Zdenka Cejka

The first characterization of a eubacterial proteasome: the 20S complex of Rhodococcus

BACKGROUND The 26S proteasome is the central protease of the ubiquitin-dependent pathway of protein degradation. The proteolytic core of the complex is formed by the 20S proteasome, a cylinder-shaped particle that in archaebacteria contains two different subunits (alpha and beta) and in eukaryotes contains fourteen different subunits (seven of the alpha-type and seven of the beta-type). RESULTS We have purified a 20S proteasome complex from the nocardioform actinomycete Rhodococcus sp. strain NI86/21. The complex has an apparent relative molecular mass of 690 kD, and efficiently degrades the chymotryptic substrate Suc-Leu-Leu-Val-Tyr-AMC in the presence or absence of 0.05% SDS. Purified preparations reveal the existence of four subunits, two of the alpha-type and two of the beta-type, the genes for which we have cloned and sequenced. Electron micrographs show that the complex has the four-ringed, cylinder-shaped appearance typical of proteasomes. CONCLUSIONS The recent description of the first eubacterial ubiquitin, and our discovery of a eubacterial proteasome show that the ubiquitin pathway of protein degradation is ancestral and common to all forms of life.

Tricorn protease - the core of a modular proteolytic system

Large macromolecular assemblies have evolved as a means of compartmentalizing reactions in organisms lacking membrane-bounded compartments. A tricorn-shaped protease was isolated from the archaeon Thermoplasma and was shown to form a multisubunit proteolytic complex. The 120-kilodalton monomer assembled to form a hexameric toroid that could assemble further into a capsid structure. Tricorn protease appeared to act as the core of a proteolytic system; when it interacted with several smaller proteins, it displayed multicatalytic activities.

Cloning, sequencing and expression of VAT, a CDC48/p97 ATPase homologue from the archaeon Thermoplasma acidophilum.

© 1997 Federation of European Biochemical Societies.

Subunit topology of the Rhodococcus proteasome

The 20S proteasome, isolated from the nocardioform actinomycete Rhodococcus erythropolis strain NI86/21, is built from two α‐type and two β‐type subunits. In order to probe the subunit topology, we have set up an expression system which allows coexpression of the genes encoding the α‐ and β‐subunits in all possible combinations. The four respective constructs obtained yielded fully assembled and proteolytically active proteasomes. Biochemical, kinetic and electron microscopy analysis allow us to rule out several of the models which were originally envisaged for the subunit topology of the Rhodococcus proteasome. The experiments further indicate that the assembly pathways of the Rhodococcus and of the Thermoplasma proteasome differ in some important details.

The molecular architecture of the extracellular hemoglobin of Ophelia bicornis : analysis of individual molecules

The dimensions and shape of extracellular hemoglobin molecules of the marine worm, Ophelia bicornis, were determined by electron microscopy using negative staining and embedding in aurothioglucose. The typical averaged double-layered hexagon has a diameter of 26.5 nm and a height of 18 nm. The three-dimensional reconstruction of the negatively stained samples, using random conical tilting, showed about 60% of the original height of the molecule due to flattening and incomplete embedding in stain. In contrast to this, the specimen embedded in aurothioglucose showed no flattening. The three-dimensional reconstructions agree with the structure found in a previous study on 2-D crystalline arrays. In particular, the main subunit shows more than three mass centers.

The Surface Protein Layer of Methanoplanus limicola: Three-dimensional Structure and Chemical Characterization

Summary Methanoplanus limocola , a member of a novel archaebacterial family, the Methanoplanaceae , possesses a hexagonally arrayed surface glycoprotein (S-layer). We have determined the molecular architecture of this surface glycoprotein via three-dimensional reconstruction from a tilt series of negatively stained preparations and surface relief reconstructions of metal shadowed preparations. The surface layer has p6 symmetry, a lattice constant of 14.7 nm and thickness of approx. 4.5 nm. The S-layer protein has an apparent molecular weight of 135 kDa as determined by SDS-polyacrylamide gel electrophoresis, but this value shifts to 115 kDa after treatment with anhydrous trifluoromethanesulfonic acid. This observation, together with quantitative estimations revealing a total neutral sugar content of 240 mg/g polypeptide suggests that the S-layer protein of Methanoplanus limicola is a glycoprotein.

The molecular architecture of the extracellular hemoglobin of Ophelia bicornis: Analysis of two-dimensional crystalline arrays

Abstract The double-layered hexagonal disks of the extracellular hemoglobin of the annelid worm Ophelia bicornis form two types of two-dimensional crystalline arrays. The hexagonal type exhibited a typical honeycomb pattern of top views with a center-to-center distance of 26.2 nm. Laterally oriented molecules formed rectangular crystals with lattice constants a = 26.7 run and b = 19.8 nm. The three-dimensional structure was determined from both crystal forms by reconstruction from images of tilt series. At the resolutions obtained, 1.8 nm for the hexagonal form and 2.5 nm for the rectangular form, flattening of the hemoglobin molecules against the support was observed. Nevertheless the two independent reconstructions provided information about the mass distribution within the main subunit and the connectivity between different parts of the molecule.

The molecular architecture of extracellular hemoglobin of Eophila tellinii

The molecular shape of the extracellular hemoglobin of the annelid worm Eophila tellinii was investigated by electron microscopy of negatively stained single molecules and of two-dimensional crystalline arrays. While the single molecules show the characteristic double hexagons, approx 28 nm in diameter and 19 nm in height, the molecules in the crystals are only 7–8 nm in height according to the 3D reconstruction. This is attributed to a dissociation of the hemoglobin complex; we present evidence that dissociation may proceed to the level of the main subunit from which half-molecules are reassembled. 3D reconstructions of two different crystal forms yield almost identical results and provide some information about the mass distribution within the main subunit. The presence or absence of the “central subunit” is tentatively interpreted in terms of a gross conformational change which entails a redistribution of mass also in the main subunit.

Three-dimensional structure of the surface layer protein of Clostridium thermohydrosulfuricum

Abstract The three-dimensional structure of the surface layer (S layer) protein of the thermophilic bacterium Clostridium thermohydrosulfuricum has been determined by electron microscopy to a resolution of about 2.5 nm. Two independent reconstructions have been obtained from the top and bottom layers of flattened S-layer sacculi after separation of the lattices in the course of correlation averaging of the projections. In spite of different levels of stain embedding, the two reconstructions agree with each other remarkably well. Six elongated spiral-shaped rods form cup-shaped complexes linked within the p6 lattice via the threefold crystallographic axes.

The Primary Structure of the HPI-Layer Polypeptide of Deinococcus radiodurans

Deinococcus radiodurans possesses a complex cell envelope which is quite unique regarding both structure and composition (Thompson et al. 1982, Brooks et al. 1980). The structure determination of its S-layer (HPI-layer) is particularly advanced (Baumeister et al. 1986, Rachel et al. 1986). As part of our objective to obtain a high-resultion 3-D structure of the HPI-layer we have determined the primary structure of the HPI-layer polypeptide.