title: The evolutionary pressure to inactivate. A subclass of synaptotagmins with an amino acid substitution that abolishes Ca2+ binding.
abstract: Synaptotagmin I is a Ca2+-binding protein of synaptic vesicles that serves as a Ca2+ sensor for neurotransmitter release and was the first member found of a large family of trafficking proteins. We have now identified a novel synaptotagmin, synaptotagmin XI, that is highly expressed in brain and at lower levels in other tissues. Like other synaptotagmins, synaptotagmin XI has a single transmembrane region and two cytoplasmic C2-domains but is most closely related to synaptotagmin IV with which it forms a new subclass of synaptotagmins. The first C2-domain of synaptotagmin I (the C2A-domain) binds phospholipids as a function of Ca2+ and contains a Ca2+-binding site, the C2-motif, that binds at least two Ca2+ ions via five aspartate residues and is conserved in most C2-domains (Shao, X., Davletov, B., Sutton, B., Südhof, T. C., Rizo, J. R. (1996) Science 273, 248-253). In the C2A-domains of synaptotagmins IV and XI, however, one of the five Ca2+-binding aspartates in the C2-motif is substituted for a serine, suggesting that these C2-domains do not bind Ca2+. To test this, we produced recombinant C2A-domains from synaptotagmins IV and XI with either wild type serine or mutant aspartate in the C2-motif. Circular dichroism showed that Ca2+ stabilizes both mutant but not wild type C2-domains against temperature-induced denaturation, indicating that the mutations restore Ca2+-binding to the wild type C2-domains. Furthermore, wild type C2A-domains of synaptotagmins IV and XI exhibited no Ca2+-dependent phospholipid binding, whereas mutant C2A-domains bound phospholipids as a function of Ca2+ similarly to wild type synaptotagmin I. These experiments suggest that a class of synaptotagmins was selected during evolution in which the Ca2+-binding site of the C2A-domain was inactivated by a single point mutation. Thus, synaptotagmins must have Ca2+-independent functions as well as Ca2+-dependent functions that are selectively maintained in distinct members of this gene family.
authors.affiliation: Department of Molecular Genetics, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235, USA.
authors.forename: C
authors.initials: C
authors.lastname: von Poser
authors.forename: K
authors.initials: K
authors.lastname: Ichtchenko
authors.forename: X
authors.initials: X
authors.lastname: Shao
authors.forename: J
authors.initials: J
authors.lastname: Rizo
authors.forename: T C
authors.initials: TC
authors.lastname: Südhof
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics*; D016415:Sequence Alignment; D017421:Sequence Analysis; D050863:Synaptotagmin I; D050857:Synaptotagmins
chemical_list: D000596:Amino Acids; D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; C494310:SYT1 protein, human; C494265:SYT11 protein, human; C494292:SYT4 protein, human; D050863:Synaptotagmin I; C494266:Syt11 protein, mouse; C494267:Syt11 protein, rat; D050857:Synaptotagmins