Bhardwaj Vidhi, Swanson Selene, Florens Laurence, Washburn Michael P, Workman Jerry L, Natarajan Krishnamurthy
Laboratory of Eukaryotic Gene Regulation, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
Stowers Institute for Medical Research, Kansas City, MO, USA.
bioRxiv. 2025 Jun 26:2025.06.23.660276. doi: 10.1101/2025.06.23.660276.
The fidelity of assembly of multiprotein complexes is essential for the formation of stable and functional protein complexes that are critical for cell growth and survival. In this context, TBP-associated factor (TAF) subunits maintain tight specificity for their integration into TFIID and SAGA complexes. In this work, using affinity purification-coupled mass spectrometry of epitope-tagged TFIID subunits TBP and TAF11, and the SAGA subunit TAF12L we identified components of the TFIID and SAGA complexes. Whereas TAF12 is a subunit of TFIID, the paralogous TAF12L is a subunit of the SAGA complex, and we further identified each of the TFIID and SAGA complex subunits with high confidence. We found that the steady-state levels of the H2B-H2A-like histone fold domain containing pairs, TAF12-TAF4 and TAF12L-Ada1 proteins, are mutually dependent on the stable expression of each other. Using RNA coimmunoprecipitation from polysome-containing extracts, we found that nascent TAF4 and Ada1 proteins interact with TAF12 and TAF12L, respectively, by a cotranslational mechanism in an ordered, sequential mode of assembly. Thus, our results indicate that heterodimerization of the TAF12 paralogs with cognate partners occur by sequential cotranslational assembly thereby ensuring both selectivity and stability of the H2A-H2B heterodimers in fungal pathogen
多蛋白复合物组装的保真度对于形成稳定且具有功能的蛋白复合物至关重要，而这些复合物对细胞生长和存活至关重要。在这种情况下，TBP相关因子（TAF）亚基对其整合到TFIID和SAGA复合物中保持严格的特异性。在这项工作中，我们使用表位标记的TFIID亚基TBP和TAF11以及SAGA亚基TAF12L的亲和纯化偶联质谱法，鉴定了TFIID和SAGA复合物的组成成分。虽然TAF12是TFIID的一个亚基，但同源的TAF12L是SAGA复合物的一个亚基，并且我们进一步高度可靠地鉴定了TFIID和SAGA复合物的每个亚基。我们发现，含有H2B - H2A样组蛋白折叠结构域的配对蛋白TAF12 - TAF4和TAF12L - Ada1蛋白的稳态水平相互依赖于彼此的稳定表达。使用来自含多核糖体提取物的RNA共免疫沉淀，我们发现新生的TAF4和Ada蛋白分别通过共翻译机制以有序的顺序组装模式与TAF12和TAF12L相互作用。因此，我们的结果表明，TAF12同源物与同源伴侣的异源二聚化通过顺序共翻译组装发生，从而确保真菌病原体中H2A - H2B异源二聚体的选择性和稳定性。
