An easily dissociated 26 S proteasome catalyzes an essential ubiquitin-mediated protein degradation pathway in Trypanosoma brucei.
The 26 S proteasome, a complex between the 20 S proteasome and 19 S regulatory units, catalyzes ATP-dependent degradation of unfolded and ubiquitinated proteins in eukaryotes. We have identified previously 20 S and activated 20 S proteasomes in Trypanosoma brucei, but not 26 S proteasome. However, the presence of 26 S proteasome in T. brucei was suggested by the hydrolysis of casein by cell lysate, a process that requires ATP but is inhibited by lactacystin, and the lactacystin-sensitive turnover of ubiquitinated proteins in the intact cells. T. brucei cDNAs encoding the six proteasome ATPase homologues (Rpt) were cloned and expressed. Five of the six T. brucei Rpt cDNAs, except for Rpt2, were capable of functionally complementing the corresponding rpt deletion mutants of Saccharomyces cerevisiae. Immunoblots showed the presence in T. brucei lysate of the Rpt proteins, which co-fractionated with the yeast 19 S proteasome complex by gel filtration and localized in the 19 S fraction of a glycerol gradient. All the Rpt and putative 19 S non-ATPase (Rpn) proteins were co-immunoprecipitated from T. brucei lysate by individual anti-Rpt antibodies. Treatment of T. brucei cells with a chemical cross-linker resulted in co-immunoprecipitation of 20 S proteasome with all the Rpt and Rpn proteins that sedimented in a glycerol gradient to the position of 26 S proteasome. These data demonstrate the presence of 26 S proteasome in T. brucei cells, which apparently dissociate into 19 S and 20 S complexes upon cell lysis. RNA interference to block selectively the expression of proteasome 20 S core and Rpt subunits resulted in significant accumulation of ubiquitinated proteins accompanied by cessation of cell growth. Expression of yeast RPT2 gene in T. brucei Rpt2-deficient cells could not rescue the lethal phenotype, thus confirming the incompatibility between the two Rpt2s. The T. brucei 11 S regulator (PA26)-deficient RNA interference cells grew normally, suggesting the dispensability of activated 20 S proteasome in T. brucei.
Li
Ziyin
Z
Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446, USA.
Zou
Chun-Bin
CB
Yao
Yi
Y
Hoyt
Martin A
MA
McDonough
Stephen
S
Mackey
Zachary B
ZB
Coffino
Philip
P
Wang
Ching C
CC
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D018076:DNA, Complementary / Q000235:genetics; D017353:Gene Deletion; D007700:Kinetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010447:Peptide Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D046988:Proteasome Endopeptidase Complex; D017510:Protein Folding; D015800:Protozoan Proteins / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics; D025801:Ubiquitin / Q000378:metabolism*
D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D025801:Ubiquitin; D000255:Adenosine Triphosphate; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease