LOCALIZATION BY SEGMENTAL DELETION ANALYSIS AND FUNCTIONAL-CHARACTERIZATION OF A 3RD ACTIN-BINDING SITE IN DOMAIN-5 OF SCINDERIN

Scinderin is a Ca2+-dependent actin filament severing protein present in a variety of secretory cells. Previous work suggests that scinderin -evoked cortical F-actin disassembly is required for secretion because local disassembly of cortical cytoskeleton allows secretory vesicle e xocytosis (Vitale, M. L., Rodriguez Del Castillo, A., Tchakarov, L., a nd Trifaro, J.-M. (1991) J. Cell Biol. 113, 1057-1067). Scinderin has six domains each containing three internal sequence motifs, two actin, and two phosphatidylinositol disphosphate-binding sites in domains 1 and 2. In this paper we report the presence of another actin-binding s ite at the NH2-terminal of domain 5 (Sc511-518). This site binds actin in a Ca2+-independent manner and a recombinant fragment (Sc5-6 or Sc5 02-715) containing this site binds to actin-DNase-I-Sepharose 4B beads , co-sediments with actin and is able to nucleate actin assembly. Reco mbinant Sc(L)5-6, a fusion protein devoid of the actin-binding site (S c519-715), did not exhibit these properties, Moreover, Sc-ABP(3), a pe ptide constructed with sequence (RLFQVRRNLASIT) identical to Sc511-523 blocked the binding of Sc5-6 to actin. Sc5-6 and Sc-ABP(3) also preve nted the actin severing activity of recombinant full-length scinderin (r-Sc) and inhibited the potentiation by r-Sc of Ca2+-evoked release o f serotonin from permeabilized platelets. On the other hand, Sc(L)5-6 failed to block the effect of r-Sc on platelet serotonin release. Sc1- 4,6, a construct devoid of domain 5, was able to sever but unable to n ucleate actin, indicating that an actin nucleation site of scinderin w as in domain 5. The results suggest that scinderin, in addition to bin ding actin on sites present in domains 1 and 2, must bind actin on a t hird site in domain 5 to sever and nucleate actin effectively.