Identification, purification, and characterization of a high molecular weight, ATP-dependent activator (PA700) of the 20 S proteasome.

M Chu-Ping,J.H Vu,R.J Proske,C.A Slaughter,G.N Demartino

doi:10.1016/s0021-9258(17)41897-7

M Chu-Ping, J.H Vu + Show 3 more

Open Access

https://doi.org/10.1016/s0021-9258(17)41897-7

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Abstract

In order to identify protein complexes consisting of the proteasome and specific proteasome regulators, crude soluble lysates of red blood cells were fractionated by gel filtration chromatography and by velocity sedimentation centrifugation. The fractionated lysates were then tested for the relative distribution of proteasome activity, proteasome protein, and protein of a known proteasome activator, PA28. At least two proteasome complexes containing PA28 were identified. One of these complexes had an apparent molecular weight of approximately 1,750,000, and appeared to have much more proteasome activity than could be accounted for by its relative concentrations of proteasome and PA28 protein. We hypothesized that this complex contained another activator of the proteasome, and we sought to purify this activator from extracts of red blood cells. A proteasome activator with an apparent molecular weight of approximately 700,000 was identified, purified, and characterized. This activator, termed PA700, greatly stimulated the peptidase activities of the proteasome in an ATP-dependent fashion. PA700 was composed of about 16 polypeptides ranging in molecular weight from 20,000 to 100,000. The ATP-dependent activation of the proteasome by PA700 was closely linked to the formation of a high molecular weight complex that required no additional ATP for activated proteolysis. These results indicate that PA700 is a regulatory protein of the proteasome and is a component of at least one high molecular weight proteasome-containing complex occurring in cell extracts.

Highlights

This work describes the identification, large scale purification, and characterization of a new protein activator of the 20
204 67 indicate thaAt TP promotes the formation of a complex between the proteasome and PA700, and thact omplex formation results in activation of the proteasome
The size of the proteasome-PA700 complex suggests that PA700 is a component of the 1,750,000-dalton proteasome complex identified in the initial part of this work

Summary

RESULTS

Any detectable effecton the purification or properties of PA700. The results reported here come from purifications that employed each type of buffer. The extract was applied to a column of Sephacryl S-300 (100 x 5 c m ) equilibrated with Buffer H supplemented with 100 m~ NaCl and eluted with the same buffer. The fractions containing the highest levels of activity were pooled and subjectedto ion-exchangechromatographyby applying phy or by velocity sedimentation centrifugation. The bound proteins were eluted from the column with a linear gradient of NaCl from 100 to 350 m~ (1000 ml) in Buffer H. The peak with most of the proteasome activity had an apparent M, of approximately 1,750,000, a value much greater than thatof the purified proteasome. This dialyzed against a buffer of 20 m~ potassium phosphate, pH 7.6. The bound proteins were eluted from ylcoumarin; PAGE, polyacrylamide gelelectrophoresis

I Proteasome

A Proteasome

Findings

DISCUSSION

Proteasome