Ubiquitin receptors are required for substrate-mediated activation of the proteasome’s unfolding ability

Abstract

The ubiquitin-proteasome system (UPS) is responsible for the bulk of protein degradation in eukaryotic cells, but the factors that cause different substrates to be unfolded and degraded to different extents are still poorly understood. We previously showed that polyubiquitinated substrates were degraded with greater processivity (with a higher tendency to be unfolded and degraded than released) than ubiquitin-independent substrates. Thus, even though ubiquitin chains are removed before unfolding and degradation occur, they affect the unfolding of a protein domain. How do ubiquitin chains activate the proteasome’s unfolding ability? We investigated the roles of the three intrinsic proteasomal ubiquitin receptors - Rpn1, Rpn10 and Rpn13 - in this activation. We find that these receptors are required for substrate-mediated activation of the proteasome’s unfolding ability. Rpn13 plays the largest role, but there is also partial redundancy between receptors. The architecture of substrate ubiquitination determines which receptors are needed for maximal unfolding ability, and, in some cases, simultaneous engagement of ubiquitin by multiple receptors may be required. Our results suggest physical models for how ubiquitin receptors communicate with the proteasomal motor proteins.

The Landscape of the Genomic Distribution and the Expression of the F-Box Genes Unveil Genome Plasticity in Hexaploid Wheat during Grain Development and in Response to Heat and Drought Stress

“…In these molecular mechanisms, cellular protein degradation and recycling processes often occur and are regulated by different conserved mechanisms in all eukaryotes [ 1 ]. One of these mechanisms is the Ubiquitin–proteasome system (UPS), an ATP-dependent highly regulated system that ensures the degradation of short-lived transcription factors, damaged or misfolded proteins and other regulatory proteins [ 2 , 3 , 4 ].…”

Mode of targeting to the proteasome determines GFP fate

“…Polyubiquitinated proteins have been shown to activate the proteasome via multiple mechanisms, including by opening the gate to the 20S core particle, increasing the ATPase activity of the proteasome, and increasing the proteasome's ability to unfold substrates (16,25,31,32). To our knowledge, no such effects have been reported for substrates containing UBL domains, but given their ability to bind to many of the same receptors that bind ubiquitin, UBL domains are likely to similarly mediate proteasomal activation.…”

“…We had previously proposed that binding of ubiquitinated proteins to Rpn13 might help activate the proteasome's unfolding ability by shifting the conformation from the s1 substratebinding state to s3-like substrate-processing states; in the kinetic model of Fig. 4A, this would correspond to increasing the initial engagement, degradation, and/or unfolding rates (16,25). The same hypothesis could explain the UBL versus Ub 4 results we see, given that Ub 4 substrates rely primarily on Rpn10, whereas UBL substrates rely primarily on Rpn13 (18).…”

“…Yeast (Saccharomyces cerevisiae) proteasome was purified from WT strain YYS40 or receptor mutant strains using a 33 FLAG-tagged copy of Rpn11 subunit of the 19S particle as described previously (16,25). The cells were grown overnight in YEPD or selective medium before being transferred to YEPD medium for large-scale growth at 30°C.…”

“…We recently showed that substrate ubiquitination can increase the proteasome's unfolding ability and that proteasomal ubiquitin receptors mediate this increase, with Rpn13 playing the largest role (16,25). Herein we set out to determine whether the degradation of GFP is affected by the mode of targeting to the proteasome, and, if so, which ubiquitin receptors are responsible.…”