Analysis of polyubiquitin conjugates reveals that the Rpn10 substrate receptor contributes to the turnover of multiple proteasome targets.
The polyubiquitin receptor Rpn10 targets ubiquitylated Sic1 to the 26S proteasome for degradation. In contrast, turnover of at least one ubiquitin-proteasome system (UPS) substrate, CPY*, is impervious to deletion of RPN10. To distinguish whether RPN10 is involved in the turnover of only a small set of cell cycle regulators that includes Sic1 or plays a more general role in the UPS, we sought to develop a general method that would allow us to survey the spectrum of ubiquitylated proteins that selectively accumulate in rpn10Delta cells. Polyubiquitin conjugates from yeast cells that express hexahistidine-tagged ubiquitin (H6-ubiquitin) were first enriched on a polyubiquitin binding protein affinity resin. This material was then denatured and subjected to IMAC to retrieve H6-ubiquitin and proteins to which it may be covalently linked. Using this approach, we identified 127 proteins that are candidate substrates for the 26S proteasome. We then sequenced ubiquitin conjugates from cells lacking Rpn10 (rpn10Delta) and identified 54 proteins that were uniquely recovered from rpn10Delta cells. These include two known targets of the UPS, the cell cycle regulator Sic1 and the transcriptional activator Gcn4. Our approach of comparing the ubiquitin conjugate proteome in wild-type and mutant cells has the resolving power to identify even an extremely in abundant transcriptional regulatory protein and should be generally applicable to mapping enzyme substrate networks in the UPS.
Mayor
Thibault
T
Howard Hughes Medical Institute, Division of Biology, MC 156-29, California Institute of Technology, Pasadena, California 91125, USA.
Lipford
J Russell
JR
Graumann
Johannes
J
Smith
Geoffrey T
GT
Deshaies
Raymond J
RJ
D002352:Carrier Proteins / Q000378:metabolism*; D002846:Chromatography, Affinity; D025821:Polyubiquitin / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D002352:Carrier Proteins; C122019:RPN10 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025821:Polyubiquitin; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease