Biology subjects

Brunger, A. T.

Publications and source records attributed to Brunger, A. T..

3 recordsLinked to original sources

bioRxiv2021

Molecular characterization of AMPA receptor trafficking vesicles

Regulated delivery of AMPA receptors (AMPARs) to the postsynaptic membrane is an essential step in synaptic strength modification, and in particular, long-term potentiation (LTP). While LTP has been extensively studied using electrophysiology and light microscopy, several questions regarding the molecular mechanisms of AMPAR delivery via trafficking vesicles remain outstanding, including the gross molecular make up of AMPAR trafficking organelles and identification and location of calcium sensors required for SNARE complex-dependent membrane fusion of such trafficking vesicles with the plasma membrane. Here, we isolated AMPAR trafficking vesicles (ATVs) from whole mouse brains via immunoprecipitation and characterized them using immunoelectron microscopy, immunoblotting, and liquid chromatography tandem mass spectrometry (LC-MS/MS). We identified several proteins on ATVs that were previously found to play a role in AMPAR trafficking, including SNARES (including synaptobrevin 2), Rabs, the SM protein Munc18-1, a calcium-sensor (synaptotagmin-1), as well as several new markers, including synaptophysin and synaptogyrin on ATV membranes. Additionally, we identified two populations of ATVs based on size and molecular composition: small-diameter, synaptobrevin-2- and GluA1-containing ATVs and larger transferrin-receptor-, GluA1-, GluA2-, GluA3-containing ATVs. The smaller population of ATVs likely represents a trafficking vesicle whose fusion is essential for LTP. These findings reveal the important role of AMPAR sorting into fusion-competent trafficking vesicles that are implicated in synaptic strength modification and reveal candidates of putative effectors and regulators of AMPAR trafficking.

neuroscience↗

bioRxiv2021

Sec17/Sec18 can support membrane fusion without help from completion of SNARE zippering

Membrane fusion requires R-, Qa-, Qb-, and Qc-family SNAREs that zipper into RQaQbQc coiled coils, driven by the sequestration of apolar amino acids. Zippering has been thought to provide all the force driving fusion. Sec17/SNAP can form an oligomeric assembly with SNAREs with the Sec17 C-terminus bound to Sec18/NSF, the central region bound to SNAREs, and a crucial apolar loop near the N-terminus poised to insert into membranes. Though Sec17 aids zippering, we now report that Sec17 and Sec18 will drive robust fusion without requiring zippering completion. Zippering-driven fusion is blocked by deleting the C-terminal quarter of any Q-SNARE domain or by replacing the apolar amino acids of the Qa-SNARE which face the center of the 4-SNARE coiled coils with polar residues. These blocks to fusion, singly or combined, are bypassed by Sec17 and Sec18, and SNARE-dependent fusion is restored without help from completing zippering.

biochemistry↗

bioRxiv2020

Conformational dynamics of auto-inhibition in the ER calcium sensor STIM1

The dimeric ER Ca2+ sensor STIM1 controls store-operated Ca2+ entry (SOCE) through the regulated binding of its CRAC activation domain (CAD) to Orai channels in the plasma membrane. In resting cells, the STIM1 CC1 domain interacts with CAD to suppress SOCE, but the structural basis of this interaction is unclear. Using single-molecule Forster resonance energy transfer (smFRET) and protein crosslinking approaches, we show that CC1 interacts dynamically with CAD in a domain-swapped configuration with an orientation predicted to sequester its Orai- binding region adjacent to the ER membrane. Following ER Ca2+ depletion and release from CAD, cysteine crosslinking indicates that the two CC1 domains become closely paired along their entire length in the active Orai-bound state. These findings provide a structural basis for the dual roles of CC1: sequestering CAD to suppress SOCE in resting cells and propelling it towards the plasma membrane to activate Orai and SOCE after store depletion.

biophysics↗