ATP‐dependent reversible association of proteasomes with multiple protein components to form 26S complexes that degrade ubiquitinated proteins in human HL‐60 cells

Abstract

The role of proteasomes in ubiquitin (LJb)-dependent protein degradation was studied by analyzing lysates of human promyelocytic leukemia HL-60 cells by glycerol density gradient centrifugation.High succinyl-Leu-Lcu-VaI-Tyr-4-methylcoumaryl-7-amide hydrolyzing activity was found in the 26s fraction, whereas the 20s fraction containing proteaomes had no activity. Addition of 0.05% sodium dodecylsulfate to the latter fraction, however, induced marked activity. The 26s. but not the 20s fraction catalyzed ATP-dependent degradation of [~251]lysozyme-Ub conjugate. Depletion from the lysate of ATP caused complete shift of the active 26s complex to the latent 20s form. whereas in the lysate prepared from ATP-depleted cells, ATP converted 20s proteasomes to 26s complexes. The immunoprecipitatcd 26s complexes were found to consist of proteasomcs and 13-l 5 other proteins ranging in size from 35 to 1 IO kDa. We conclude that in the lysate. latent proteasomes undergo reversible. ATP-dependent association with multiple protein components to form 26s complexes "Ihat catalyze ATP-dependent degradation of Ub-protein conjugates.

Electron microscopy of 26 S complex containing 20 S proteasome

“…The 26 S complex of rat liver was purified by density gradient centrifugation of 10-40% glycerol in Tris-HC1 buffer containing 2 mM ATP according to the method ofOrino et al [5] (on a human leukemia cell system) with modifications. Details of the modifications will be published by Tanaka together with biochemical properties of the complex including its noted stability in the presence and absence of ATP.…”

“…Uppsala, Sweden) in the final stage of purification. The purified complex with the activity to degrade ubiquitinated lysozyme in the presence of ATP as determined according to [5] was applied to a TSK G4000SW column (Tosoh, Tokyo) and eluted Fig. 1 gives an example of the electron micrographs of the molecular species contained in the peak fraction ofTSK G4000SW column elution.…”

“…It has recently been shown that one of the enzyme complexes of this type. called 26 S complex, is associated with a multiprotease complex known as proteasome [3][4][5]. Association between 20 S proteasome and the rest of the complex is labile under the purification conditions so far employed and the structure of the intact 26 S complex has remained elusive.…”

“…Association between 20 S proteasome and the rest of the complex is labile under the purification conditions so far employed and the structure of the intact 26 S complex has remained elusive. We have recently been successful in obtaining the complex from rat liver as a stable entity in the presence of glycerol and ATP [5] (with modifications to be published by Tanaka) and the present report deals with the electron micrograph of the complex in the closest form to the intact one.…”

Effect of a tumour-produced lipid-mobilizing factor on protein synthesis and degradation

“…C 2 C 12 myoblasts were treated with different concentrations of LMF for 24 h and the activity of the 26S proteasome was determined according to the method of Orino et al (1991). Cellular supernatants were prepared in 20 mM Tris.…”