Kourmouli N, Theodoropoulos P A, Dialynas G, Bakou A, Politou A S, Cowell I G, Singh P B, Georgatos S D
Department of Basic Sciences, The University of Crete School of Medicine, 71 110 Heraklion, Crete, Greece.
EMBO J. 2000 Dec 1;19(23):6558-68. doi: 10.1093/emboj/19.23.6558.
To study the dynamics of mammalian HP1 proteins we have microinjected recombinant forms of mHP1alpha, M31 and M32 into the cytoplasm of living cells. As could be expected from previous studies, the three fusion proteins were efficiently transported into the nucleus and targeted specific chromatin areas. However, before incorporation into these areas the exogenous proteins accumulated in a peripheral zone and associated closely with the nuclear envelope. This transient association did not occur when the cells were treated with deacetylase inhibitors, indicating an acetylation-inhibited interaction. In line with these observations, recombinant HP1 proteins exhibited saturable binding to purified nuclear envelopes and stained the nuclei of detergent-permeabilized cells in a rim-like fashion. Competition experiments with various M31 mutants allowed mapping of the nuclear envelope-binding site within an N-terminal region that includes the chromodomain. A His(6)-tagged peptide representing this region inhibited recruitment of LAP2beta and B-type lamins around the surfaces of condensed chromosomes, suggesting involvement of HP1 proteins in nuclear envelope reassembly.
为了研究哺乳动物HP1蛋白的动力学，我们已将mHP1α、M31和M32的重组形式显微注射到活细胞的细胞质中。正如先前研究所预期的那样，这三种融合蛋白被有效地转运到细胞核中，并靶向特定的染色质区域。然而，在整合到这些区域之前，外源蛋白在外围区域积累，并与核膜紧密相关。当细胞用脱乙酰酶抑制剂处理时，这种短暂的关联并未发生，表明存在乙酰化抑制的相互作用。与这些观察结果一致，重组HP1蛋白对纯化的核膜表现出饱和结合，并以边缘样方式对去污剂通透的细胞的细胞核进行染色。用各种M31突变体进行的竞争实验允许在包含色域的N端区域内定位核膜结合位点。代表该区域的His(6)标记肽抑制了LAP2β和B型核纤层蛋白在浓缩染色体表面周围的募集，表明HP1蛋白参与了核膜的重新组装。
