Abstract
Bassoon is a core scaffold protein of the presynaptic active zone. In brain synapses, the C-terminus of Bassoon is oriented toward the plasma membrane and its N-terminus is oriented toward synaptic vesicles. At the Golgi-apparatus, Bassoon is thought to assemble active zone precursor structures, but whether it is arranged in an orderly fashion is unknown. Understanding the topology of this large scaffold protein is important for models of active zone biogenesis. Using stimulated emission depletion nanoscopy in cultured hippocampal neurons, we found that an N-terminal intramolecular tag of recombinant Bassoon, but not C-terminal tag, colocalized with markers of the trans-Golgi network (TGN). The N-terminus of Bassoon was located between 48 and 69 nm away from TGN38, while its C-terminus was located between 100 and 115 nm away from TGN38. Sequences within the first 95 amino acids of Bassoon were required for this arrangement. Our results indicate that, at the Golgi-apparatus, Bassoon is oriented with its N-terminus toward and its C-terminus away from the trans Golgi network membrane. Moreover, they suggest that Bassoon is an extended molecule at the trans Golgi network with the distance between amino acids 97 and 3,938, estimated to be between 46 and 52 nm. Our data are consistent with a model, in which the N-terminus of Bassoon binds to the membranes of the trans-Golgi network, while the C-terminus associates with active zone components, thus reflecting the topographic arrangement characteristic of synapses also at the Golgi-apparatus.
Highlights
Scaffold proteins recruit and anchor molecules to subcellular sites
In the course of these studies, we found that Bassoon, in addition to being a CAZ protein, is associated with the Golgi-apparatus, and that associating with the Golgi-apparatus is a prerequisite for the subsequent trafficking of Bassoon to synapses (Dresbach et al, 2006)
We realized serendipitously that this diffusely distributed green fluorescence occurred when the EGFP-coding sequence was attached out of frame to the 3 end of Bassoon, suggesting that a cryptic ribosomal entry site exists somewhere near the 3 -region of Bassoon or in the linker located between Bassoon and EGFP
Summary
Scaffold proteins recruit and anchor molecules to subcellular sites. Due to their multi-domain and modular structure, they bind and regulate multiple proteins to coordinate biochemical reactions in space and time. One way by which the presynaptic machinery acts is through RIMs, which recruit both voltagegated calcium channels and Munc13s, a family of proteins essential for making synaptic vesicles tethered at the active zone fusion competent (Südhof, 2012; Imig et al, 2014; Acuna et al, 2016). Bassoon regulates this core transmitter release machinery, at least at some synapses, by selectively recruiting the P/Q type of voltage-gated calcium channels and by speeding up synaptic vesicle reloading to release sites during ongoing activity (Hallermann et al, 2010; Davydova et al, 2014; Mendoza Schulz et al, 2014). At the electron microscopy level, the multimolecular complex of presynaptic scaffold proteins manifests as a meshwork of filamentous structures termed the presynaptic particle web (Phillips et al, 2001) or cytomatrix of active zones, i.e., CAZ (Cases-Langhoff et al, 1996; Garner et al, 2000; Dresbach et al, 2001)
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