Molecular determinants of synapsin targeting to presynaptic terminals.
Although synapsins are abundant synaptic vesicle proteins that are widely used as markers of presynaptic terminals, the mechanisms that target synapsins to presynaptic terminals have not been elucidated. We have addressed this question by imaging the targeting of green fluorescent protein-tagged synapsins in cultured hippocampal neurons. Whereas all synapsin isoforms targeted robustly to presynaptic terminals in wild-type neurons, synapsin Ib scarcely targeted in neurons in which all synapsins were knocked-out. Coexpression of other synapsin isoforms significantly strengthened the targeting of synapsin Ib in knock-out neurons, indicating that heterodimerization is required for synapsin Ib to target. Truncation mutagenesis revealed that synapsin Ia targets via distributed binding sites that include domains B, C, and E. Although domain A was not necessary for targeting, its presence enhanced targeting. Domain D inhibited targeting, but this inhibition was overcome by domain E. Thus, multiple intermolecular and intramolecular interactions are required for synapsins to target to presynaptic terminals.
Gitler
Daniel
D
Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Xu
Yimei
Y
Kao
Hung-Teh
HT
Lin
Dayu
D
Lim
Sangmi
S
Feng
Jian
J
Greengard
Paul
P
Augustine
George J
GJ
D000818:Animals; D002478:Cells, Cultured; D019281:Dimerization; D018390:Gene Targeting; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000378:metabolism*; D010766:Phosphorylation; D017729:Presynaptic Terminals / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016704:Synapsins / Q000235:genetics / Q000378:metabolism*; D013572:Synaptic Vesicles / Q000378:metabolism; D014162:Transfection
D008164:Luminescent Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D016704:Synapsins; D049452:Green Fluorescent Proteins