Dual functions of the Hsm3 protein in chaperoning and scaffolding regulatory particle subunits during the proteasome assembly
Authors: Jean-Baptiste Charbonnier
Chloe Godard
Benoît Le Tallec
Erwann Rousseau
Nicolas Richet
Pierre Legrand
Anne Peyroche
Marie-Bénédicte Barrault
Françoise Ochsenbein
Marie-Hélène Le Du
Raphaël Guérois
Brice Murciano
Journal: Proceedings of the National Academy of Sciences of the United States of America (2012)
Discipline: Multidisciplinary
Keywords: 26S proteasome; regulated protein degradation; proteolytic core particle; 19S regulatory particles; base subcomplex
Affiliations: Commissariat à l'Energie Atomique (CEA),  Institut de Biologie et Technologies de Saclay (iBiTEC-S), Service de Biologie Intégrative et Génétique Moléculaire, Laboratoire du Métabolisme des Acides Nucléiques et Réponses aux Génotoxiques, F-91191 Gif-sur-Yvette,  France.

Abstract:  The 26S proteasome, a molecular machine responsible for regulated protein degradation, consists of a proteolytic core particle (20S CP) associated with 19S regulatory particles (19S RPs) subdivided into base and lid subcomplexes. The assembly of 19S RP base subcomplex is mediated by multiple dedicated chaperones. Among these, Hsm3 is important for normal growth and directly targets the carboxyl-terminal (C-terminal) domain of Rpt1 of the Rpt1-Rpt2-Rpn1 assembly intermediate. Here, we report crystal structures of the yeast Hsm3 chaperone free and bound to the C-terminal domain of Rpt1. Unexpectedly, the structure of the complex suggests that within the Hsm3-Rpt1-Rpt2 module, Hsm3 also contacts Rpt2. We show that in both yeast and mammals, Hsm3 actually directly binds the AAA domain of Rpt2. The Hsm3 C-terminal region involved in this interaction is required in vivo for base assembly, although it is dispensable for binding Rpt1. Although Rpt1 and Rpt2 exhibit weak affinity for each other, Hsm3 unexpectedly acts as an essential matchmaker for the Rpt1-Rpt2-Rpn1 assembly by bridging both Rpt1 and Rpt2. In addition, we provide structural and biochemical evidence on how Hsm3/S5b may regulate the 19S RP association to the 20S CP proteasome. Our data point out the diverse functions of assembly chaperones. 

Retracted: No

PubPeer comments:
 One gel from Fig. 6G ![file] , 
 #1 The central splice is obvious, whatever its (in)significance. In the original figure, there appear to be similar splices in the two gels to the left of that illustrated here. Maybe the  cuts around the edges simply result from a slight rotation? , 
 Here are the panels from Figure 6G, where I have adjusted contrast of the third one (Anti-Rpt1). The lower third of the bands are retouched, airbrushed, cloned. ![file] Closeup: ![file] , 
 Figure 6C after contrast adjustment. ![file] , 
 Two top panels of Figure 6F, adjusted to show spliced lanes. ![file] , 
 My mistake. Both panels in Fig 6C appear to be spliced. ![file] , 
 Undisclosed splicing in Figure 6B. ![file] , 
 #1 As noted above, the AntiRpt1 panel displays peculiar discontinuities and visual echoes across the bottom of the lanes. The accompanying AntiRpt2 panel is also suggestive of discontinuity: ![file] Here I have defaced the image (already contrast-enhanced) with lines marking and hiding the discontinuity: ![file] Am I imagining this? Feedback is welcome. , 
 #1 Response to comments 1,3 8 Here there are the original full-length autoradiographs. As you can see on the original images below,  u0394C and  Myc samples were spaced apart from each other in the gel in order to have a clear signal and the central space was removed for the sake of clarity, resulting in the observed central cut. ![file] ![file] , 
 Thank you for taking the time to post the original data. This clears any doubt I may have had. , 
 Response to comments 3 8 Here there are the original full-length autoradiographs that have been rescanned. There are no issues about background. Different exposures are shown below. Anomalies noticed here probably correspond to scanning anomalies. ![file] ![file] ![file] Response to comment 5 Here there are original autoradiographs for anti-Myc, anti-Rpt1, anti-Rpt2 and anti-Rpn1 revelations. For anti-Myc revelation, IP Myc u2206C 30u00b0C and IP Myc FL 30u00b0C were loaded in a different order as indicated. For sake of clarity, lanes were presented in the same order in the figure explaining the spliced lanes. We should have mentioned that. ![file] Response to comment 7 We were used to perform extensive experiments with many internal controls and assays. Here there is the original gel. Due to limited space available and for sake of clarity, several lanes were removed. We should have mentioned that. ![file] , 
 Many thanks to the authors for providing the originals! , 
 Supplementary Figure 6, with legend u201cNative gel comparing WT, hsm3u0394C(u0394C), and hsm3u0394(u0394) strains grown in minimal medium at 37 u00b0Cu201c has undisclosed vertical and horizontal splicing. ![file] ,