ORIGINAL PAPER Arabidopsis sensitivity to protein synthesis inhibitors depends on 26S proteasome activity Jasmina Kurepa • Console ´e Karangwa • Liliana Sfichi Duke • Jan A. Smalle Received: 24 December 2009 / Revised: 3 January 2010 / Accepted: 7 January 2010 / Published online: 20 January 2010 Ó Springer-Verlag 2010 Abstract The 26S proteasome (26SP), the central prote- ase of the ubiquitin-dependent proteolysis pathway, con- trols the regulated proteolysis of functional proteins and the removal of misfolded and damaged proteins. In Arabid- opsis, cellular and stress response phenotypes of a number of mutants with partially impaired 26SP function have been reported. Here, we describe the responses of proteasome mutants to protein synthesis inhibitors. We show that the rpt2a-3, rpn10-1 and rpn12a-1 mutants are hypersensitive to the antibiotic hygromycin B, and tolerant to the trans- lation inhibitor cycloheximide (CHX) and herbicide L- phosphinothricin (PPT). In addition to the novel mecha- nism for herbicide tolerance, our data suggests that the combination of hygromycin B, CHX and PPT growth- response assays could be used as a facile diagnostic tool to detect altered 26SP function in plant mutants and trans- genic lines. Keywords Arabidopsis thaliana Á Cycloheximide Á Herbicide tolerance Á Hygromycin B Á L-Phosphinothricin Á Proteolysis Introduction The ubiquitin-proteasome system (UPS) regulates the degradation of most cellular proteins. It controls the proteolysis of key components of numerous signaling pathways, and thus regulates many—if not all—cellular processes (Smalle and Vierstra 2004; Vierstra 2009). The UPS is also essential for the removal of misfolded proteins that are generated either by mutations, translational errors or by the actions of cellular stressors (Goldberg 2003). These non-functional proteins need to be removed from the cell before they become proteotoxic, i.e., before they accumulate, aggregate and induce cell death. The main components of the UPS are ubiquitin (Ub), the enzymes that catalyze the attachment of Ub chains to target proteins, the enzymes that catalyze the hydrolysis of Ub chains from target proteins and finally, the 26S proteasome, a multi- subunit, multicatalytic protease that degrades the poly- ubiquitinated protein targets (Smalle and Vierstra 2004; Vierstra 2009). The 26S proteasome (26SP) is composed of two subcomplexes: the proteolytically active 20S protea- some (20SP) and the regulatory particle (RP) that recog- nizes, unfolds and channels most of the UPS targets into the 20SP for degradation (Kurepa and Smalle 2008). Protein synthesis and protein degradation by the 26SP are the two main phases in the life cycle of many proteins, and they are networked at a number of levels (Chuang et al. 2005; Guerrero et al. 2008; Reits et al. 2000; Schubert et al. 2000). On the genomic level, we see a footprint of the link between translation and degradation in the structure of genes encoding Ub itself (Finley et al. 1989). In Arabidopsis for instance, from 14 Ub genes, five encode a single Ub peptide fused to a ribosomal subunit (Callis et al. 1995, 1990). Another link between translation and the UPS is inferred from the ubiquitination of many components of the trans- lation machinery such as ribosomal subunits, ribosome associated proteins, and several translational initiation and elongation factors (Saracco et al. 2009; Strunk and Karbstein 2009). Proteasome-mediated co-translational proteolysis is Communicated by J. Register. J. Kurepa Á C. Karangwa Á L. S. Duke Á J. A. Smalle (&) Plant Physiology, Biochemistry, Molecular Biology Program, Department of Plant and Soil Sciences, College of Agriculture, University of Kentucky, 1401 University Drive, Lexington, KY 40546-0236, USA e-mail: jsmalle@uky.edu 123 Plant Cell Rep (2010) 29:249–259 DOI 10.1007/s00299-010-0818-8