Proteasome activator 200: the heat is on.

Abstract

Proteasomes play a key regulatory role in all eukaryotic cells by removing proteins in a timely manner. There are two predominant forms: The 20S core particle (CP) can hydrolyze peptides and certain unstructured proteins, and the 26S holoenzyme is able to proteolyse most proteins conjugated to ubiquitin. The 26S complex consists of a CP barrel with a 19S regulatory particle (RP; a.k.a PA700) attached to its outer surface. Several studies purified another proteasome activator with a MW of 200 kDa (PA200) that attaches to the same outer ring of the CP. A role for PA200 has been demonstrated in spermatogenesis, in response to DNA repair and in maintenance of mitochondrial inheritance. Enhanced levels of PA200-CP complexes are observed under conditions in which either activated or disrupted CP prevail, suggesting it participates in regulating overall proteolytic activity. PA200, or its yeast ortholog Blm10, may also incorporate into 26S proteasomes yielding PA200-CP-RP hybrids. A three-dimensional molecular structure determined by x-ray crystallography of Blm10-CP provides a model for activation. The carboxy terminus of Blm10 inserts into a dedicated pocket in the outer ring of the CP surface, whereas multiple HEAT-like repeats fold into an asymmetric solenoid wrapping around the central pore to stabilize a partially open conformation. The resulting hollow domelike structure caps the entire CP surface. This asymmetric structure may provide insight as to how the 19S RP, with two HEAT repeatlike subunits (Rpn1, Rpn2) alongside six ATPases (Rpt1-6), attaches to the same surface of the CP ring, and likewise, induces pore opening.

Fig. 1.

PA200/Blm10 association with different proteasome species, and related roles. Because most evidence for proteasome complexes with PA200 was obtained in yeast, we use the yeast nomenclature, Blm10, in this figure: A, Biological functions of Blm10. Blm10 is involved in different cellular functions such as enhancing hydrolysis of small peptides (–23), maintenance of mitochondrial functions (24), response to DNA repair (21, 34), and in proteasome maturation and assembly (–43). Whether it has an inhibitory or activating role in these processes is still a matter of debate. B, Blm10 and proteasome assembly. PA200/Blm10 was detected in inactive proteasome assembly precursors (39, 40), as well as in complexes with immature or nascent 20S CP (30). Following dimerization of half proteasomes (1/2 CP; a.k.a 15S proteasomes) the β-subunits are processed to yield the active mature form of the 20S CP. These active, yet latent species can be activated upon binding of proteasome activators such as the 19S RP (PA700) or by attachment of non-ATPase activators such as PA200/Blm10. Complexes of PA200 with activated 20S CP were found as symmetric or asymmetric versions (Blm10-CP and (Blm10)₂-CP) (22, 35, 41). These species were labeled CPx in (22)). PA200 is also found as an opposing appendage in hybrid complexes in the form of Blm10-CP-19S (22, 41).

Fig. 2.

Structure of Blm10–20S complexes as revealed by x-ray crystallography (24). A, Side view of the symmetric Blm10-CP-Blm10 complex. The four-ringed 20S CP (each α or β ring is color coded separately) capped on both ends by Blm10 in white. Blm10 forms a dome-like structure, with a lateral opening. B, Top view of Blm10-CP complex. Blm10 (in red) contacts the α-ring surface of the 20S CP and opens the axial gates of each of the seven α-subunits of the 20S (in white). The cartoon presentation of Blm10 highlights the repetitive α-helical repeats that interact with all seven α-subunits below encircling the central pore. C, Docking of Blm10 onto 20S CP α-ring. The three C-terminal amino acids of Blm10 (red tail jutting downward from dome) insert into a pocket between proteasome subunits α5 and α6 (the “lys66” pocket). Binding of Blm10 reorients Pro17 of α5, leading to rearrangement of α5 N-terminal residues away from central pore and toward Blm10 cavity (white tail pointing upwards into Blm10 cavity). The resulting partially open gate (see also panel B) may alleviate some hindrance to substrate traffic. D, Solenoid fold of blm10, Top view. Blm10 forms a solenoid structure formed by the 32 HEAT repeats, each consisting of a α-turn-α of variable length (29). The helixes are color coded from Red at the C terminus (docking into the 20S; panel C) to Blue at the N terminus (peak of dome) to highlight the super helical nature of Blm10.