Abstract
Synaptojanin is an Src homology 3 domain-binding inositol 5-phosphatase that is thought to function in synaptic vesicle endocytosis. It is encoded by a cDNA with two open reading frames separated by an in-frame stop codon. The first open reading frame encodes a 145-kDa form of the protein, whereas a 170-kDa isoform appears to be composed of both open reading frames and contains additional Src homology 3 domain-binding consensus sequences. Here, we demonstrate that the two synaptojanin isoforms are generated by the alternative use of an exon containing the stop codon. Whereas the 145-kDa isoform is highly enriched in adult brain, the 170-kDa isoform is excluded from this tissue and has a widespread distribution in non-neuronal cells. Unlike the 145-kDa isoform, which can be removed from membranes by a low salt wash, the 170-kDa isoform remains membrane-associated, even in the presence of 1 salt. Further, the 170-kDa form, but not the 145-kDa form, can be isolated from membranes as part of a large molecular weight complex. These properties may allow the 170-kDa isoform of synaptojanin to play a unique and perhaps more general role in endocytosis as compared with the 145-kDa isoform.
Highlights
Synaptic vesicles are highly specialized organelles that neurons use to secrete non-peptide neurotransmitters at the synapse
Has a similar subcellular distribution as dynamin including both a soluble and particulate pool, and undergoes dephosphorylation in parallel with dynamin following nerve terminal depolarization [8]. Dynamin and synaptojanin both interact with the Src homology 3 (SH3) domains of amphiphysin [9, 10], a nerve terminal protein that is implicated in synaptic vesicle endocytosis (10 –12)
Synaptojanin Isoform Expression in Developing Brain—In order to examine the developmental expression of the 170-kDa synaptojanin isoform, we enriched for SH3 domain-binding proteins from extracts made from rat E12 heads, as well as E16, E18, and adult brains by Grb2 affinity chromatography and blotted with an affinity purified polyclonal antibody that recognizes both synaptojanin isoforms [8]
Summary
SH3 Domain Purification of Synaptojanin Isoforms—Various tissues were homogenized at 1:5 (w/v) in buffer A (10 mM HEPES-OH, pH 7.4, 1 mM EGTA, 0.32 M sucrose, 0.83 mM benzamidine, 0.23 mM phenylmethylsulfonyl fluoride) using a polytron or a glass Teflon homogenizer. Rat genomic DNA (generous gift of Dr Phil Barker, Montreal Neurological Institute), prepared as described [28] was used in PCR reactions with a 60 °C annealing temperature with non-degenerate forward and reverse primers, corresponding to nucleotides 4003– 4023 and 4154 – 4173, respectively, of clone-9 [9]. Sucrose Density Gradient Centrifugation—Proteins of P2 fractions (approximately 10 mg) from E18 brains or PC-12 cells, prepared as described above, were solubilized by resuspension in buffer B (10 mM HEPES-OH, pH 7.4, 1% Triton X-100, 0.83 mM benzamidine, 0.23 mM phenylmethylsulfonyl fluoride) followed by incubation for 30 min at 4 °C. S2 fractions (2 ml) from PC-12 cells and E18 brains, prepared as described above, were diluted 1:1 in 10 mM HEPES-OH, pH 7.4, 2% Triton X-100 and were loaded on 5–20% linear sucrose gradients prepared in buffer B. Antibodies—Affinity purified antibodies against the 145-kDa synaptojanin isoform, the 170-kDa synaptojanin isoform, and dynamin were previously described (8 –10)
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