The Axial Channel of the 20 S Proteasome Opens Upon Binding of the PA200 Activator

doi:10.1016/j.jmb.2004.12.049

Journal of Molecular Biology

Volume 346, Issue 5, 11 March 2005, Pages 1221-1227

https://doi.org/10.1016/j.jmb.2004.12.049 Get rights and content

Proteasomes consist of a proteolytic core called the 20 S particle and ancillary factors that regulate its activity in various ways. PA200 has been identified as a large (200 kDa) nuclear protein that stimulates proteasomal hydrolysis of peptides. To characterize its interaction with the 20 S core, we have visualized PA200-20 S complexes by electron microscopy. Monomers of PA200 bind to one or both ends of the 20 S core. Reconstructed in three dimensions to 23 Å resolution from cryo-electron micrographs of the singly bound complex, PA200 has an asymmetric dome-like structure with major and minor lobes. Taking into account previous bioinformatic analysis, it is likely to represent an irregular folding of an α-helical solenoid composed of HEAT-like repeats. PA200 makes contact with all α-subunits except α7, and this interaction induces an opening of the axial channel through the α-ring. Thus, the activation mechanism of PA200 is expressed via its allosteric effects on the 20 S core particle, perhaps facilitating release of digestion products or the entrance of substrates.

Introduction

Intracellular proteolysis serves in several capacities as a major mechanism of homeostasis. The lifespans of many regulatory proteins, among them, cyclins, transcription factors, and components of signal transduction pathways,1, 2 are controlled by programmed degradation. Misfolded proteins that are potentially toxic to the cell are eliminated in protein quality control.³ Moreover, the immune system relies on a supply of immunocompetent peptides generated by the dissection of foreign antigens.4, 5

The proteasome is the major proteolytic enzyme in eukaryotic cells.6, 7 Its architecture serves to prevent indiscriminate degradation of proteins by sequestering the proteolytic active sites inside a barrel-shaped particle, a steric-blocking stratagem that is also employed in other intracellular proteases.8, 9, 10, 11 The 14 α-subunits and 14 β-subunits of the 20 S proteasome are stacked in four heptameric rings in the order αββα to form a bipolar barrel.12, 13 The two β-rings contain the proteolytic active sites, which face an internal chamber. In eukaryotic cells, only three of the seven β-subunits are proteolytically active, and they have distinct specificities. Electron microscopy provided direct evidence that the axial channel is the port of entry for substrates.¹⁴ However, since the N-terminal regions of the α-subunits ordinarily close off the axial pathway,15, 16 some mechanism is needed for opening this channel to allow the passage of substrates.

Two kinds of complexes have been described that bind to the ends of the 20 S core particle and activate it. One is the 19 S regulatory particle that enables the proteasome to degrade ubiquitinated proteins by an ATP-dependent mechanism.² The second group of regulatory complexes, known as 11 S REG or PA28, stimulates peptidase activity in the 20 S particle but does not promote the degradation of folded proteins.¹⁷ Upon binding of PA28, conformational changes are induced in the α-subunits that open the gate to the axial channel.¹⁸

As part of a systematic search for proteasome modulators, we recently identified PA200, a 200-kDa protein bound to 20 S proteasomes isolated from bovine testis.¹⁹ PA200 was found to stimulate hydrolysis of small fluorogenic peptides, but not of folded proteins. However, several basic questions remained. What is the structure of PA200? How does it bind to the 20 S proteasome? Does this binding induce structural changes that contribute to activation? To address these questions, we have undertaken electron microscopy studies combined with two-dimensional image averaging and three-dimensional reconstruction of PA200-20 S complexes.

Section snippets

PA200 binds to the ends of the 20 S proteasome

We first examined PA200-20 S complexes by negative staining. To obtain a preponderance of side views, which provide the most readily interpretable images of cylindrical macromolecules, the EM grids were treated with polylysine.20, 21 Three kinds of particles were observed (Figure 1(a)). They represent the 20 S core particle, with its distinctive set of four transverse striations, alone, and with a rounded cap at one or both ends. They were found to occur in ratios of approximately 50 : 40: 10. To

Discussion

Our data indicate that PA200 activates the 20 S proteasome primarily by opening the axial gate of the α-rings. In this respect, its mode of action resembles that of the PA28 activator, as visualized in a crystal structure,¹⁸ despite the fact that PA28 binds as a heptamer of 28-kDa subunits and PA200 as a monomer of 200 kDa. The internal structure of the 20 S proteasome with bound 19 S regulatory complex has not yet been visualized in detail but there are strong indications that their binding

Purification of PA200-20 S proteasome complexes

Complexes were isolated from bovine testis and purified through the two DEAE columns as described.¹⁹ However, instead of employing gel filtration to obtain free PA200, the second DEAE pool was sedimented on 15 ml 10% to 30% (w/v) glycerol gradients for 22 hours at 25,000 rpm. Fractions (0.5 ml) were collected by bottom puncture, and some fractions contained PA200-proteasome complexes at >90% purity as assayed by staining of SDS-PAGE gels. These highly purified PA200-proteasome complexes were used

Acknowledgements

We thank Drs D. Winkler, N. Cheng and T. Ishikawa for help with EM, Dr S. Ludtke for help with EMAN, Drs D. Belnap and B. Trus for advice on computational matters, and Dr P. Zwickl for insightful comments on the manuscript.

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Published by Elsevier Ltd.

Journal of Molecular Biology

The Axial Channel of the 20 S Proteasome Opens Upon Binding of the PA200 Activator

Introduction

Section snippets

PA200 binds to the ends of the 20 S proteasome

Discussion

Purification of PA200-20 S proteasome complexes

Acknowledgements

Trends Biochem. Sci.

Adv. Protein Chem.

Cell

Cell

Mol. Cell

FEBS Letters

Structure (Camb)

J. Mol. Biol.

J. Struct. Biol.

Mol. Cell

J. Struct. Biol.

Cell

Cell

Mol. Cell

J. Struct. Biol.

J. Struct. Biol.

Ultramicroscopy

Ultramicroscopy