The proteasome is the major ATP-dependent protease in eukaryotic cells, but limited structural information strongly restricts a mechanistic understanding of its activities. The proteasome regulatory particle, consisting of the lid and base subcomplexes, recognizes and processes poly-ubiquitinated substrates. We used electron microscopy and a newly-developed heterologous expression system for the lid to delineate the complete subunit architecture of the regulatory particle. Our studies reveal the spatial arrangement of ubiquitin receptors, deubiquitinating enzymes, and the protein unfolding machinery at subnanometer resolution, outlining the substrate’s path to degradation. Unexpectedly, the ATPase subunits within the base unfoldase are arranged in a spiral staircase, providing insight into potential mechanisms for substrate translocation through the central pore. Large conformational rearrangements of the lid upon holoenzyme formation suggest allosteric regulation of deubiquitination. We provide a structural basis for the ability of the proteasome to degrade a diverse set of substrates and thus regulate vital cellular processes.

The 26S proteasome is the major eukaryotic ATP-dependent protease, yet the detailed mechanisms utilized by the proteasomal heterohexameric AAA+ unfoldase to drive substrate degradation remain poorly understood. To perform systematic mutational analyses of individual ATPase subunits, we heterologously expressed unfoldase subcomplex from Saccharomyces cerevisiae in Escherichia coli and reconstituted the proteasome in vitro. Our studies demonstrate that the six ATPases play distinct roles in degradation, corresponding to their positions in spiral staircases adopted by the AAA+ domains in the absence and presence of substrate. ATP hydrolysis in subunits at the top of the staircases is critical for substrate engagement and translocation. While the unfoldase relies on this vertical asymmetry for substrate processing, interaction with the peptidase exhibits three-fold symmetry with contributions from every other subunit. These diverse functional asymmetries highlight how the 26S proteasome deviates from simpler, homomeric AAA+ proteases.

The 26S proteasome is the major ATP-dependent protease in eukaryotes and thus involved in regulating a diverse array of vital cellular processes. Three subcomplexes form this massive degradation machine: the lid, the base, and the core. While assembly of base and core has been well-studied, the detailed molecular mechanisms involved in formation of the nine-subunit lid remain largely unknown. Here, we reveal that helices found at the C terminus of each lid subunit form a helical bundle that directs the ordered self-assembly of the lid subcomplex. Furthermore, we use an integrative modeling approach to gain critical insights into the bundle topology and provide an important structural framework for our biochemical data. We show that the helical bundle serves as a hub through which the last-added subunit Rpn12 monitors proper lid assembly before incorporation into the proteasome. Finally, we predict that the assembly of the COP9 signalosome depends on a similar helical bundle.

controls their function is not understood. Here, we study the contribution of individual CH domains to the actin-binding function of utrophin's tandem CH domain. Co-sedimentation assays indicate that the C-terminal CH2 domain binds weakly to F-actin when compared with the full-length tandem CH domain, consistent with the published results on tandem CH domains. However, the surprise came from the CH1 domain. Isolated CH1 binds strongly to F-actin when compared with the full-length tandem CH domain. These results indicate that CH2 has a negative influence on actin-binding when it is linked with CH1. Thus, the obvious question that arises is why tandem CH domains require CH2, when CH2 is reducing their actin-binding efficiency. To answer, we probed the thermodynamic stabilities of individual CH domains. Isolated CH1 domain is unstable and is prone to serious aggregation. Isolated CH2 is very stable, even more stable than that of the fulllength tandem CH domain. This makes utrophin's tandem CH domain as the first example where an isolated domain is more stable than the fulllength protein. These results indicate that the main function of CH2 is to stabilize CH1 at the expense of decreasing the actin-binding efficiency. Consistently, the proposed structure of utrophin's tandem CH domain based on earlier X-ray studies indicates a close proximity between the C-terminal helix of CH2 and the N-terminal helix of CH1, and this helix in CH2 becomes more dynamic in the full-length protein when compared with that in the absence of CH1, suggesting a mechanism by which CH2 stabilizes CH1 despite the decrease in actin-binding function. Rhodopsin is the light-activated receptor located in the disc membranes of rod photoreceptor cells in the retina and initiates vision via the phototransduction signaling cascade. There are divergent views on rhodopsin's quaternary organization within native disc membranes. The classical view posits that rhodopsin molecules function as freely diffusing monomers. However, recent evidence suggests that rhodopsin oligomerizes and forms higher order structures within the membrane. An accurate description of signaling events in phototransduction and of associated disease mechanisms is reliant on a comprehensive understanding of how rhodopsin is organized within native disc membranes. The aim of the current study was to determine and quantify the physiological arrangement of several vertebrate rhodopsins within their native disc membranes using atomic force microscopy (AFM). AFM is a microscopic method that allows for the imaging of membrane proteins in their native environment under physiological conditions. Disc membranes, with 90% of the total protein content comprised of rhodopsin, were isolated from human, mouse, and frog ocular tissue. AFM images of single-bilayer disc membranes revealed that these vertebrate disc membranes have similar topographies. Topographic features in these images indicate that rhodopsin is organized into microdomains and that the formation of these microdomains is not an effec...

The eukaryotic 26S proteasome utilizes a complex set of coordinated processing steps for the ATP‐dependent degradation of ubiquitin‐tagged substrates. Our cryo‐EM studies reveal important features of the proteasome regulatory particle, including a pronounced spiral‐staircase arrangement of its heterohexameric ATPase ring, that facilitate substrate engagement and degradation initiation. Substrate engagement induces a translocation‐competent conformation, in which the de‐ubiquitinating subunit Rpn11 is repositioned to function as a gatekeeper at the entrance of the processing pore and the ATPase ring adopts a distinct spiral‐staircase configuration, suggesting that highly coordinated ATP‐hydrolysis events drive substrate translocation. Optical tweezers single‐molecule studies of a related protease, ClpXP, further support such a coordinated ATP‐hydrolysis mechanism. Systematic mutational analyses of the proteasome ATPase ring, using a heterologous expression system and in‐vitro reconstitution of 26S holoenzymes, indicate that the six ATPase subunits play distinct roles in substrate engagement and translocation, corresponding to their positions in the spiral‐staircase arrangements of the ATPase ring. Furthermore, structural and mutational studies of Rpn11 provide new mechanistic insights into the translocation‐dependence and regulation of substrate de‐ubiquitination.