V-ATPase V0 Sector Subunit a1 in Neurons Is a Target of Calmodulin

Wei Zhang,Dong Wang,Elzi Volk,Hugo J Bellen,Peter Robin Hiesinger,Florante A Quiocho

doi:10.1074/jbc.m708058200

Abstract

The V(0) complex forms the proteolipid pore of a vesicular ATPase that acidifies vesicles. In addition, an independent function in membrane fusion has been suggested in vacuolar fusion in yeast and synaptic vesicle exocytosis in fly neurons. Evidence for a direct role in secretion has also recently been presented in mouse and worm. The molecular mechanisms of how the V(0) components might act or are regulated are largely unknown. Here we report the identification and characterization of a calmodulin-binding site in the large cytosolic N-terminal region of the Drosophila protein V100, the neuron-specific V(0) subunit a1. V100 forms a tight complex with calmodulin in a Ca(2+)-dependent manner. Mutations in the calmodulin-binding site in Drosophila lead to a loss of calmodulin recruitment to synapses. Neuronal expression of a calmodulin-binding deficient V100 uncovers an incomplete rescue at low levels and cellular toxicity at high levels. Our results suggest a vesicular ATPase V(0)-dependent function of calmodulin at synapses.

Highlights

Calmodulin (CaM)4 is a small, ubiquitous Ca2ϩ-binding protein that has been implicated in the Ca2ϩ-dependent regulation of a plethora of cell biological processes
A search for the presence of a CaM-binding domain (CBD) in the primary sequences of all V0 sector protein components of the Drosophila neuron V-ATPase hinted at the large N-terminal cytosolic region (V100-N) of subunit a1, suggesting that subunit a1 might be a direct target of CaM regulation
In the presence of EDTA (Fig. 1B), the peak fraction 1 is ascribed to V100-N because it matches exactly that of V100-N alone (Fig. 1A)

Summary

Introduction

Calmodulin (CaM)4 is a small, ubiquitous Ca2ϩ-binding protein that has been implicated in the Ca2ϩ-dependent regulation of a plethora of cell biological processes. We report the identification and characterization of a calmodulin-binding site in the large cytosolic N-terminal region of the Drosophila protein V100, the neuron-specific V0 subunit a1. V100 Contains a Specific Site for Tight Binding of Ca2ϩ1⁄7 CaM—A critical regulatory role of CaM in V0-dependent membrane fusion has been shown only in yeast vacuolar fusion [18, 19].

Results

Conclusion