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J. Biol. Chem., Vol. 281, Issue 14, 9569-9575, April 7, 2006

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20S Proteasomes Have the Potential to Keep Substrates in Store for Continual Degradation^*

Michal Sharon¹, Susanne Witt, Karin Felderer, Beate Rockel, Wolfgang Baumeister, and Carol V. Robinson²

From the Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom and Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany

The 20S core of the proteasome, which together with the regulatory particle plays a major role in the degradation of proteins in eukaryotic cells, is traversed by an internal system of cavities, namely two antechambers and one central proteolytic chamber. Little is known about the mechanisms underlying substrate binding and translocation of polypeptide chains into the interior of 20S proteasomes. Specifically, the role of the antechambers is not fully understood, and the number of substrate molecules sequestered within the internal cavities at any one time is unknown. Here we have shown that by applying both electron microscopy and tandem mass spectrometry (MS) approaches to this multisubunit complex we obtain precise information regarding the stoichiometry and location of substrates within the three chambers. The dissociation pattern in tandem MS allows us to conclude that a maximum of three green fluorescent protein and four cytochrome c substrate molecules are bound within the cavities. Our results also show that >95% of the population of proteasome molecules contain the maximum number of partially folded substrates. Moreover, we deduce that one green fluorescent protein or two cytochrome c molecules must reside within the central proteolytic chamber while the remaining substrate molecules occupy, singly, both antechambers. The results imply therefore an additional role for 20S proteasomes in the storage of substrates prior to their degradation, specifically in cases where translocation rates are slower than proteolysis. More generally, the ability to locate relatively small protein ligands sequestered within the 28-subunit core particle highlights the tremendous potential of tandem MS for deciphering substrate binding within large macromolecular assemblies.

Received for publication, November 7, 2005 , and in revised form, January 3, 2006.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental text and supplemental Fig. S1.

¹ Supported by the European Molecular Biology Organization and a Wingate scholarship.

² Funded by the Walters Kundert Trust. To whom correspondence should be addressed. Tel.: 44-1223-763864; E-mail: cvr24{at}cam.ac.uk.

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				M. Sharon, S. Witt, E. Glasmacher, W. Baumeister, and C. V. Robinson Mass Spectrometry Reveals the Missing Links in the Assembly Pathway of the Bacterial 20 S Proteasome J. Biol. Chem., June 22, 2007; 282(25): 18448 - 18457. [Abstract] [Full Text] [PDF]