Abstract
In Archaea, an hexameric ATPase complex termed PAN promotes proteins unfolding and translocation into the 20 S proteasome. PAN is highly homologous to the six ATPases of the eukaryotic 19 S proteasome regulatory complex. Thus, insight into the mechanism of PAN function may reveal a general mode of action mutual to the eukaryotic 19 S proteasome regulatory complex. In this study we generated a three-dimensional model of PAN from tomographic reconstruction of negatively stained particles. Surprisingly, this reconstruction indicated that the hexameric complex assumes a two-ring structure enclosing a large cavity. Assessment of distinct three-dimensional functional states of PAN in the presence of adenosine 5'-O-(thiotriphosphate) and ADP and in the absence of nucleotides outlined a possible mechanism linking nucleotide binding and hydrolysis to substrate recognition, unfolding, and translocation. A novel feature of the ATPase complex revealed in this study is a gate controlling the "exit port" of the regulatory complex and, presumably, translocation into the 20 S proteasome. Based on our structural and biochemical findings, we propose a possible model in which substrate binding and unfolding are linked to structural transitions driven by nucleotide binding and hydrolysis, whereas translocation into the proteasome only depends upon the presence of an unfolded substrate and binding but not hydrolysis of nucleotide.
Highlights
In eukaryotic cells most protein breakdown in the cytosol and nucleus is catalyzed by the 26 S proteasome
Based on our structural and biochemical findings, we propose a possible model in which substrate binding and unfolding are linked to structural transitions driven by nucleotide binding and hydrolysis, whereas translocation into the proteasome only depends upon the presence of an unfolded substrate and binding but not hydrolysis of nucleotide
PAN recognizes the bacterial targeting sequence ssrA and efficiently unfolds and translocates globular substrates, like green fluorescent protein, when tagged with ssrA [15]. In both PAN and the S proteasome regulatory complexes, ATP is essential for substrate unfolding and translocation and for opening of the gated channel in the ␣ ring through which substrates enter the S particle [15,16,17]
Summary
PAN was purified according to Navon and Goldberg [15]. The ␣ and  subunits of the MJ 20 S proteasome were cloned into the pET22 plasmid downstream of the T7 promoter. The MJ 20 S complex was expressed in E. coli BL21 and purified by. Heating the resuspended bacterial pellet (50 mM Hepes, pH 7.5) and removing the aggregated E. coli proteins by centrifugation. The cleared lysate was loaded onto an anion exchange column (Mono Q), and PAN-containing fractions were pooled, concentrated, and dialyzed against 50 mM Hepes, pH 7.5
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