裂殖酵母SAGA各亚基亚细胞荧光定位分析

SAGA(Spt-Ada-Gcn5 Acetyltransferase complex)是一个多亚基保守的转录复合物, 在裂殖酵母(Schizosaccharomyces pombe)里由19个亚基组成, 调控体内10%基因的转录。文章通过构建原位整合荧光菌株, 完整地分析了SAGA所有亚基的亚细胞荧光定位。荧光数据显示这些亚基的定位可分为4种类型, 提示SAGA亚基除共同参与转录调控之外, 可能还有其他功能。SAGA亚基Sgf73是联系去泛素化模块与SAGA其他模块的桥梁, 它的缺失不仅明显减少了去泛素化亚基Ubp8、Sgf11、Sus1核内的定位, 同时也影响了乙酰化亚基Gcn5、Sgf29、Ngg1以及核心结构亚基Spt7在细胞核内的定位, 这提示Sgf73对维持SAGA的酶学功能和稳定性至关重要。另外, sgf73+的缺失还造成了胞质分裂的缺陷, 导致细胞出现多核多膈膜表型。在△sgf73里过量表达膈膜降解途径中的关键基因ace2+和mid2+的回补结果表明, ace2+不能回补sgf73+缺失造成的缺陷, 而mid2+也仅能部分回补, 提示Sgf73可能还通过其他途径影响了胞质分裂。     

The ADA complex is a distinct histone acetyltransferase complex in Saccharomyces cerevisiae.

We have identified two Gcn5-dependent histone acetyltransferase (HAT) complexes from Saccharomyces cerevisiae, the 0.8-MDa ADA complex and the 1.8-MDa SAGA complex. The SAGA (Spt-Ada-Gcn5-acetyltransferase) complex contains several subunits which also function as part of other protein complexes, including a subset of TATA box binding protein-associated factors (TAFIIs) and Tra1. These observations raise the question of whether the 0.8-MDa ADA complex is a subcomplex of SAGA or whether it is a distinct HAT complex that also shares subunits with SAGA. To address this issue, we sought to determine if the ADA complex contained subunits that are not present in the SAGA complex. In this study, we report the purification of the ADA complex over 10 chromatographic steps. By a combination of mass spectrometry analysis and immunoblotting, we demonstrate that the adapter proteins Ada2, Ada3, and Gcn5 are indeed integral components of ADA. Furthermore, we identify the product of the S. cerevisiae gene YOR023C as a novel subunit of the ADA complex and name it Ahc1 for ADA HAT complex component 1. Biochemical functions of YOR023C have not been reported. However, AHC1 in high copy numbers suppresses the cold sensitivity caused by particular mutations in HTA1 (I. Pinto and F. Winston, personal communication), which encodes histone H2A (J. N. Hirschhorn et al., Mol. Cell. Biol. 15:1999-2009, 1995). Deletion of AHC1 disrupted the integrity of the ADA complex but did not affect SAGA or give rise to classic Ada(-) phenotypes. These results indicate that Gcn5, Ada2, and Ada3 function as part of a unique HAT complex (ADA) and represent shared subunits between this complex and SAGA.