An Abundance of Free Proteasomal Regulatory (19S) Particles Regulate Neuronal Synapses Independent of the Proteasome

The major protein-degradation machine, the proteasome, functions at brain synapses and regulates long-term information storage. Here we found that the two essential subcomplexes of the proteasome, the regulatory (19S) and catalytic (20S) particles that assemble to recognize and degrade substrates, were differentially distributed within individual rat cortical neurons. By detecting and quantifying 19 and 20S particles at single-molecule resolution, we discovered a surprising abundance of free regulatory particles (19S) near synapses. Furthermore, we found that the free neuronal 19S binds and deubiquitylates Lys63-ubiquitin, a distinct ubiquitin linkage that does not target substrates to the proteasome. Pull-down assays for Lys63 or Lys48 ubiquitin revealed a significant over-representation of synaptic molecules as Lys63 interactors, while conventional proteasome-related proteins dominated the Lys48 interactome. Inhibition of 19S deubiquitylase (DUB) activity significantly altered spontaneous 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.