Medalia Noa, Sharon Michal, Martinez-Arias Rosa, Mihalache Oana, Robinson Carol V, Medalia Ohad, Zwickl Peter
Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
J Struct Biol. 2006 Oct;156(1):84-92. doi: 10.1016/j.jsb.2006.03.015. Epub 2006 Apr 25.
We have cloned the proteasome and the proteasome activating nucleotidase (PAN) genes from the mesophilic archaeon Methanosarcina mazei and produced the respective proteins in Escherichia coli cultures. The recombinant complexes were purified to homogeneity and characterized biochemically, structurally, and by mass spectrometry. We found that the degradation of Bodipy-casein by Methanosarcina proteasomes was activated by Methanosarcina PAN. Notably, the Methanosarcina PAN unfolded GFP-SsrA only in the presence of Methanosarcina proteasomes. Structural analysis by 2D averaging electron microscopy of negatively stained complexes displayed the typical structure for the proteasome, namely four-striped side-views and sevenfold-symmetric top-views, with 15 nm height and 11 nm diameter. The structural analysis of the PAN preparation revealed also four-striped side-views, albeit with a height of 18 nm and sixfold-symmetric top-views with a diameter of 15 nm, which corresponds most likely to a dimer of two hexameric complexes. Mass spectrometric analysis of both the Methanosarcina and the Methanocaldococcus PAN proteins indicated hexameric complexes. In summary, we performed a functional and structural characterization of the PAN and proteasome complexes from the archaeon M. mazei and described unique new structural and functional features.
我们已从嗜温古菌马氏甲烷八叠球菌中克隆出蛋白酶体和蛋白酶体激活核苷酸酶（PAN）基因，并在大肠杆菌培养物中表达了相应的蛋白质。将重组复合物纯化至同质，并通过生化、结构和质谱分析进行表征。我们发现，马氏甲烷八叠球菌蛋白酶体对Bodipy-酪蛋白的降解被马氏甲烷八叠球菌PAN激活。值得注意的是，马氏甲烷八叠球菌PAN仅在存在马氏甲烷八叠球菌蛋白酶体的情况下才会使GFP-SsrA解折叠。通过对负染复合物进行二维平均电子显微镜结构分析，显示出蛋白酶体的典型结构，即四条带的侧视图和七重对称的顶视图，高度为15 nm，直径为11 nm。对PAN制剂的结构分析还显示出四条带的侧视图，尽管高度为18 nm，顶视图为六重对称，直径为15 nm，这很可能对应于两个六聚体复合物的二聚体。对马氏甲烷八叠球菌和嗜热栖热菌PAN蛋白的质谱分析均表明为六聚体复合物。总之，我们对来自马氏甲烷八叠球菌的PAN和蛋白酶体复合物进行了功能和结构表征，并描述了独特的新结构和功能特征。
