Characterization of Dynamic UbR-Proteasome Subcomplexes by In vivo Cross-linking (X) Assisted Bimolecular Tandem Affinity Purification (XBAP) and Label-free Quantitation.
Proteasomes are protein degradation machines that exist in cells as heterogeneous and dynamic populations. A group of proteins function as ubiquitin receptors (UbRs) that can recognize and deliver ubiquitinated substrates to proteasome complexes for degradation. Defining composition of proteasome complexes engaged with UbRs is critical to understand proteasome function. However, because of the dynamic nature of UbR interactions with the proteasome, it remains technically challenging to capture and isolate UbR-proteasome subcomplexes using conventional purification strategies. As a result, distinguishing the molecular differences among these subcomplexes remains elusive. We have developed a novel affinity purification strategy, in vivo cross-linking (X) assisted bimolecular tandem affinity purification strategy (XBAP), to effectively isolate dynamic UbR-proteasome subcomplexes and define their subunit compositions using label-free quantitative mass spectrometry. In this work, we have analyzed seven distinctive UbR-proteasome complexes and found that all of them contain the same type of the 26S holocomplex. However, selected UbRs interact with a group of proteasome interacting proteins that may link each UbR to specific cellular pathways. The compositional similarities and differences among the seven UbR-proteasome subcomplexes have provided new insights on functional entities of proteasomal degradation machineries. The strategy described here represents a general and useful proteomic tool for isolating and studying dynamic and heterogeneous protein subcomplexes in cells that have not been fully characterized.
From the ‡Department of Physiology & Biophysics, University of California, Irvine, California 92697;
Clinton
C
Yu
From the ‡Department of Physiology & Biophysics, University of California, Irvine, California 92697;
Yingying
Y
Yang
From the ‡Department of Physiology & Biophysics, University of California, Irvine, California 92697;
Xiaorong
X
Wang
§Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143;
Shenheng
S
Guan
From the ‡Department of Physiology & Biophysics, University of California, Irvine, California 92697;
Lei
L
Fang
¶Protein Processing Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702;
Fen
F
Liu
¶Protein Processing Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702;
Kylie J
KJ
Walters
‖Department of Biological Chemistry, University of California, Irvine, California 92697.
Peter
P
Kaiser
From the ‡Department of Physiology & Biophysics, University of California, Irvine, California 92697; lanhuang@uci.edu.
Lan
L
Huang
D002846:Chromatography, Affinity / Q000379:methods; D003432:Cross-Linking Reagents / Q000378:metabolism; D006801:Humans; D046988:Proteasome Endopeptidase Complex / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods; D025801:Ubiquitin / Q000378:metabolism*
D003432:Cross-Linking Reagents; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease