Sorting Nexin 9 Interacts with Dynamin 1 and N-WASP and Coordinates Synaptic Vesicle Endocytosis

Sang-Gun Ahn,Narae Shin,Soo-A Kim,Sunghoe Chang,Suho Lee,Namhui Ahn,Zee YongPark

doi:10.1074/jbc.m700283200

Abstract

Sorting nexin 9 (SNX9) is a member of the sorting nexin family of proteins, each of which contains a characteristic Phox homology domain. SNX9 is widely expressed and plays a role in clathrin-mediated endocytosis, but it is not known if it is present in neuronal cells. We report that SNX9 is expressed in the presynaptic compartment of cultured hippocampal neurons, where it binds to dynamin-1 and N-WASP. Overexpression of full-length SNX9 or a C-terminal truncated version caused severe defects in synaptic vesicle endocytosis during, as well as after, stimulation. Knockdown of SNX9 with short interfering RNA also reduced synaptic vesicle endocytosis, and the W39A mutation of SNX9 abolished the inhibitory effect of SNX9 on endocytosis. Rescue experiments showed that most of the effect of SNX9 on endocytosis results from its interaction with dynamin 1, although its interaction with N-WASP contributes in some degree. We further showed that SNX9 dimerizes through its C-terminal domain, suggesting that it may interact simultaneously with dynamin 1 and N-WASP. We propose that SNX9 interacts with dynamin-1 and N-WASP in presynaptic terminals, where it links actin dynamics and synaptic vesicle endocytosis.

Highlights

We report that Sorting nexin 9 (SNX9) is expressed in the presynaptic compartment of cultured hippocampal neurons where it associates with dynamin-1 as well as N-WASP, and that it plays a regulatory role in synaptic vesicle endocytosis
We used three different culture conditions to test the expression of SNX9 as follows: a pure glial cell culture obtained by 2 h of treatment with 200 ␮M of glutamate to remove neurons; a serum-free Neurobasal culture completely depleted of glial cells with Ara-C, and a Neurobasal culture without Ara-C treatment, containing mostly neurons and only a few glial cells
D and E, GST fusions of SNX9, SNX9-⌬SH3, SNX9-SH3, or GST alone were incubated with rat brain lysates. 10% of each lysate was used for the pulldown, and the complexes were resolved by SDS-PAGE, and immunoblotted with anti-dynamin-1 and anti-N-WASP antibodies

Summary

Introduction

The presence of SNX9 in neuronal cells and the functional interplay between it and its presynaptic binding partners in synaptic vesicle endocytosis have never been studied. We report that SNX9 is expressed in the presynaptic compartment of cultured hippocampal neurons where it associates with dynamin-1 as well as N-WASP, and that it plays a regulatory role in synaptic vesicle endocytosis.

Results

Conclusion