Analysis of Drosophila 26 S proteasome using RNA interference.
We have utilized double-stranded RNA interference (RNAi) to examine the effects of reduced expression of individual subunits of the 26 S proteasome in Drosophila S2 cells. RNAi significantly decreased mRNA and protein levels of targeted subunits of both the core 20 S proteasome and the PA700 regulatory complex. Cells deficient in any of several 26 S proteasome subunits (e.g. d beta 5, dRpt1, dRpt2, dRpt5, dRpn2, and dRpn12) displayed decreased proteasome activity (as judged by hydrolysis of succinyl-Leu-Leu-Val-Tyr-aminomethylcoumarin), increased apoptosis, decreased cell proliferation without a specific block of the cell cycle, and accumulation of ubiquitinated cellular proteins. RNAi of many individual 26 S proteasome subunits promoted increased expression of many non-targeted subunits. This effect was not mimicked by chemical proteasome inhibitors such as lactacystin. Reduced expression of most targeted subunits disrupted the assembly of the 26 S proteasome. RNAi of six of eight targeted PA700 subunits disrupted that structure and caused accumulation of increased levels of uncapped 20 S proteasome. Notable exceptions included RNAi of dRpn10, a polyubiquitin binding subunit, and dUCH37, a ubiquitin isopeptidase. dRpn10-deficient cells showed a significant increase in succinyl-Leu-Leu-Val-Tyr-aminomethylcoumarin hydrolyzing activity of the 26 S proteasomes but accumulated polyubiquitinated proteins. d beta 5-Deficient cells had a phenotype similar to that of most PA700-deficient cells but also accumulated low molecular mass complexes containing subunits of the 20 S proteasome, probably representing unassembled precursors of the 20 S proteasomes. Cells deficient in several of the 26 S proteasome subunits were more resistant to otherwise toxic concentrations of various proteasome inhibitors. Our data suggest that those cells adapted to grow in conditions of impaired ubiquitin and proteasome-dependent protein degradation.
Wójcik
Cezary
C
Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9040, USA.
DeMartino
George N
GN
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D017931:DNA Primers; D004331:Drosophila melanogaster / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010447:Peptide Hydrolases / Q000235:genetics*; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D012330:RNA, Double-Stranded / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic
D017931:DNA Primers; D010446:Peptide Fragments; D011480:Protease Inhibitors; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease