1. Mechanism of Substrate Unfolding and Translocation by the Regulatory Particle of the Proteasome from Methanocaldococcus jannaschii 
Fan Zhang, Zhuoru Wu, Ping Zhang, Geng Tian, Daniel Finley, Yigong Shi 
Molecular Cell 
Volume 34, Issue 4, Pages (May 2009)
DOI: /j.molcel
Copyright © 2009 Elsevier Inc. Terms and Conditions

2. Figure 1 Functional Characterization of the PAN Complex
(A) The PAN complex fully unfolds and releases the globular protein GFP-ssrA in the absence of the 20S CP. GFP alone undergoes minor, reversible conformational changes at 65°C (dotted line). Increasing molar ratios of PAN hexamer over GFP-ssrA results in increased rates of unfolding.
(B) The presence of 20S CP greatly accelerates the unfolding of substrate.
(C) The 20S CP does not increase the rate of ATP hydrolysis by the PAN complex during unfolding reactions.
(D) Improved substrate unfolding by the 20S CP is negated by inhibition of the protease activity. Curves shown in (B) and (C) are representative of at least three independent experiments with standard deviation (SD) of less than 5%. Error bars represent mean ± SD of at least three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

3. Figure 2
Role of the Axial Channel of Subcomplex I in the Function of the PAN Complex
(A) A schematic diagram of the structure of subcomplex I, with the axial channel highlighted. Residues in the distal ring, the proximal ring, and the intervening hydrophobic surface are shown in close-up views.
(B) Impact of mutations of the axial channel on unfolding and degradation activities of the PAN complex. Error bars represent mean ± SD of at least three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

4. Figure 3
Role of the Coiled Coils of Subcomplex I in the Function of the PAN Complex
(A) A schematic diagram of the structure of subcomplex I, with the coiled coils highlighted. Surface residues in the coiled coils are shown in a close-up view.
(B) Impact of mutations of the coiled coils on unfolding and degradation activities of the PAN complex. Error bars represent mean ± SD of at least three independent experiments.
(C) Removal of negative charges in the ssrA tag resulted in accelerated unfolding rates.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

5. Figure 4
Role of the Axial Channel of Subcomplex II in the Function of the PAN Complex
(A) A schematic diagram of the modeled structure of subcomplex II, with the axial channel highlighted. Residues in the Ar-Φ loop and the pore 2 loop are shown in close-up views. Sequence alignment between PAN and the Rpt subunits is shown in the Ar-Φ and the pore 2 loops.
(B) Impact of mutations of the axial channel on unfolding and degradation activities of the PAN complex. Error bars represent mean ± SD of at least three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

6. Figure 5 Role of the Interface between Subcomplexes I and II
(A) A schematic diagram of the interface between subcomplexes I and II, with interface residues and structural motifs shown in a close-up view.
(B) A schematic diagram of the distal face of subcomplex II, with interface amino acids labeled.
(C) Impact of mutations of the interface residues on unfolding and degradation activities of the PAN complex. The mutants 150G and 150G2 denote insertion of one and two Gly residues after amino acid 150, respectively. The same nomenclature applies to 157G and 157G2. Error bars represent mean ± SD of at least three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

7. Figure 6 A Working Model for the PAN Complex
In this model, folded substrate protein is bound to the distal face of subcomplex I. The degradation tag is then recognized by surface motifs in subcomplex II, likely by the Ar-Φ loop. Cycles of ATP hydrolysis result in a pulling force that is exerted on the folded substrate protein. Translocation is hindered when the folded substrate is pulled flush against the CC-OB domain because the entry port is smaller than the folded protein domain. Continued pulling forces from subcomplex II generate tension on the leading end of the substrate, leading to its unfolding (step 2) and the resumption of translocation. Degradation of the unfolded polypeptide by the 20S CP enhances PAN activity by providing an efficient mechanism for product release.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions