loop II and β-strand IV contribute to binding to unilamelar pS vesicles. In the presence of Cu²⁺, additional residues located in Loops I and III appear to stabilize the protein-lipid interactions. The results of this study provide valuable information towards understanding the molecular mechanism of the Cu²⁺-induced non-classical secretion of hFGF-1.
S100A13-C2A binary complex structure-a key component in the acidic fibroblast growth factor for the non-classical pathway
2009, Biochemical and Biophysical Research Communications
Citation Excerpt :
Our results confirm that the C-terminal end of S100A13 is involved in the C2A binding, which is the protein partner in the FGF-1 releasing complex. The S100A13-C2A complex acts as a template for multiprotein complex formation, which is the prerequisite for the FGF releasing [3,4,6]. The above results can be useful for understanding the molecular mechanism of the acidic fibroblast growth factor non-classical pathway and also provide clues on how to stop the formation of the multiprotein complex formation.
Fibroblast growth factors (FGFs) are key regulators of cell proliferation, differentiation, tumor-induced angiogenesis and migration. FGFs are essential for early embryonic development, organ formation and angiogenesis. They play important roles in tumor formation, inflammation, wound healing and restenosis. The biological effects of FGFs are mediated through the activation of the four transmembrane phosphotyrosine kinase receptors (FGFRs) in the presence of heparin sulfate proteoglycans (HSPGs) and therefore require the release of FGFs into the extracellular space. However, FGF-1 lacks the signal peptide required for the releasing of these proteins through the classical endoplasmic reticulum (ER)-Golgi secretary pathway. Maciag et al. demonstrated that FGF-1 is exported through a non-classical release pathway involving the formation of a specific multiprotein complex [M. Landriscina, R. Soldi, C. Bagala, I. Micucci, S. Bellum, F. Tarantini, I. Prudovsky, T. Maciag, S100A13 participates in the release of fibroblast growth factor 1 in response to heat shock in vitro, J. Biol. Chem. 276 (2001) 22544–22552; C.M. Carreira, T.M. LaVallee, F. Tarantini, A. Jackson, J.T. Lathrop, B. Hampton, W.H. Burgess, T. Maciag, S100A13 is involved in the regulation of fibroblast growth factor-1 and p40 synaptotagmin-1 release in vitro, J. Biol. Chem. 273 (1998) 22224–22231; T.M. LaValle, F. Tarantini, S. Gamble, C.M. Carreira, A. Jackson, T. Maciag, Synaptotagmin-1 is required for fibroblast growth factor-1 release, J. Biol. Chem. 273 (1998) 22217–22223; C. Bagalá, V. Kolev, A. Mandinova, R. Soldi, C. Mouta, I. Graziani, I, Prudovsky, T. Maciag, The alternative translation of synaptotagmin 1 mediates the non-classical release of FGF1, Biochem. Biophys. Res. Commun. 310 (2003) 1041–1047]. The protein constituents of this complex include FGF-1, S100A13 (a Ca²⁺-binding protein), and the p40 form of synaptotagmin 1 (Syt1). To understand the molecular events in the FGF-1 releasing pathway, we have studied the interactions of S100A13 with C2A by ¹H-¹⁵N HSQC titration and 3D-filtered NOESY experiments. We characterized the binary complex structure of S100A13-C2A by using a variety of multi-dimensional NMR experiments. This complex acts as a template for FGF-1 dimerization and multiprotein complex formation.
The cytoplasmic C2A domain of synaptotagmin shows sequence specific interaction with its own mRNA
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Synaptotagmin-1 (Syt1) is essential in Ca²⁺-dependent neurotransmitter release, but its expression regulation is unknown. Here we report that the cytoplasmic Syt1 fragment forms ribonucleoprotein complex by interacting with the 3′ untranslated region (3^′UTR) of its own mRNA. Two protein-binding domains, GU₁₅ repeat and GUCAAUG, within the Syt 3′UTR and the C2 domains in Syt1, especially C2A, are essential in this ribonucleoprotein complex formation. Furthermore, in in vitro assay the translation efficiency of Syt1 mRNA was downregulated in presence of 3′UTR. These results demonstrate for the fist time that the soluble fraction of Syt1 can interact with its own mRNA in a highly sequence specific manner.

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^☆: Abbreviations: ECMV, encephalomyocarditis virus; Capn, calpain; FGF, fibroblast growth factor; IL, interleukin; IRES, internal ribosome entry site; SDS–PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Syt, synaptotagmin.

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The alternative translation of synaptotagmin 1 mediates the non-classical release of FGF1☆

Abstract

Section snippets

Results and discussion

Materials and methods

Acknowledgements

J. Biol. Chem.

Cell

J. Biol. Chem.

J. Biol Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Cell

Biochim. Biophys. Acta

FEBS Lett.

Gene

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

Role of synaptotagmin in Ca2+-triggered exocytosis

Biochem. J.

Role of synaptotagmin in Ca²⁺-triggered exocytosis