In this scenario, a presynaptic targeting signal might reside outside this region, between amino acids 1 and 52. generating an antibody against phosphorylated Mover and Western blot analysis of fractionated rat brain, we found that Mover is a phospho-protein. The localization of Mover to synaptic vesicles is phosphorylation dependent; treatment with a phosphatase caused Mover to dissociate from synaptic vesicles. A yeast-2-hybrid screen, co-immunoprecipitation and cell-based optical assays of homomerization revealed that Mover undergoes homophilic interaction, and regions within both the N- Acacetin and C- terminus of the protein are required for this interaction. Deleting a region required for homomeric interaction abolished presynaptic targeting of recombinant Mover in cultured neurons. Together, these data prove that Mover is associated with synaptic vesicles, and implicate phosphorylation and multimerization in targeting of Mover to synaptic vesicles and presynaptic sites. Introduction Neurotransmitter release at fast chemical synapses relies on sets of evolutionarily conserved proteins that mediate the regulated exocytosis, retrieval and re-use of transmitter containing synaptic vesicles (SVs). With remarkably few exceptions, the molecules mediating SV exocytosis at active zones are structurally and functionally conserved between vertebrates and invertebrates with nervous systems, such as Drosophila and SV protein. To determine if Mover exists in a phosphorylated form, we generated an antibody against Mover phosphorylated at threonin 13 (T13). We found that Mover is indeed phosphorylated at this site and virtually all T13-phosphorylated-Mover is on SVs. This is consistent with Mover T13 phosphorylation discovered in a screen for synaptosomal proteins that are phosphorylated during activity [14]. In a yeast 2-hybrid screen, co-immunoprecipitation experiments, and optical assays of homomerization, we found that Mover undergoes homophilic Rabbit polyclonal to alpha Actin interaction, consistent with another screen for self-interacting proteins, in which Mover was found [13]. Thus, we proved, using a number of different assays, that Mover is indeed a phosphoprotein associated with SVs and that Mover undergoes homomeric interaction. In addition, our data revealed four novel features of Mover. First, Mover is expressed as early as E14, well before synaptophysin, which has a steep onset of expression Acacetin at P0. Second, phosphatase treatment causes Mover to dissociate Acacetin from SVs. Third, Mover remains associated with SVs in response to depolarization. Fourth, a 39-amino acid region of Mover is required for both homomeric interaction and targeting to SVs. Our data reveal that Mover shares certain similarities with synapsin, one of the most abundant SV proteins: both are peripheral membrane proteins that are phosphorylated and undergo homomeric interaction. Dimerization of synapsins has been proposed to mediate SV clustering [32], raising the possibility that Mover may act in a similar manner. In addition to these similarities we found striking differences between these two proteins. First, synapsin dissociates from SVs upon depolarization [33]C[36]. Mover, on the other hand, does not dissociate from SVs in response to depolarization, and thus likely remains attached to SVs throughout their life cycle. Acacetin Second, phosphorylation causes synapsin to dissociate from SVs [37], but dephosphorylation causes Mover to dissociate from SVs. Our fractionation data (Figures 4 and ?and5)5) indicate that virtually all Mover is Acacetin associated with SVs, suggesting that Mover predominantly exists in the phosphorylated form. Thus, despite striking similarities, Mover appears to behave differently in response to depolarization and phosphorylation compared to synapsin. Mover dissociates from SVs upon phosphatase treatment, which caused dephosphorylation of Mover at T13, but should also lead to general dephosphorylation of SV proteins. This raises two possibilities for the mechanisms by which Mover is attached to SVs: 1) phosphorylation of Mover itself- at T13 or any of the other predicted phosphorylation sites- may be necessary for its association with SVs, or 2) phosphorylation of interacting partners of Mover may be necessary for its association with SVs. Although we cannot distinguish between these two possibilities at this point, the fact that SV bound Mover is phosphorylated at T13 is consistent with the former scenario. Future experiments using viral expression of a T13 phospho-deficient mutant in a Mover knockout background would allow a direct test of this notion. Working in a knockout background will be essential for future experiments because our yeast 2-hybrid, co-immunoprecipitation and Vero cell data reveal a strong tendency of Mover to form homomers. In a wild-type background loss of function mutants lacking SV targeting signals may piggyback on endogenous Mover and thereby be targeted to SVs. We have identified a region between amino acids 52.
In this scenario, a presynaptic targeting signal might reside outside this region, between amino acids 1 and 52
