Reconstitution of the 26S Proteasome Reveals Functional Asymmetries in its Heterohexameric AAA+ Unfoldase

Abstract

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...

Proteasome Structure and Assembly

“…Additionally, the ATPase domains of the Rpt subunits in the ring adopt a spiral staircase-like configuration in the order of Rpt3-Rpt4-Rpt5-Rpt1-Rpt2 from top to bottom relative to the CP, with Rpt6 at about the same height as Rpt5 and bridging the vertical displacement between the highest (Rpt3) and lowest (Rpt2) subunits[84,88,89]. When the ring binds to a substrate, the conformation of the ATPase ring transitions to a more planar configuration with alignment of the RP ATPase and CP central channels[90].…”

“…A CP-independent base-assembly pathway has been inferred primarily from the ability to reconstitute the full yeast base complex in the absence of CP in E. coli [89]. This functional base complex was reconstituted by co-expressing all six Rpt subunits, Rpn1, Rpn2, Rpn13 and four base assembly chaperones Hsm3, Nas2, Nas6 and Rpn14[89].…”

“…This functional base complex was reconstituted by co-expressing all six Rpt subunits, Rpn1, Rpn2, Rpn13 and four base assembly chaperones Hsm3, Nas2, Nas6 and Rpn14[89]. Although full base complex can be assembled independently of CP in vitro , it does not necessarily rule out a CP-templating mechanism for enhancing base assembly.…”