Wolfgang Baumeister

The Proteasome: Paradigm of a Self-Compartmentalizing Protease

The work of one of us (W. B.) was supported by a grant of the Human Frontiers Science Program. We wish to thank Drs. C. P. Hill (Salt Lake City) and J. M. Flanagan (Brookhaven) for making available to us structural data prior to publication. We are grateful to Drs. F. U. Hartl and M. Kania for critically reading the manuscript.

A subcomplex of the proteasome regulatory particle required for ubiquitin-conjugate degradation and related to the cop9-signalosome and eif3

The proteasome consists of a 20S proteolytic core particle (CP) and a 19S regulatory particle (RP), which selects ubiquitinated substrates for translocation into the CP. An eight-subunit subcomplex of the RP, the lid, can be dissociated from proteasomes prepared from a deletion mutant for Rpn10, an RP subunit. A second subcomplex, the base, contains all six proteasomal ATPases and links the RP to the CP. The base is sufficient to activate the CP for degradation of peptides or a nonubiquitinated protein, whereas the lid is required for ubiquitin-dependent degradation. By electron microscopy, the base and the lid correspond to the proximal and distal masses of the RP, respectively. The lid subunits share sequence motifs with components of the COP9/signalosome complex and eIF3, suggesting that these functionally diverse particles have a common evolutionary ancestry.

From words to literature in structural proteomics.

Technical advances on several frontiers have expanded the applicability of existing methods in structural biology and helped close the resolution gaps between them. As a result, we are now poised to integrate structural information gathered at multiple levels of the biological hierarchy — from atoms to cells — into a common framework. The goal is a comprehensive description of the multitude of interactions between molecular entities, which in turn is a prerequisite for the discovery of general structural principles that underlie all cellular processes.

The genome sequence of the thermoacidophilic scavenger Thermoplasma acidophilum.

Thermoplasma acidophilum is a thermoacidophilic archaeon that thrives at 59 °C and pH 2, which was isolated from self-heating coal refuse piles and solfatara fields. Species of the genus Thermoplasma do not possess a rigid cell wall, but are only delimited by a plasma membrane. Many macromolecular assemblies from Thermoplasma , primarily proteases and chaperones, have been pivotal in elucidating the structure and function of their more complex eukaryotic homologues. Our interest in protein folding and degradation led us to seek a more complete representation of the proteins involved in these pathways by determining the genome sequence of the organism. Here we have sequenced the 1,564,905-base-pair genome in just 7,855 sequencing reactions by using a new strategy. The 1,509 open reading frames identify Thermoplasma as a typical euryarchaeon with a substantial complement of bacteria-related genes; however, evidence indicates that there has been much lateral gene transfer between Thermoplasma and Sulfolobus solfataricus, a phylogenetically distant crenarchaeon inhabiting the same environment. At least 252 open reading frames, including a complete protein degradation pathway and various transport proteins, resemble Sulfolobus proteins most closely.

Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach

The 26S proteasome is at the executive end of the ubiquitin-proteasome pathway for the controlled degradation of intracellular proteins. While the structure of its 20S core particle (CP) has been determined by X-ray crystallography, the structure of the 19S regulatory particle (RP), which recruits substrates, unfolds them, and translocates them to the CP for degradation, has remained elusive. Here, we describe the molecular architecture of the 26S holocomplex determined by an integrative approach based on data from cryoelectron microscopy, X-ray crystallography, residue-specific chemical cross-linking, and several proteomics techniques. The “lid” of the RP (consisting of Rpn3/5/6/7/8/9/11/12) is organized in a modular fashion. Rpn3/5/6/7/9/12 form a horseshoe-shaped heterohexamer, which connects to the CP and roofs the AAA-ATPase module, positioning the Rpn8/Rpn11 heterodimer close to its mouth. Rpn2 is rigid, supporting the lid, while Rpn1 is conformationally variable, positioned at the periphery of the ATPase ring. The ubiquitin receptors Rpn10 and Rpn13 are located in the distal part of the RP, indicating that they were recruited to the complex late in its evolution. The modular structure of the 26S proteasome provides insights into the sequence of events prior to the degradation of ubiquitylated substrates.

Chaperonin-mediated protein folding: GroES binds to one end of the GroEL cylinder, which accommodates the protein substrate within its central cavity.

The mechanism of GroEL (chaperonin)‐mediated protein folding is only partially understood. We have analysed structural and functional properties of the interaction between GroEL and the co‐chaperonin GroES. The stoichiometry of the GroEL 14mer and the GroES 7mer in the functional holo‐chaperonin is 1:1. GroES protects half of the GroEL subunits from proteolytic truncation of the approximately 50 C‐terminal residues. Removal of this region results in an inhibition of the GroEL ATPase, mimicking the effect of GroES on full‐length GroEL. Image analysis of electron micrographs revealed that GroES binding triggers conspicuous conformational changes both in the GroES adjacent end and at the opposite end of the GroEL cylinder. This apparently prohibits the association of a second GroES oligomer. Addition of denatured polypeptide leads to the appearance of irregularly shaped, stain‐excluding masses within the GroEL double‐ring, which are larger with bound alcohol oxidase (75 kDa) than with rhodanese (35 kDa). We conclude that the functional complex of GroEL and GroES is characterized by asymmetrical binding of GroES to one end of the GroEL cylinder and suggest that binding of the substrate protein occurs within the central cavity of GroEL.

Snapshots of nuclear pore complexes in action captured by cryo-electron tomography

Nuclear pore complexes reside in the nuclear envelope of eukaryotic cells and mediate the nucleocytoplasmic exchange of macromolecules. Traffic is regulated by mobile transport receptors that target their cargo to the central translocation channel, where phenylalanine-glycine-rich repeats serve as binding sites. The structural analysis of the nuclear pore is a formidable challenge given its size, its location in a membranous environment and its dynamic nature. Here we have used cryo-electron tomography to study the structure of nuclear pore complexes in their functional environment, that is, in intact nuclei of Dictyostelium discoideum. A new image-processing strategy compensating for deviations of the asymmetric units (protomers) from a perfect eight-fold symmetry enabled us to refine the structure and to identify new features. Furthermore, the superposition of a large number of tomograms taken in the presence of cargo, which was rendered visible by gold nanoparticles, has yielded a map outlining the trajectories of import cargo. Finally, we have performed single-molecule Monte Carlo simulations of nuclear import to interpret the experimentally observed cargo distribution in the light of existing models for nuclear import.

Electron tomography of molecules and cells

Abstract Electron tomography is the most widely applicable method for obtaining three-dimensional information by electron microscopy. It is, in fact, the only method suitable for investigating pleomorphic structures, such as many supramolecular assemblies, organelles and cells. With the recent development of automated low-dose data-acquisition schemes, it is now possible to study molecules and cells embedded in vitreous ice. This opens up new horizons for investigating the functional organization of cellular components with minimal perturbation of the cellular context.

2D crystallization: from art to science

The techniques as well as the principles of the 2D crystallization of membrane and water-soluble proteins for electron crystallography are reviewed. First, the biophysics of the interactions between proteins, lipids and detergents is surveyed. Second, crystallization of membrane proteins in situ and by reconstitution methods is discussed, and the various factors involved are addressed. Third, we elaborate on the 2D crystallization of water-soluble proteins, both in solution and at interfaces, such as lipid monolayers, mica, carbon film or mercury surfaces. Finally, techniques and instrumentations that are required for 2D crystallization are described.

The multicatalytic proteinase (prosome) is ubiquitous from eukaryotes to archaebacteria

From the thermoacidophilic archaebacterium, Thermoplasma acidophilum, a proteolytically active particle has been isolated which is almost identical in size and shape with the multicatalytic proteinase (prosome) from rat. This result indicates that prosomes have been developed early in evolution and that they possibly serve functions common to all living cells.