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The HEAT repeat protein Blm10 regulates the yeast proteasome by capping the core particle

Nature Structural & Molecular Biology volume 12, pages 294–303 (2005)Cite this article

Abstract

Proteasome activity is fine-tuned by associating the proteolytic core particle (CP) with stimulatory and inhibitory complexes. Although several mammalian regulatory complexes are known, knowledge of yeast proteasome regulators is limited to the 19-subunit regulatory particle (RP), which confers ubiquitin-dependence on proteasomes. Here we describe an alternative proteasome activator from Saccharomyces cerevisiae, Blm10. Synthetic interactions between blm10Δ and other mutations that impair proteasome function show that Blm10 functions together with proteasomes in vivo. This large, internally repetitive protein is found predominantly within hybrid Blm10–CP–RP complexes, representing a distinct pool of mature proteasomes. EM studies show that Blm10 has a highly elongated, curved structure. The near-circular profile of Blm10 adapts it to the end of the CP cylinder, where it is properly positioned to activate the CP by opening the axial channel into its proteolytic chamber.

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Figure 1: Identification of a novel S. cerevisiae proteasome subcomplex containing core particle subunits and Blm10.
Figure 2: Blm10 interaction with the CP results in enhanced peptidase activity.
Figure 3: Structural analysis of Blm10–CP complexes.
Figure 4: Structural characterization of Blm10.
Figure 5: Blm10 forms a distinct proteasomal subcomplex composed of Blm10–CP–RP in fresh unfractionated cell lysates.
Figure 6: Effects of BLM10 deletion on cells lacking RPN4 and ECM29.
Figure 7: The conserved C terminus of Blm10 is required for nuclear import.

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Acknowledgements

This work was supported by a grant from the Deutsche Forschungsgemeinschaft to M.S. (SCHM 884/3-1), and US National Institutes of Health (NIH) grants to D.F. (GM65592) and S.G. (GM67945). We thank B. Petre for collecting the EM images. The molecular EM facility at Harvard Medical School was established by a generous donation from the Giovanni Armenise Harvard Center for Structural Biology and is maintained by funds from NIH grant GM62580 to T.W. We are also grateful to the Harvard Medical School Nikon Imaging Center for technical assistance and access to their instruments, to R. Li for providing us with an antibody against Arc15, and to S. Elsasser, J. Hanna and J. Roelofs for critical reading of the manuscript. We thank R. Gali, and the Bauer Center for Genomics Research, Harvard University, for help with preliminary results of microarray analysis on the Rosetta Resolver microarray analysis platform. We furthermore are grateful to C. Hill for communicating results prior to publication and to A.L. Goldberg for his permission to include PA28-20S electron micrographs in our study.

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  1. Patricia G Santamaria

    Present address: Department of Pathology, MSB#599, NYU School of Medicine, 550 First Avenue, New York, New York, 10016, USA

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  1. Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, 02115, Massachusetts, USA

    Marion Schmidt, Wilhelm Haas, Bernat Crosas, Patricia G Santamaria, Steven P Gygi, Thomas Walz & Daniel Finley

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Supplementary information

Supplementary Fig. 1

Identification of CP-bound Blm10 by mass spectrometry. (PDF 20 kb)

Supplementary Fig. 2

Purification of CP from a blm10Δ strain. (PDF 316 kb)

Supplementary Fig. 3

Identification of RP-CP bound Blm10 by mass spectrometry. (PDF 39 kb)

Supplementary Fig. 4

Sequence alignment of the conserved Blm10 C terminus. (PDF 73 kb)

Supplementary Table 1

Primer used in this study. (PDF 12 kb)

Supplementary Methods (PDF 26 kb)

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Schmidt, M., Haas, W., Crosas, B. et al. The HEAT repeat protein Blm10 regulates the yeast proteasome by capping the core particle. Nat Struct Mol Biol 12, 294–303 (2005). https://doi.org/10.1038/nsmb914

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