Abstract
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.
Highlights
From the ‡Department of Physiology & Biophysics, University of California, Irvine, California 92697; §Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143; ¶Protein Processing Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702; ʈDepartment of Biological Chemistry, University of California, Irvine, California 92697
One of the key elements in this strategy is the construction of two affinity tags that can be fused to two selected baits for sequential purification of protein subcomplexes containing the two desired baits, i.e. a Ubiquitin receptors (UbRs) protein and a proteasome subunit
Among the five ubiquilin proteins, Ubqln1, Ubqln2 and Ubqln4 have been found in all tissues, whereas Ubqln3 and Ubqlnl appear to be enriched in testis
Summary
Chemicals and Reagents—General chemicals for buffers and cell culture media were purchased from Fisher (Waltham, MA) or VWR (Radnor, PA). The obtained PCR fragment was confirmed by DNA sequencing, digested by PacI and EcoR I and inserted into the same treated pQCXIP-HisFlag to get pQCXIP-HisFlag-ADRM1. The obtained UbR PCR fragments were confirmed by DNA sequencing, digested by PacI and EcoR I (BamH1 for Ubqln1) and inserted into the same treated pQCXIP-HisFlag-Rpn13/ADRM1 to get pQCXIP-HisFlag-UbR. The obtained fragment was confirmed by DNA sequencing, digested by PacI and EcoR I and inserted into the same treated Rpn11-pQCXIH to get Rpn11-TB-pQCXIH. Experimental Design and Statistical Rationale—To isolate UbRproteasome subomplexes for XBAP experiments, 293HF-UbR/Rpn11-TB cells were grown to confluence in DMEM medium containing 10% FBS and 1% Pen/strep, trypsinized and washed 3 times with PBS buffer. Three biological replicates were performed, and each of them was analyzed with three technical replicates
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