Brooks P, Fuertes G, Murray R Z, Bose S, Knecht E, Rechsteiner M C, Hendil K B, Tanaka K, Dyson J, Rivett J
Department of Biochemistry, University of Bristol, School of Medical Sciences, Bristol BS8 1TD, U.K.
Biochem J. 2000 Feb 15;346 Pt 1(Pt 1):155-61.
Proteasomes can exist in several different molecular forms in mammalian cells. The core 20S proteasome, containing the proteolytic sites, binds regulatory complexes at the ends of its cylindrical structure. Together with two 19S ATPase regulatory complexes it forms the 26S proteasome, which is involved in ubiquitin-dependent proteolysis. The 20S proteasome can also bind 11S regulatory complexes (REG, PA28) which play a role in antigen processing, as do the three variable gamma-interferon-inducible catalytic beta-subunits (e.g. LMP7). In the present study, we have investigated the subcellular distribution of the different forms of proteasomes using subunit specific antibodies. Both 20S proteasomes and their 19S regulatory complexes are found in nuclear, cytosolic and microsomal preparations isolated from rat liver. LMP7 was enriched approximately two-fold compared with core alpha-type proteasome subunits in the microsomal preparations. 20S proteasomes were more abundant than 26S proteasomes, both in liver and cultured cell lines. Interestingly, some significant differences were observed in the distribution of different subunits of the 19S regulatory complexes. S12, and to a lesser extent p45, were found to be relatively enriched in nuclear fractions from rat liver, and immunofluorescent labelling of cultured cells with anti-p45 antibodies showed stronger labelling in the nucleus than in the cytoplasm. The REG was found to be localized predominantly in the cytoplasm. Three- to six-fold increases in the level of REG were observed following gamma-interferon treatment of cultured cells but gamma-interferon had no obvious effect on its subcellular distribution. These results demonstrate that different regulatory complexes and subpopulations of proteasomes have different distributions within mammalian cells and, therefore, that the distribution is more complex than has been reported for yeast proteasomes.
蛋白酶体在哺乳动物细胞中可以以几种不同的分子形式存在。包含蛋白水解位点的核心20S蛋白酶体，在其圆柱形结构的末端结合调节复合物。它与两个19S ATP酶调节复合物一起形成26S蛋白酶体，参与泛素依赖性蛋白水解。20S蛋白酶体也可以结合11S调节复合物（REG，PA28），其在抗原加工中发挥作用，三种可变的γ-干扰素诱导的催化β亚基（例如LMP7）也是如此。在本研究中，我们使用亚基特异性抗体研究了不同形式蛋白酶体的亚细胞分布。在从大鼠肝脏分离的核、胞质和微粒体制剂中都发现了20S蛋白酶体及其19S调节复合物。在微粒体制剂中，LMP7比核心α型蛋白酶体亚基富集约两倍。在肝脏和培养的细胞系中，20S蛋白酶体比26S蛋白酶体更丰富。有趣的是，在19S调节复合物的不同亚基的分布中观察到一些显著差异。发现S12以及程度较轻的p45在大鼠肝脏的核级分中相对富集，用抗p45抗体对培养细胞进行免疫荧光标记显示，细胞核中的标记比细胞质中的更强。发现REG主要定位于细胞质中。用γ-干扰素处理培养细胞后，观察到REG水平增加了三到六倍，但γ-干扰素对其亚细胞分布没有明显影响。这些结果表明，蛋白酶体的不同调节复合物和亚群在哺乳动物细胞内具有不同的分布，因此，其分布比酵母蛋白酶体的报道更为复杂。
