Proteasome storage granules protect proteasomes from autophagic degradation upon carbon starvation
Authors: Richard D Vierstra
Richard S Marshall
Journal: eLife (2018)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: A. thaliana; S. cerevisiae; autophagy; biochemistry; cell biology
Affiliations: Department of Biology, Washington University in St. Louis, St. Louis, United States.

Abstract:  26S proteasome abundance is tightly regulated at multiple levels, including the elimination of excess or inactive particles by autophagy. In yeast, this proteaphagy occurs upon nitrogen starvation but not carbon starvation, which instead stimulates the rapid sequestration of proteasomes into cytoplasmic puncta termed proteasome storage granules (PSGs). Here, we show that PSGs help protect proteasomes from autophagic degradation. Both the core protease and regulatory particle sub-complexes are sequestered separately into PSGs via pathways dependent on the accessory proteins Blm10 and Spg5, respectively. Modulating PSG formation, either by perturbing cellular energy status or pH, or by genetically eliminating factors required for granule assembly, not only influences the rate of proteasome degradation, but also impacts cell viability upon recovery from carbon starvation. PSG formation and concomitant protection against proteaphagy also occurs in Arabidopsis, suggesting that PSGs represent an evolutionarily conserved cache of proteasomes that can be rapidly re-mobilized based on energy availability. 

Retracted: No

PubPeer comments:
 Panel D The lower GFP bands look the same including the backgrounds. ![file] , 
 Dear Colleagues, Like you, we are committed to publishing accurate and meaningful scientific data and became concerned about a possible error in the above publication upon receiving a notification from PubPeer. We have now research the outlined concerns and associated raw data. We affirm that the conclusions of the paper are correct and, fortunately, feel that the error implied by PubPeer was not in fact a mistake by the authors. Figure 4, Figure Supplement 1, panel D For the indicated immunoblot panels, the attached JPEG file shows the published data, original TIF scans for the anti-GFP immunoblots implicated, and brightness and contrast enhanced TIF scans for each blot. We agree that the bottom portions of these blots do look somewhat similar, but we think a close inspection of the raw data will show that they are not duplicates. For example, the white background lines on the right (which are artifacts of the scanner we were using at the time), are of two different angles, passing through the center of the second band from the right in the upper PRE10-GFP panel, but mostly to the left of the band in the lower RPN5-GFP panel. This white band also confirms that the images in the paper were from a contiguous scan of the blots, and not two images pieced together. Similarly, there are a number of background speckles in the upper PRE10-GFP panel that are not present in the lower RPN5-GFP panel (highlighted with red circles). We are therefore satisfied that the data presented in the paper are accurate. Yours sincerely, Prof Richard D. Vierstra and Dr. Richard S. Marshall , 
 see attached figure file ![file] , 
 Panel F Between the two different blots, the lower and upper bands look the same including the background. Could the authors please clarify? ![file] , 
 #4 I agree that in this case similarities are particularly high. , 
 Dear PubPeer: Thanks for your attention and concern about Figure 3F published in: Marshall, R.S., and R.D. Vierstra (2018) Proteasome storage granules protect 26S proteasomes from autophagic clearance during carbon starvation. eLife 2018;7:e34532. doi:10.7554 eLife.34532. tPMCID: PMC5947986. The Figure 3F. From comparisons of the published figure to the original x-ray film images of the two blots (see appended figure), we conclude that the figure is correct as is despite some apparent similarities in band appearances for free GFP. The two panels were actually derived from two separate SDS-PAGE gels from samples derived from two separate yeast strains designed to analyze the autophagic degradation of the Pre10-GFP and Rpn5-GFP reporters of the RP and CP subcomplexes of the proteasome. As can be seen from the two original images, they are clearly distinct based not only on variations in the signal densities of the undigested Pre10-GFP and Rpn5-GFP reporters in several lanes and various imperfections in the X-ray films (see circles), but also on the positions of the molecular mass markers that are indicated on each film (see circles). The Pre10-GFP protein electrophoreses as a 60-kDa species while the Rpn5-GFP protein electrophoreses as an 80-kDa species consistent with the size differences of the two proteins. We are therefore confident that the images in these two panels are not duplicates. ![file] , 
 I want to thank PubPeer for bringing forward potential problems with this 2018 paper in eLife. As an FYI, a probe was begun into the posed challenges that included the Journal and the Scientific Integrity Office at WashU to make sure that the issues were fully understood. Sorry that this probe has taken longer than expected, but each step required its own deliberative process. Fortunately, I have found much of the original raw images, but not all, which I have passed onto the Journal. I have had several subsequent correspondences with eLife, with my last one in the beginning of July. I am currently awaiting their response. Again, sorry for the delay. I hope to officially resolve this issue soon. , 
 Retraction notice https: elifesciences.org articles 82988 As the corresponding author I retract the eLife paper cited above based on unexplained irregularities associated with a collection of western blots and inappropriate placement of several confocal fluorescence micrographs generated by the first author Dr. Richard S. Marshall who was responsible for all the experiments and the first preparation and final drafts of the figures. As first identified by reports on PubPeer, I have thoroughly investigated the allegations and confirmed both the duplication of several micrographs and duplicated horizontal narrow bands in the background regions of a number of western blots, and further discovered poor archiving of the experimental data and associated yeast strains. The modifications of the western blots first flagged by PubPeer were particularly perplexing from my analysis of the original x-ray films of the blots as they appear as replicated narrow slices across the blots positioned often between the GFP fusion and free GFP released by autophagic turnover. These bands impacted only background regions and did not appear to be designed to cover any extraneous bands nor to splice two different western blot images together. Dr. Marshall has not yet offered an explanation for these duplications. While the main premise of the eLife report, i.e. to demonstrate a role of proteasome stress granules in protecting proteasomes from autophagic turnover during carbon and or energy starvation, appears to remain intact from my analysis of the data and subsequent independent studies by us and others related to key aspects of the study, I am not sure that all aspects of the studies therein are valid. We are currently repeating several parts of the paper and hope to provide appropriate and rigorous confirmatory data in the near future. Consequently, I feel that it is prudent at this time to retract the paper to avoid confusion in the literature. I apologize to the readers for these errors irregularities. I, as the last author, was responsible for ensuring scientific integrity for this publication but clearly failed as a u2018gatekeeperu2019 in this case. The corresponding author Richard D. Vierstra agrees to this retraction. The following author was also contacted but he did not provided comments nor explanations for the errors: Richard S. Marshall. , 
 Here is my detailed image forensics analysis of the immunoblot figures in this paper, done in May 2022 in my role as a member of the eLife Ethics Committee for which I receive an annual stipend. In most of these figure panels, repetitive, horizontal strips appear to have been duplicated, as shown with boxes of the same color. Figure 1 - Supplement 1 - Panel D: ![file] Detail left panel: ![file] Figure 2B and 2D: ![file] Figure 2 Supplement 1 Panel C ![file] Figure 3B: ![file] Figure 3F: ![file] Figure 3 Supplement 1 panel B: ![file] Figure 4F: ![file] Figure 4 Supplement 1 Panel D: ![file] Figure 5C 5D: ![file] Figure 7C: ![file] Figure 9A: ![file] , 
 Dear PubPeer: Thanks for helping bring to the surface a number of image problems associated with this eLife paper by Dr. Richard Marshall and myself, which was first published in 2018. After a thorough review of the publication by me, eLife and others, I recently issued a retraction of the paper, the details of which can found at https: doi.org 10.7554 eLife.82988. Sorry for any confusion this paper and its retraction has generated. Sincerely, Richard D. Vierstra ,