Abstract
Endophilin 1 is proposed to participate in synaptic vesicle biogenesis through SH3 domain-mediated interactions with the polyphosphoinositide phosphatase synaptojanin and the GTPase dynamin. Endophilin family members have also been identified as binding partners for a number of diverse cellular proteins. We define here the endophilin 1-binding site within synaptojanin 1 and show that this sequence independently and selectively purifies from brain extracts endophilin 1 and a closely related protein, endophilin 2. Endophilin 2, like endophilin 1, is highly expressed in brain, concentrated in nerve terminals, and found in complexes with synaptojanin and dynamin. Although a fraction of endophilins 1 and 2 coexist in the same complex, the distribution of these endophilin isoforms among central synapses only partially overlaps. Endophilins 1 and 2 are found predominantly as stable dimers through a predicted coiled-coil domain in their conserved NH2-terminal moiety. Dimerization may allow endophilins to link a number of different cellular targets to the endocytic machinery.
Highlights
Neurotransmitter release requires the fusion of small neurotransmitter-containing vesicles with the presynaptic plasma membrane
Nerve terminals, which are spatially segregated from the biosynthetic machinery in the neuronal cell body, must locally recycle synaptic vesicle membrane components to maintain a functional pool of secretory vesicles [1, 2]
Many protein factors that participate in clathrin-mediated synaptic vesicle recycling have been identified as direct or indirect interactors of either components of the clathrin coat or the GTPase dynamin, a protein required for the maturation and scission of nascent coated pits
Summary
Antibodies—Affinity purified antibodies against endophilin 1, endophilin 2, and anti-synaptojanin 1 monoclonal antibodies have been described previously (19 –21). 5 g of affinity purified antibodies were incubated for 1 h at 4 °C in 500 g of either total brain cytosol (see below), Triton X-100-extracted total brain homogenate, or cell lysate. 50 g of fusion protein immobilized on glutathione-Sepharose (Amersham Pharmacia Biotech) was added to 5 mg of Triton X-100 extracted total brain homogenate and incubated at 4 °C for 3 h. Cross-linking of Detergent Extracts of Brain Homogenate and Recombinant Endophilins—Full-length endophilin 1 and endophilin 2 cDNAs or various deletion mutants were subcloned into the pGex6p1 expression vector (Amersham Pharmacia Biotech), expressed in the bacterial strain BL21 and purified on glutathione-Sepharose according to the manufacturer’s instructions. Gel filtration chromatographies of total brain cytosol and recombinant proteins were performed on a Sephadex G100 column and a Superdex 200 column equilibrated with buffer A, respectively. Endophilin Dimers in Nerve Terminals dase-conjugated goat anti-rabbit or goat anti-mouse IgGs (Jackson) and an ECL detection system (Pierce)
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