TY - JOUR
T1 - The regulatory complex of Drosophila melanogaster 26S proteasomes
T2 - Subunit composition and localization of a deubiquitylating enzyme
AU - Hölzl, Harald
AU - Kapelari, Barbara
AU - Kellermann, Josef
AU - Seemüller, Erika
AU - Sümegi, Máté
AU - Udvardy, Andor
AU - Medalia, Ohad
AU - Sperling, Joseph
AU - Müller, Shirley A.
AU - Engel, Andreas
AU - Baumeister, Wolfgang
PY - 2000/7/10
Y1 - 2000/7/10
N2 - Drosophila melanogaster embryos are a source for homogeneous and stable 26S proteasomes suitable for structural studies. For biochemical characterization, purified 26S proteasomes were resolved by two-dimensional (2D) gel electrophoresis and subunits composing the regulatory complex (RC) were identified by amino acid sequencing and immunoblotting, before corresponding cDNAs were sequenced. 17 subunits from Drosophila RCs were found to have homologues in the yeast and human RCs. An additional subunit, p37A, not yet described in RCs of other organisms, is a member of the ubiquitin COOH-terminal hydrolase family (UCH). Analysis of EM images of 26S proteasomes-UCH-inhibitor complexes allowed for the first time to localize one of the RC's specific functions, deubiquitylating activity. The masses of 26S proteasomes with either one or two attached RCs were determined by scanning transmission EM (STEM), yielding a mass of 894 kD for a single RC. This value is in good agreement with the summed masses of the 18 identified RC subunits (932 kD), indicating that the number of subunits is complete.
AB - Drosophila melanogaster embryos are a source for homogeneous and stable 26S proteasomes suitable for structural studies. For biochemical characterization, purified 26S proteasomes were resolved by two-dimensional (2D) gel electrophoresis and subunits composing the regulatory complex (RC) were identified by amino acid sequencing and immunoblotting, before corresponding cDNAs were sequenced. 17 subunits from Drosophila RCs were found to have homologues in the yeast and human RCs. An additional subunit, p37A, not yet described in RCs of other organisms, is a member of the ubiquitin COOH-terminal hydrolase family (UCH). Analysis of EM images of 26S proteasomes-UCH-inhibitor complexes allowed for the first time to localize one of the RC's specific functions, deubiquitylating activity. The masses of 26S proteasomes with either one or two attached RCs were determined by scanning transmission EM (STEM), yielding a mass of 894 kD for a single RC. This value is in good agreement with the summed masses of the 18 identified RC subunits (932 kD), indicating that the number of subunits is complete.
KW - ATP-dependent proteolysis
KW - Electron microscopy
KW - Protein degradation
KW - Ubiquitin
KW - Ubiquitin hydrolase
UR - https://www.scopus.com/pages/publications/0342297875
U2 - 10.1083/jcb.150.1.119
DO - 10.1083/jcb.150.1.119
M3 - Article
AN - SCOPUS:0342297875
SN - 0021-9525
VL - 150
SP - 119
EP - 129
JO - Journal of Cell Biology
JF - Journal of Cell Biology
IS - 1
ER -