YEAST CYCLOHEXIMIDE-RESISTANT CRL MUTANTS ARE PROTEASOME MUTANTS DEFECTIVE IN PROTEIN-DEGRADATION

In 1988 McCusker and Haber generated a series of mutants which are res istant to the minimum inhibitory concentration of the protein synthesi s inhibitor cycloheximide. These cycloheximide-resistant, temperature- sensitive (crl) mutants, in addition, exhibited other pleiotropic phen otypes, e.g., incorrect response to starvation, hypersensitivity again st amino acid analogues, and other protein synthesis inhibitors. Tempe rature sensitivity of one of these mutants, crl3-2, had been found to be suppressed by a mutation, SCL1-1, which resided in an a-type subuni t of the 20S proteasome. We cloned the CRL3 gene by complementation an d found CRL3 to be identical to the SUG1/CIM3 gene coding for a subuni t of the 19S cap complex of the 26S proteasome. Another mutation, crl2 1, revealed to be allelic with the 20S proteasomal gene PRE3, crl3-2 a nd crl21 mutant cells show significant defects in proteasome-dependent proteolysis, whereas the SCL1-1 suppressor mutation causes partial re storation of crl3-2-induced proteolytic defects. Notably, cycloheximid e resistance was also detected for other proteolytically deficient pro teasome mutants (pre1-1, pye2-1, pre3-1, pre4-1). Moreover, proteasoma l genes were found within genomic sequences of 9 of 13 chromosomal loc i to which crl mutations had been mapped. We therefore assume that mos t if not all crl mutations reside in the proteasome and that phenotype s found are a result of defective protein degradation.