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.

Distinct prion strains often exhibit different incubation periods and patterns of neuropathological lesions. Strain characteristics are generally retained upon intraspecies transmission, but may change on transmission to another species. We investigated the inactivation of two related prions strains: BSE prions from cattle and mouse-passaged BSE prions, termed 301V. Inactivation was manipulated by exposure to sodium dodecyl sulfate (SDS), variations in pH, and different temperatures. Infectivity was measured using transgenic mouse lines that are highly susceptible to either BSE or 301V prions. Bioassays demonstrated that BSE prions are up to 1,000-fold more resistant to inactivation than 301V prions while Western immunoblotting showed that short acidic SDS treatments reduced protease-resistant PrPSc from BSE prions and 301V prions at similar rates. Our findings argue that despite being derived from BSE prions, mouse 301V prions are not necessarily a reliable model for cattle BSE prions. Extending these comparisons to human sporadic Creutzfeldt-Jakob disease and hamster Sc237 prions, we found that BSE prions were 10- and 106-fold more resistant to inactivation, respectively. Our studies contend that any prion inactivation procedures must be validated by bioassay against the prion strain for which they are intended to be used.

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 26S proteasome is the major protease in eukaryotic cells responsible for selective protein degradation to mediate protein quality control and regulation, yet the detailed mechanisms by which the proteasomal heterohexameric AAA+ unfoldase drives ATP‐dependent protein degradation remain poorly understood. Delineating the roles of the six distinct ATPase subunits in substrate processing has been hindered by limitations in working with endogenous proteasomes due to misassembly or lethal degradation defects. We therefore developed a heterologous expression system to produce the unfoldase subcomplex from Saccharomyces cerevisiae in Escherichia coli and reconstituted the proteasome in vitro to perform systematic mutational analyses of the individual ATPase subunits. Our studies demonstrate that the six ATPases have distinct functions in degradation, corresponding to their positions in the spiral staircases adopted by their large AAA+ domains in the absence or presence of substrate. ATP hydrolysis in subunits at the top of the staircases is critical for substrate engagement and translocation. Whereas the unfoldase relies on this vertical asymmetry for substrate processing, interaction with the peptidase exhibits a pronounced three‐fold symmetry. Only three ATPase subunits, arranged in alternate positions within the unfoldase ring, contain a conserved C‐terminal hydrophobic/aromatic/unspecified (HbYX) motif that is critical for both peptidase binding and gate‐opening, whereas the C‐terminal tails of the interjacent ATPase subunits are dispensable. Our study provides an initial glimpse into the potential importance of the spiral staircase configurations of proteasomal ATPase subunits in substrate processing and highlights how the 26S proteasome may deviate from simpler, homomeric AAA+ proteases. Grant Funding Source: Supported by US National Institutes of Health grant R01‐GM094497‐01A1