An abundance of free regulatory (19 S ) proteasome particles regulates neuronal synapses

Abstract

The proteasome, the major protein-degradation machine in cells, regulates neuronal synapses and long-term information storage. Here, using super-resolution microscopy, we found that the two essential subcomplexes of the proteasome, the regulatory (19 S ) and catalytic (20 S ) particles, are differentially distributed within individual rat cortical neurons. We discovered an unexpected abundance of free 19 S particles near synapses. The free neuronal 19 S particles bind and deubiquitylate lysine 63–ubiquitin (Lys 63 -ub), a non–proteasome-targeting ubiquitin linkage. Pull-down assays revealed a significant overrepresentation of synaptic molecules as Lys 63 -ub interactors. Inhibition of the 19 S deubiquitylase activity significantly altered excitatory synaptic transmission and reduced the synaptic availability of AMPA receptors at multiple trafficking points in a proteasome-independent manner. Together, these results reveal a moonlighting function of the regulatory proteasomal subcomplex near synapses.

Single-Molecule-Resolution Approaches in Synaptic Biology

“…However, the free 19S we observed targets a type of ubiquitin signal unrecognized by the proteasome: ubiquitin chains linked at the Lysine 63 residue. 39 This clearly distinguishes the synaptic function of free 19S particles from the local proteasomes. Furthermore, synaptic proteins were identified as prominent interactors of this atypical ubiquitin signal and potential substrates for free neuronal 19S particles.…”

“…Local protein degradation is found to be an important mechanism for modifying synaptic composition. − For example, the major protein complex for cytosolic protein degradation, the proteasome, possesses two essential subcomplexes: (i) the regulatory (19S) particles that recognize substrates and (ii) the catalytic (20S) particles that degrade substrates . Using single-molecule localization microscopy ( e.g ., DNA-PAINT), we recently observed that these two essential subcomplexes are not present in equal amounts near synapses . Instead, there is a surprising abundance of free regulatory (19S) particles near synapses.…”

“…A recent study further quantified the copy numbers of AMPA receptors at individual neuronal synapses . Emerging studies using various types of super-resolution microscopy have begun to move beyond neurotransmitter receptors to elucidate how cell biological events are organized spatially at synapses. ,,, …”

“…38 Using single-molecule localization microscopy (e.g., DNA-PAINT), we recently observed that these two essential subcomplexes are not present in equal amounts near synapses. 39 Instead, there is a surprising abundance of free regulatory (19S) particles near synapses. Why do synapses possess "solitary" 19S proteasome particles in their neighborhoods?…”

“…Secondary antibodies conjugated to quantum dots are commercially available and have been used to tag primary antibodies that target, e.g., the extracellular epitope of AMPA receptors. 39 Via single-particle tracking, the mobile and immobilized fractions of surface AMPA receptors have been measured, lending insights into activity-dependent regulation of AMPA receptor dynamics at synapses as a critical mechanism for memory and learning. 64,70 ■ CONCLUSION As perhaps the most complex structure known to us, the brain harnesses its computational power from over 100 trillion small subcellular compartments, the synapses.…”