Addition and removal of ubiquitin regulates the abundance of glutamate receptors at synapses. We previously showed that the conserved deubiquitinating enzyme (DUB) Ubiquitin Specific Protease-46 (USP-46) regulates the abundance of the glutamate receptor GLR-1 in C. elegans (1).
usp-46 loss-of-function mutants have decreased levels of GLR-1 in the ventral nerve cord (VNC) and corresponding defects in glutamatergic behavior. We showed that USP-46 deubiquitinates GLR-1 and protects it from lysosomal degradation. While an increasing number of neuronal DUB substrates are beginning to be defined, the mechanisms that regulate DUBs are poorly understood. USP-46 function appears to be tightly controlled in vivo because overexpression of
usp-46 by itself does not increase GLR-1 abundance. We, and others, showed that two WD40-repeat proteins, WDR-48 and WDR-20, can interact with USP-46 and stimulate its catalytic activity (2-4). Furthermore, we found that overexpression of WDR-48, WDR-20 and USP-46 in neurons results in increased levels of GLR-1 and glutamatergic behavior (4). Here we show that USP-46, WDR-48, and WDR-20 act together and are required to regulate surface levels of GLR-1 in the VNC based on analysis of GLR-1 tagged with pH-sensitive superecliptic pHluorin (5). We found that WDR-48 promotes USP-46 protein levels in HEK293 cells and in GLR-1-expressing interneurons. We show that USP-46 is ubiquitinated and degraded in the proteasome and that WDR-48 promotes USP-46 stability by preventing USP-46 ubiquitination. This effect is mediated by direct binding of WDR-48 to USP-46, because point mutations that disrupt this interaction abrogate WDR-48 regulation of USP-46 and GLR-1 surface abundance. Interestingly, overexpression of
wdr-20, but not
wdr-48, promotes GLR-1 surface abundance in an
usp-46-dependent manner suggesting that regulation of
wdr-20 expression may provide a novel mechanism to control synaptic GLR-1. Together, these data suggest a model where WDR-48 increases the stability of USP-46 and, together with WDR-20, promotes USP-46 activity, surface levels of GLR-1, and glutamatergic behavior. (1) Kowalski et al. (2011) J Neurosci 31:1341-1354. (2) Kee et al. (2010) J Biol Chem 285:11252-11257. (3) Sowa et al. (2009) Cell 138:389-403. (4) Dahlberg and Juo. (2014). J Biol Chem 289:3444-3456. (5) Hoerndli et al. (2013). Neuron 80:1421-1437.