Information processing by the nervous system depends on neurotransmitter release from synaptic vesicles (SVs) at the presynaptic active zone. Molecular components of the cytomatrix at the active zone (CAZ) regulate the final stages of the SV cycle preceding exocytosis and thereby shape the efficacy and plasticity of synaptic transmission. Part of this regulation is reflected by a physical association of SVs with filamentous CAZ structures via largely unknown protein interactions. The very C-terminal region of Bruchpilot (Brp), a key component of the Drosophila melanogaster CAZ, participates in SV tethering. Here, we identify the conserved SNARE regulator Complexin (Cpx) in an in vivo screen for molecules that link the Brp C terminus to SVs. Brp and Cpx interact genetically and functionally. Both proteins promote SV recruitment to the Drosophila CAZ and counteract short-term synaptic depression. Analyzing SV tethering to active zone ribbons of cpx3 knockout mice supports an evolutionarily conserved role of Cpx upstream of SNARE complex assembly.
Protein insertion into membranes is a process occurring in every cell and every cellular compartment. Yet, many thermodynamic aspects of this fundamental biophysical process are not well understood. We investigated physicochemical parameters that influence protein insertion using the model protein KcsA, a 2-transmembrane ion channel. To understand what drives insertion and to identify individual steps of protein integration into a highly apolar environment, we investigated the contribution of electrostatic interactions and lipid composition on protein insertion on a single molecule level. We show that insertion of KcsA is spontaneous and directional as the cytosolic part of the protein does not translocate across the membrane barrier. Surprisingly, not hydrophobic residues but charged amino acids are crucial for the insertion of the unfolded protein into the membrane. Our results demonstrate the importance of electrostatic interactions between membrane and protein during the insertion process of hydrophobic polypeptides into the apolar membrane. On the basis of the observation that negatively charged lipids increase insertion events while high ionic strength in the surrounding aqueous phase decreases insertion events, a two-step mechanism is proposed. Here, an initial electrostatic attraction between membrane and protein represents the first step prior to insertion of hydrophobic residues into the hydrocarbon core of the membrane.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.