Proteomic analysis of affinity-purified 26S proteasomes identifies a suite of assembly chaperones in Arabidopsis
Authors: Qingzhen Zhao
Kwang-Hee Lee
Fionn McLoughlin
Lloyd M Smith
Weiming Hu
Richard S Marshall
Mark Scalf
Richard D Vierstra
David C Gemperline
Journal: Journal of Biological Chemistry (2019)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Arabidopsis; chaperone; core protease; mass spectrometry (MS); proteasome
Affiliations: Department of Genetics, University of Wisconsin, Madison, Wisconsin 53706., Department of Biology, Washington University in St. Louis, St. Louis, Missouri 63130., Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706., Department of Genetics, University of Wisconsin, Madison, Wisconsin 53706 .

Abstract:  The 26S proteasome is an essential protease that selectively eliminates dysfunctional and short-lived regulatory proteins in eukaryotes. To define the composition of this proteolytic machine in plants, we tagged either the core protease (CP) or the regulatory particle (RP) sub-complexes in Arabidopsis to enable rapid affinity purification followed by mass spectrometric analysis. Studies on proteasomes enriched from whole seedlings, with or without ATP needed to maintain the holo-proteasome complex, identified all known proteasome subunits but failed to detect isoform preferences, suggesting that Arabidopsis does not construct distinct proteasome sub-types. We also detected a suite of proteasome-interacting proteins, including likely orthologs of the yeast and mammalian chaperones Pba1, Pba2, Pba3, and Pba4 that assist in CP assembly; Ump1 that helps connect CP half-barrels; Nas2, Nas6, and Hsm3 that assist in RP assembly; and Ecm29 that promotes CP-RP association. Proteasomes from seedlings exposed to the proteasome inhibitor MG132 accumulated assembly intermediates, reflecting partially built proteasome sub-complexes associated with assembly chaperones, and the CP capped with the PA200/Blm10 regulator. Genetic analyses of Arabidopsis UMP1 revealed that, unlike in yeast, this chaperone is essential, with mutants lacking the major UMP1a and UMP1b isoforms displaying a strong gametophytic defect. Single ump1 mutants were hypersensitive to conditions that induce proteotoxic, salt and osmotic stress, and also accumulated several proteasome assembly intermediates, consistent with its importance for CP construction. Insights into the chaperones reported here should enable study of the assembly events that generate the 26S holo-proteasome in Arabidopsis from the collection of 64 or more subunits. 

Retracted: No

PubPeer comments:
 Figure 10, panels seem to duplicate (red boxes) could the authors please check? ![file] , 
 Figure 10C, panels seem to duplicate (red boxes), could the authors please check? ![file] , 
 Figure 2, much more similar than expected (red boxes). Could the authors please check? ![file] , 
 Figure 3, much more similar than expected (green boxes). Could the authors please check? and provide the original scans? ![file] , 
 Dear Colleagues, Like you, we are committed to publishing accurate and meaningful scientific data and became concerned about possible errors in the above publication upon receiving a notification from PubPeer. We have now researched the outlined concerns and associated raw data and, although we affirm that the conclusions of the paper are correct, we did confirm two of the errors in Figure 2 and 10C highlighted above that involved the incorrect placement of immunoblots during construction of Figures containing glycerol gradients. Figures 2 and 10C In both Figure 2 and 10C of the above paper, a pair of immunoblots were inadvertently duplicated within each Figure, as shown in the PubPeer comments above. It is clear that we made errors during Figure preparation, as the panels for WT PBA1 and FLAG-RPT4a rpt4a-1 PBF1 are the same in Figure 2, while the blots for RPN3 and RPT2 are the same in Figure 10C. We very much apologize for these unintentional duplications, which likely occurred during preparation of the Figures, when templates and panels were being duplicated for use as placeholders to ensure uniformity in the presentation of each gradient. In the attached JPEG files, we highlight the duplicated panels and show the original TIF files for the implicated panels, along with corrected versions with the duplicated panels replaced. As these blots were part of a series of ten for each glycerol gradient, these duplications did not appreciably impact the conclusions of the data. However, they are certainly an unacceptable error, for which we apologize. We do however feel that the additional issue highlighted by PubPeer for Figure 3B is not justified. Analysis of the raw data for the three panels highlighted in Figure 3, where there is concern about the similar shape of some bands, confirms that these are indeed separate blots. The attached JPEG file shows that the RPN12a and PBA1 panels actually come from the same blot that was probed with both antibodies simultaneously, whereas the RPT4 panel is from a separate blot that likely came from the same piece of membrane cut at an appropriate place been the positions of RPT4 (44 kDa) and RPN12 (30 kDa). The unusual shape of the bands is likely an artifact caused by the sample well from the specific gel that was run on that day. Adjustment of the levels, curves and or brightness for each panel likewise confirms that they are continuous blots, and have not had bands pasted in. We are therefore satisfied that the data presented in these blots are accurate. Please find the corrected versions of Figures 2 and 10C in the attached JPEG files. We apologize for these mistakes and regret that they were not detected by us prior to publication. The errors will be corrected in an upcoming issue of the Journal of Biological Chemistry in the near future. Yours sincerely, Prof Richard D. Vierstra and Dr. Richard S. Marshall , 
 see attached figure files ![file] ![file] ![file] ![file] , 
 #6 Dear Authors, thank you for your reply, for taking the time to clarify the issues, and for going to correct the literature record. I do agree that concerns raised in comment #4 are unjustified, it was clear from the start actually. Regards. ,