The novel human platelet septin SEPT8 is an interaction partner of SEPT4

Summary

Septins are a family of GTP-binding proteins, which are essential for active membrane movement such as cytokinesis and vesicle trafficking. In non-dividing cells (such as platelets and neurons) septins are implicated in exocytosis. Platelets from a SEPT5 knockout mouse showed an altered serotonin secretion and platelet aggregation, suggesting that SEPT5 is involved in secretion in platelets. Septins form complexes consisting of multiple septin polypeptides. Using the yeast two-hybrid system we had demonstrated that SEPT5 partners with SEPT8.The aim of this study was to identify other interaction partners of the human platelet septin SEPT8. Using the yeast two-hybrid system with SEPT8 as bait protein we identified the human septin SEPT4 as an interaction partner of SEPT8. The interaction between SEPT4 and SEPT8 was confirmed by immunoprecipitation. Expression analysis revealed that SEPT4 is also expressed in human platelets. Thus, SEPT4 is the third described platelet septin besides SEPT5 and SEPT8.Transmission electron microscopy of platelets revealed that SEPT8 and SEPT4 are localized surrounding α-granules (as it had been shown for the septin SEPT5) suggesting that the three septins may be components of the septin complex in platelets and contribute in such a way to platelet biology. Activation of platelets by agonists resulted in the translocation of SEPT4 and SEPT8 to the platelet surface indicating a possible functional role of these proteins in platelet granular secretion.

Keywords

Septins - SEPT4 - SEPT8 - Platelets - GTP-binding proteins

• References

• 1 Kinoshita M, Noda M. Roles of septins in the mammalian cytokinesis machinery. Cell Struct Funct 2001; 26: 667-70.
• 2 Faty M, Fink M, Barral Y. Septins: a ring to part mother and daughter. Curr Genet 2002; 41: 123-31.
• 3 Macara IG, Baldarelli R, Field CF. et al. Mammalian septins nomenclature. Mol Biol Cell 2002; 13: 4111-3.
• 4 Zieger B, Tran H, Hainmann I. et al. Characterization and expression analyses of two human septin genes, PNUTL1 and PNUTL2. Gene 2000; 261: 197-203.
• 5 Bläser S, Jersch K, Hainmann I. et al. Human septin-septin interaction: CDCrel-1 partners with KIAA0202. FEBS Lett 2002; 519: 169-72.
• 6 Zieger B, Hashimoto Y, Ware J. Alternative expression of platelet glyocoprotein Ibβ mRNA from an adjacent 5´gene with an imperfect polyadenylation signal sequence. J Clin Invest 1997; 99: 520-5.
• 7 Kartmann B, Roth D. Novel roles for mammalian septins: from vesicle trafficking to oncogenesis. J Cell Sci 2001; 114: 839-44.
• 8 Hsu SC, Hazuka CD, Roth R. et al. Subunit composition, protein interactions, and structures of the mammalian brain sec6/8 complex and septin filaments. Neuron 1998; 20: 1111-22.
• 9 Dent J, Kato K, Peng X-R. et al. A prototypic platelet septin and its participation in secretion. Proc Nat Acad Sci 2002; 99: 3064-9.
• 10 Zhang Y, Gao J, Chung KK. et al. Parkin functions as an E2-dependent ubiquitin-protein ligase and promotes the degradation of the synaptic vesicle-associated protein, CDCrel-1. Proc Natl Acad Sci U S A 97: 13354-9.
• 11 Beites CL, Xie H, Bowser R. et al. The septin CDCrel-1 binds syntaxin and inhibits exocytosis. Nat. Neurosci 1999; 02: 434-9.
• 12 Bläser S, Jersch K, Hainmann I. et al. Isolation of new isoforms, characterization and expression analysis of the human septin SEPT8 (KIAA0202). Gene 2003; 312: 313-20.
• 13 Bartel PL, Chien C-T, Sternglanz R, Fields S. In: Cellular Interactions and Development: A Practical Approach. Hartley DA. 1993. Oxford University Press; Oxford.:
• 14 Lehrach H, Diamond D, Wozney JM, Boedtker H. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry 1977; 16: 4743-51.
• 15 Thomas PS. Hybridization of denatured RNA transferred or dotted to nitrocellulose paper. Methods Enzymol 1983; 100: 255-66.
• 16 Studier FW. Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J Mol Biol 1991; 219: 37-44.
• 17 Harboe LH, Inghild A. Immunization, isolation of immunoglobulins, estimation of antibody titre. Scand J Immunol 1973; 02 (Suppl. 01) 161-4.
• 18 Nurden P, Poujol C, Winckler J. et al. Immunolocalization of P2Y1 and TPα receptors in platelets showed a major pool associated with the membranes of α-granules and the open canalicular system. Blood 2003; 101: 1400-8.
• 19 Field CM, Kellogg D. Septins: cytoskeletal polymers or signalling GTPases?. Trends Cell Biol 1999; 09 (10) 387-94.
• 20 Sheffield PJ, Oliver CJ, Kremer BE. et al. Borg/septin interactions and the assembly of mammalian septin heterodimers, trimers, and filaments. J Biol Chem 2003; 278: 3483-8.
• 21 Trimble WS. Septins: a highly conserved family of membrane-associated GTPases with functions in cell division and beyond. J Membr Biol 1999; 169 (02) 75-81.
• 22 Kinoshita M, Kumar S, Mizoguchi A. et al. Nedd5, a mammalian septin, is a novel cytoskeletal component interacting with actinbased structures. Genes Dev 1997; 11 (12) 1535-47.
• 23 Flaumenhaft R, Croce K, Chen E. et al. Proteins of the exocytotic core complex mediate platelet alpha-granule secretion Roles of vesicle-associated membrane protein, SNAP-23, and syntaxin 4. J Biol Chem 1999; 274 (04) 2492-501.
• 24 Reed GL, Fitzgerald ML, Polgar J. Molecular mechanisms of platelet exocytosis: insights into the “secrete” life of thrombocytes. Blood 2000; 96 (10) 3334-42.