THE EVOLUTIONARY PRESSURE TO INACTIVATE - A SUBCLASS OF SYNAPTOTAGMINS WITH AN AMINO-ACID SUBSTITUTION THAT ABOLISHES CA2+ BINDING

Synaptotagmin I is a Ca2+-binding protein of synaptic vesicles that se rves as a Ca2+ sensor for neurotransmitter release and was the first m ember found of a large family of trafficking proteins. We have now ide ntified a novel synaptotagmin, synaptotagmin XI, that is highly expres sed in brain and at lower levels in other tissues. Like other synaptot agmins, synaptotagmin XI has a single transmembrane region and two cyt oplasmic C-2-domains but is most closely related to synaptotagmin TV w ith which it forms a new subclass of synaptotagmins. The first C-2-dom ain of synaptotagmin I (the C(2)A-domain) binds phospholipids as a fun ction of Ca2+ and contains a Ca2+-binding site, the C-2-motif, that bi nds at least two Ca2+ ions via five aspartate residues and is conserve d in most C-2-domains (Shao, X., Davletov, B., Sutton, B., Sudhof, T. C., Rizo, J. R. (1996) Science 273, 248-253). In the C(2)A-domains of synaptotagmins IV and XI, however, one of the five Ca2+-binding aspart ates in the C-2-motif is substituted for a serine, suggesting that the se C-2-domains do not bind Ca2+. To test this, we produced recombinant C(2)A-domains from synaptotagmins IV and XI with either wild type ser ine or mutant aspartate in the C-2-motif. Circular dichroism showed th at Ca2+ stabilizes both mutant but not wild type C-2-domains against t emperature-induced denaturation, indicating that the mutations restore Ca2+-binding to the wild type C-2-domains. Furthermore, wild type C(2 )A-domains of synaptotagmins IV and XI exhibited no Ca2+-dependent pho spholipid binding, whereas mutant C(2)A-domains bound phospholipids as a function of Ca2+ similarly to wild type synaptotagmin I. These expe riments suggest that a class of synaptotagmins was selected during evo lution in which the Ca2+-binding site of the C(2)A-domain was inactiva ted by a single point mutation. Thus, synaptotagmins must have Ca2+-in dependent functions as well as Ca2+-dependent functions that are selec tively maintained in distinct members of this gene family.