Lid Assembly Research Articles

In-depth Analysis of the Lid Subunits Assembly Mechanism in Mammals

The 26S proteasome is a key player in the degradation of ubiquitinated proteins, comprising a 20S core particle (CP) and a 19S regulatory particle (RP). The RP is further divided into base and lid subcomplexes, which are assembled independently from each other. We have previously demonstrated the assembly pathway of the CP and the base by observing assembly intermediates resulting from knockdowns of each proteasome subunit and the assembly chaperones. In this study, we examine the assembly pathway of the mammalian lid, which remains to be elucidated. We show that the lid assembly pathway is conserved between humans and yeast. The final step is the incorporation of Rpn12 into the assembly intermediate consisting of two modular complexes, Rpn3-7-15 and Rpn5-6-8-9-11, in both humans and yeast. Furthermore, we dissect the assembly pathways of the two modular complexes by the knockdown of each lid subunit.

Base-CP proteasome can serve as a platform for stepwise lid formation

26S proteasome, a major regulatory protease in eukaryotes, consists of a 20S proteolytic core particle (CP) capped by a 19S regulatory particle (RP). The 19S RP is divisible into base and lid sub-complexes. Even within the lid, subunits have been demarcated into two modules: module 1 (Rpn5, Rpn6, Rpn8, Rpn9 and Rpn11), which interacts with both CP and base sub-complexes and module 2 (Rpn3, Rpn7, Rpn12 and Rpn15) that is attached mainly to module 1. We now show that suppression of RPN11 expression halted lid assembly yet enabled the base and 20S CP to pre-assemble and form a base-CP. A key role for Regulatory particle non-ATPase 11 (Rpn11) in bridging lid module 1 and module 2 subunits together is inferred from observing defective proteasomes in rpn11–m1, a mutant expressing a truncated form of Rpn11 and displaying mitochondrial phenotypes. An incomplete lid made up of five module 1 subunits attached to base-CP was identified in proteasomes isolated from this mutant. Re-introducing the C-terminal portion of Rpn11 enabled recruitment of missing module 2 subunits. In vitro, module 1 was reconstituted stepwise, initiated by Rpn11–Rpn8 heterodimerization. Upon recruitment of Rpn6, the module 1 intermediate was competent to lock into base-CP and reconstitute an incomplete 26S proteasome. Thus, base-CP can serve as a platform for gradual incorporation of lid, along a proteasome assembly pathway. Identification of proteasome intermediates and reconstitution of minimal functional units should clarify aspects of the inner workings of this machine and how multiple catalytic processes are synchronized within the 26S proteasome holoenzymes.

Solution Structure of Yeast Rpn9: INSIGHTS INTO PROTEASOME LID ASSEMBLY

The regulatory particle (RP) of the 26 S proteasome functions in preparing polyubiquitinated substrates for degradation. The lid complex of the RP contains an Rpn8-Rpn11 heterodimer surrounded by a horseshoe-shaped scaffold formed by six proteasome-COP9/CSN-initiation factor (PCI)-containing subunits. The PCI domains are essential for lid assembly, whereas the detailed molecular mechanisms remain elusive. Recent cryo-EM studies at near-atomic resolution provided invaluable information on the RP architecture in different functional states. Nevertheless, atomic resolution structural information on the RP is still limited, and deeper understanding of RP assembly mechanism requires further studies on the structures and interactions of individual subunits or subcomplexes. Herein we report the high-resolution NMR structures of the PCI-containing subunit Rpn9 from Saccharomyces cerevisiae. The 45-kDa protein contains an all-helical N-terminal domain and a C-terminal PCI domain linked via a semiflexible hinge. The N-terminal domain mediates interaction with the ubiquitin receptor Rpn10, whereas the PCI domain mediates interaction with the neighboring PCI subunit Rpn5. The Rpn9-Rpn5 interface highlights two structural motifs on the winged helix module forming a hydrophobic center surrounded by ionic pairs, which is a common pattern for all PCI-PCI interactions in the lid. The results suggest that divergence in surface composition among different PCI pairs may contribute to the modulation of lid assembly.

Formation of an Intricate Helical Bundle Dictates the Assembly of the 26S Proteasome Lid

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.

Incorporation of the Rpn12 Subunit Couples Completion of Proteasome Regulatory Particle Lid Assembly to Lid-Base Joining

The 26S proteasome, the central eukaryotic protease, comprises a core particle capped by a 19S regulatory particle (RP). The RP is divisible into base and lid subcomplexes. Lid biogenesis and incorporation into the RP remain poorly understood. We report several lid intermediates, including the free Rpn12 subunit and a lid particle (LP) containing the remaining eight subunits, LP2. Rpn12 binds LP2 in vitro, and each requires the other for assembly into 26S proteasomes. Stable Rpn12 incorporation depends on all other lid subunits, indicating that Rpn12 distinguishes LP2 from smaller lid subcomplexes. The highly conserved C terminus of Rpn12 bridges the lid and base, mediating both stable binding to LP2 and lid-base joining. Our data suggest a hierarchical assembly mechanism where Rpn12 binds LP2 only upon correct assembly of all other lid subunits, and the Rpn12 tail then helps drive lid-base joining. Rpn12 incorporation thus links proper lid assembly to subsequent assembly steps.

Fusion neutron flux of center hole in LiD assembly under CARR reactor

A LiD assembly was proposed to produce fusion neutrons from thermal neutrons in D2O region of the China Advanced Research Reactor (CARR). The transportation of produced fusion neutrons in the LiD assembly can obtain the fusion neutron flux in its central hole. The neutron flux and the distribution of 6Li(n,α) reaction rate in the LiD assembly were calculated by a computer simulation method with MCNP code. The calculation results show that the fusion neutron flux in the central hole of LiD assembly, Φ, is dependent on the size and structure of assembly, 6Li enrichment, tritium concentration in LiD rods and the physical state of LiD. In the case of LiD rods containing 5% tritium and 6Li enrichment 20%, Φ value reaches to 5.4×1013cm−2s−1, when the assembly was placed at the thermal neutron flux about 8.0×1014cm−2s−1 in CARR.

Dissection of the assembly pathway of the proteasome lid in Saccharomyces cerevisiae

The 26S proteasome is a highly conserved multisubunit protease that degrades ubiquitinated proteins in eukaryotic cells. It comprises a 20S core particle and two 19S regulatory particles that are further divided into the lid and base complexes. The lid is a nine subunits complex that is structurally related to the COP9 signalosome and the eukaryotic initiation factor 3. Although the assembly pathway of the 20S and the base are well described, that of the lid is still unclear. In this study, we dissected the lid assembly using yeast lid mutant cells, rpn7-3, Δ rpn9, and rpn12-1. Using mass spectrometry, we identified a number of lid subassemblies, such as Rpn3–Rpn7 pair and a lid-like complex lacking Rpn12, in the mutants. Our analysis suggests that the assembly of the lid is a highly ordered and multi-step process; first, Rpn5, 6, 8, 9, and 11 are assembled to form a core module, then a second module, consisting of Rpn3, 7, and Sem1, is attached, followed by the incorporation of Rpn12 to form the lid complex.

Fission–fusion neutron source

In order to meet the requirements of fusion power reactors and nuclear waste treatment, a concept of fission–fusion neutron source is proposed, which consists of a LiD assembly located in the heavy water region of the China Advanced Research Reactor. This assembly of LiD fuel rods will be irradiated with slow neutrons and will produce fusion neutrons in the central hole via the reaction 6Li(n, α). More precisely, tritium ions with a high energy of 2.739 MeV will be produced in LiD by the impinging slow neutrons. The tritium ions will in turn bombard the deuterium ions present in the LiD assembly, which will induce fusion reaction and then the production of 14 MeV neutrons. The fusion reaction rate will increase with the accumulation of tritium in LiD by the reaction between tritium and deuteron recoils produced by the 14 MeV neutrons. When the concentration of tritium reaches 0.5 · 10 22 and the fraction of fusion reactions between tritium and deuteron recoils approaches 1, the 14 MeV neutron flux is doubled and redoubled, an so forth, approaching saturation in which the tritium produced at a time t is exhausted by the fusion reactions to keep constant the tritium concentration in LiD.

Automation of the car battery lid assembly operation

Automation of an assembly operation in an automotive lead/acid battery production plant is described in the paper. The operation—assembly of the polypropylene lids on the battery containers—is automated using robot technology. During the process of lid assembly, it is necessary for two plate stack electrodes to slide into two hollow lead terminals in the lid. In the part mating task the sticking is possible to occur bringing the assembly process into the halt. To design an automatic assembly system the lid assembly operation is considered as a problem of multiple peg-in-hole insertions. The paper investigates the basic principles of a multiple part mating task with special regard to the lid assembly. The theoretical and experimental findings are presented enabling improved understanding of the lid assembly process. On this basis, the robot workcell is built and assembly strategy proposed. Presented are the design of the workcell peripheral devices and incorporation of the workcell into the existing automotive battery production line.

Implementation of the Robot Workcell in the Production of the Lead/Acid Batteries

Automation of a manual operation in the automotive lead/acid battery assembly line is described in the paper. The working operation - setting of the polypropylene lids on the battery containers is automated using a small-size industrial robot. Active and passive compliance strategies are considered as possible approaches to the robot lid setting. Furthermore, the design of the workcell peripheral devices - two lid feeders and development of special robot gripper intended for sensory supported lid assembly are presented.

Design and Test of a Field Sampler for Ammonia Volatilization

AbstractA system is needed to measure ammonia volatilization from N fertilizer applied under field conditions. This study was undertaken to develop a device for making these measurements and still maintain a field environment. The basic system developed consists of a vacuum pump, a chemical trap to capture ammonia, and the volatilization chamber.The volatilization chamber consists of a steel cylinder 21.8‐cm I.D. by 15‐cm long that is forced into the soil with its top flush with the soil surface to provide a microplot. The lid assembly attached to the cylinder includes a hinge and reversible electric motor that allows the lid to be automatically rotated from the microplot. The volatilization chamber was designed with the removable lid so that the lid is closed only for short intervals during the day while ammonia loss is measured. The total ammonia loss is calculated by integrating the rates of loss over time. Between measurements, the lid is open to allow normal environmental conditions of temperature, wind movement, etc., on the micro‐plot. The volatilization chamber has five air intake ports and one exhaust port. Ammonia loss was unaffected by air flow rate if air movement through the chamber exceeded 15 exchange volumes/min.Our complete system consisted of eight volatilization chambers with a system of multiple chemical traps that can operate automatically without attention for 24 hours. When tested in the field, duplicate measurements of ammonia loss from ammonium sulfate agreed quite closely, both in the rates of loss and the total ammonia loss. In another test, ammonia loss from ammonium chloride agreed reasonably well with other laboratory results.