Brown C E, Tarun S Z, Boeck R, Sachs A B
Department of Molecular and Cell Biology, University of California at Berkeley 94720, USA.
Mol Cell Biol. 1996 Oct;16(10):5744-53. doi: 10.1128/MCB.16.10.5744.
The Pab1p-dependent poly(A) nuclease (PAN) from Saccharomyces cerevisiae copurifies with polypeptides of approximately 127 and 76 kDa. Previously, it was demonstrated that the 127-kDa Pan2 protein is required for PAN activity (R. Boeck, S. Tarun, M. Reiger, J. Deardorff, S. Müller-Auer, and A.B. Sachs, J. Biol. Chem. 271:432-438, 1996). Here we demonstrate that the 76-kDa protein, encoded by the nonessential PAN3 gene, is also required for enzymatic activity. Deletion of PAN3 resulted in the loss of PAN activity in yeast extracts, and immunodepletion of Pan3p from purified PAN fractions abolished enzymatic activity. We show by coimmunoprecipitation and directed two-hybrid studies that the Pan2 and Pan3 proteins physically interact. In addition, we demonstrate that a deletion of PAN2, PAN3, or both resulted in similar increases in mRNA poly(A) tail lengths in vivo. These data strongly suggest that both Pan2p and Pan3p are required subunits of the PAN enzyme and that PAN functions in vivo to shorten mRNA poly(A) tails.
来自酿酒酵母的Pab1p依赖性聚腺苷酸核酸酶（PAN）与大约127 kDa和76 kDa的多肽共纯化。以前的研究表明，127 kDa的Pan2蛋白是PAN活性所必需的（R. Boeck、S. Tarun、M. Reiger、J. Deardorff、S. Müller-Auer和A.B. Sachs，《生物化学杂志》271:432 - 438，1996年）。在此我们证明，由非必需的PAN3基因编码的76 kDa蛋白也是酶活性所必需的。删除PAN3导致酵母提取物中PAN活性丧失，从纯化的PAN组分中免疫去除Pan3p消除了酶活性。我们通过共免疫沉淀和直接的双杂交研究表明，Pan2和Pan3蛋白存在物理相互作用。此外，我们证明删除PAN2、PAN3或两者在体内均导致mRNA聚腺苷酸尾巴长度出现类似增加。这些数据有力地表明，Pan2p和Pan3p都是PAN酶的必需亚基，并且PAN在体内发挥作用缩短mRNA聚腺苷酸尾巴。
