Repertoire of Proteasomal Protein Degradation
A cell and its world of molecular machines are challenged by stress and environmental insults, but work remarkably well due to the existence of quality control mechanisms. Quality control ensures the homeostasis of biological processes and acts at different levels of macromolecules, e,g, chaperones guide protein folding. If chaperones´ folding capacities are saturated, misfolded proteins accumulate, unless they are degraded by proteases. Failures in protein degradation result in protein aggregations, typical features of neurodegenerative diseases. The key protease responsible for eliminating misfolded and potentially toxic proteins is the proteasome (for review see Hershko and Ciechanover (1998) Annu. Rev. Biochem. 67, 425; Goldberg (2003) Nature 426, 895).

Proteasomes are macromolecular machines composed of multiple subunits. Integrating quality control in proteasome assembly is advised to maintain proteasome functions and to adjust proteasome configurations to cellular requirements. All proteasome configurations have a proteolytically active core particle (CP). CP association with one or two regulatory particles (RP) and accessory proteins allows the plasticity of proteasome configuration, the fine-tuning of enzyme activity and substrate recognition.

Quality Control of Proteasome Assembly
The basic concept of CP assembly is understood which is assisted by several CP-dedicated chaperones. Four stacked seven-membered subunit rings arranged in a alpha1-7beta1-7beta1-7alpha1-7 configuration form the CP. Gated channels within each outer alpha ring control the access of protein substrates into the CP. The proteolytic cavity is located within both inner beta rings exposing the active sites after propeptide cleavage. Concomitantly with CP maturation CP-dedicated chaperones are degraded (for review see Ramos and Dohmen (2008) Structure 16, 1296).

Little is known about the fate of alternatively and aberrantly configured proteasomes. Useful and useless proteasome configurations need to be distinguished by quality control mechanisms. In this context we discovered two conserved HEAT-like repeat proteins, named Blm10 and Ecm29 in yeast.According to genome-wide transcription profiles BLM10 and ECM29 expression is induced upon stress, e.g. unfolded protein response, proteasome inhibition, oxidative stress and DNA damage (Yeast Genome Database).

We found that Blm10 preferentially binds pre-activated proteasomes (Lehmann et al. (2008) EMBO Rep. 9, 1237).

Ecm29 recognizes incompletely matured RP-CP assemblies (Lehmann et al. (2010) Mol. Cell 38, 879).

Proteasome configurations under stress conditions
Based on our studies on Blm10 and Ecm29 we want to understand how cells manage quality control of proteasomal protein degradation under stress and different growth conditions. For this purpose, we developed a native polyacrylamide gel electrophoresis system exploiting GFP labelling techniques (Enenkel (2011) in preparation). By this means we investigate proteasome configurations which occur in insignificant amounts under normal conditions but accumulate under stress conditions. Biochemical means including mass spectrometry are applied to identify their subunit compositions and enzyme activities. Furthermore, our approach allows us to purify unusually configured proteasome species under native conditions.

Reconstitution assays are used to investigate whether unusually configured proteasome species can be remodelled into regular enzymes and which factors are limiting. Cryo-electron microscopy and X-ray crystallography will further elucidate their structural features.

Proteasome dynamics
Live cell imaging of proliferating mammalian cells and growing yeast revealed that GFP-labelled proteasomes are primarily located in the nucleus, where they are assembled from inactive precursor complexes.

In non-dividing cells, at least in yeast, proteasomes are sequestered into motile cytosolic clusters (MCC) and mobilized upon request (Kaganovich et al., (2008) Nature 454, 1088; Laporte et al. (2008) J. Cell Biol. 181, 737).

Proteasome functions in motile cytosolic clusters
We want to understand the physiological role of MCC formation and will address the following questions:

(1) What are the ultrastructural features of MCC ?

(2) Which proteasome configurations are sequestered in MCC ?

(3) Which proteins are involved in MCC formation and clearance ?

(4) Which proteins are responsible for proteasome translocations between the nucleus and cytoplasm ?

(5) Do MCC present major sites of proteolysis or are they used as storage sites ?

(6) What are the phenotypes of yeast mutants lacking MCC ?

Despite genetic evidence supporting the hypothesis that cell death in neurodegenerative diseases is a consequence of protein aggregation, there is still a controversy about the toxicity of protein aggregrates.

Our working hypothesis is that MCC formation prevents rather than promotes the progression of irreversible protein aggregations. Under physiological conditions MCC formation is mainly observed in non-dividing cells, G0-arrested cells, the most common cell state in our bodies. Difficulties in handling G0-arrested mammalian cells have made understanding of proteasomal protein degradation during G0-phase almost impossible. We hope that our basic research in yeast facilitates future approaches in mammalian systems.

Invited Reviews and Book Chapters

Enenkel, C. (2006) Yeast Proteasome Structure and Biogenesis. In the textbook “The Proteasome in Neurodegeneration” (Eds. L. Stefanis and J. Keller, Springer Publisher), p. 1-16.

Enenkel, C. (2011) Using native gel electrophoresis and phosphofluoroimaging to analyze GFP-tagged proteasomes. In the series of "Methods in Molecular Biology" (submitted to Humana Press Publisher).

Peer-reviewed Journal Articles

Lehmann, A., Janek, K., Braun, B., Kloetzel, P.-M., and Enenkel, C. (2002) 20S proteasomes are imported as precursor complexes into the nucleus of yeast. J. Mol. Biol. 317, 401-413.

Fehlker, M., Wendler, P., Lehmann, A., and Enenkel, C. (2003) Blm3 is part of nascent proteasomes and is involved in a late stage of nuclear proteasome assembly. EMBO rep. 4, 559-563.

Wendler, P., Lehmann, A., Janek, K., Baumgart, S., and Enenkel, C. (2004) The bipartite nuclear localisation sequence of Rpn2 is required for nuclear import of proteasomal base complexes via karyopherin alpha beta and proteasome functions. J. Biol. Chem. 279, 37751-37762.

Lehmann, A., Jechow, K., and Enenkel, C. (2008) Blm10 binds to pre-activated proteasome core particles with open gate conformation. EMBO rep. 9, 1237-1243.

Bech-Otschir. D., Helfrich, A., Enenkel, C., Consiglieri, G., Seeger, M., Holzhütter, H., Dahlmann, B., and Kloetzel, P.-M. (2009) Poly-ubiquitin substrates allosterically activate their own degradation by the 26S proteasome Nat. Struct. Mol. Biol. 16, 219-225.

Lehmann, A., Niewienda, A., Jechow, K., Janek, K. and Enenkel, C. (2010) Ecm29 fulfils quality control functions in proteasome assembly Mol. Cell 38, 879-888.