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Abstract：
A recently-discovered protein post-translational modification, lysine polyphosphorylation (K-PPn), consists of the covalent attachment of inorganic polyphosphate (polyP) to lysine residues. The nonenzymatic nature of K-PPn means that the degree of this modification depends on both polyP abundance and the amino acids surrounding the modified lysine. K-PPn was originally discovered in budding yeast (Saccharomyces cerevisiae), in which polyP anabolism and catabolism are well-characterized. However, yeast vacuoles accumulate large amounts of polyP, and upon cell lysis, the release of the vacuolar polyP could nonphysiologically cause K-PPn of nuclear and cytosolic targets. Moreover, yeast vacuoles possess two very active endopolyphosphatases, Ppn1 and Ppn2, that could have opposing effects on the extent of K-PPn. Here, we characterized the contribution of vacuolar polyP metabolism to K-PPn of two yeast proteins, Top1 (DNA topoisomerase 1) and Nsr1 (nuclear signal recognition 1). We discovered that whereas Top1-targeting K-PPn is only marginally affected by vacuolar polyP metabolism, Nsr1-targeting K-PPn is highly sensitive to the release of polyP and of endopolyphosphatases from the vacuole. Therefore, to better study K-PPn of cytosolic and nuclear targets, we constructed a yeast strain devoid of vacuolar polyP by targeting the exopolyphosphatase Ppx1 to the vacuole and concomitantly depleting the two endopolyphosphatases (ppn1Δppn2Δ, vt-Ppx1). This strain enabled us to study K-PPn of cytosolic and nuclear targets without the interfering effects of cell lysis on vacuole polyP and of endopolyphosphatases. Furthermore, we also define the fundamental nature of the acidic amino acid residues to the K-PPn target domain.
摘要：
最近发现的蛋白质翻译后修饰，赖氨酸多磷酸化（K-PPn），由无机多磷酸盐（polyP）与赖氨酸残基的共价连接组成。K-PPn的非酶性质意味着这种修饰的程度取决于polyP丰度和修饰的赖氨酸周围的氨基酸。K-PPn最初是在出芽酵母（酿酒酵母）中发现的，其中polyP合成代谢和分解代谢被充分表征。然而,酵母液泡积累了大量的polyP，细胞裂解后，液泡polyP的释放可以非生理地引起核和胞浆靶的K-PPn。此外，酵母液泡具有两种非常活跃的内聚磷酸酶，Ppn1和Ppn2，可能对K-PPn的程度有相反的影响。这里,我们表征了两种酵母蛋白的液泡聚P代谢对K-PPn的贡献，Top1(DNA拓扑异构酶1)和Nsr1(核信号识别1)。我们发现，而Top1靶向K-PPn仅受到液泡polyP代谢的轻微影响，靶向Nsr1的K-PPn对从液泡中释放polyP和内切多磷酸酶高度敏感。因此,为了更好地研究细胞溶质和核靶标的K-PPn，我们通过将外聚磷酸酶Ppx1靶向液泡并同时消耗两种内聚磷酸酶（ppn1Δppn2Δ，vt-Ppx1）。该菌株使我们能够研究细胞溶质和核靶标的K-PPn，而不会干扰细胞裂解对液泡polyP和内切多聚磷酸酶的影响。此外，我们还定义了K-PPn靶结构域的酸性氨基酸残基的基本性质。
