Alvarez-Tabarés Isabel, Grallert Agnes, Ortiz Jose-Miguel, Hagan Iain M
CRUK Cell Division Group, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, M20 4BX, UK.
J Cell Sci. 2007 Oct 15;120(Pt 20):3589-601. doi: 10.1242/jcs.007567. Epub 2007 Sep 25.
PP1 holoenzymes are composed of a small number of catalytic subunits and an array of regulatory, targeting, subunits. The Schizosaccharomyces pombe genome encodes two highly related catalytic subunits, Dis2 and Sds21. The gene for either protein can be individually deleted, however, simultaneous deletion of both is lethal. We fused enhanced green fluorescent protein (EGFP) coding sequences to the 5' end of the endogenous sds21(+) and dis2(+) genes. Dis2.NEGFP accumulated in nuclei, associated with centromeres, foci at cell tips and endocytic vesicles. This actin-dependent endocytosis occurred between nuclei and growing tips and was polarised towards growing tips. When dis2(+) was present, Sds21.NEGFP was predominantly a nuclear protein, greatly enriched in the nucleolus. When dis2(+) was deleted, Sds21.NEGFP levels increased and Sds21.NEGFP was then clearly detected at centromeres, endocytic vesicles and cell tips. Dis2.NEGFP was recruited to cell tips by the formin binding, stress pathway scaffold Wsh3 (also known as Tea4). Wsh3/Tea4 modulates polarised tip growth in unperturbed cell cycles and governs polarised growth following osmotic stress. Mutating the PP1 recruiting RVXF motif in Wsh3/Tea4 blocked PP1 binding, altered cell cycle regulated growth to induce branching, induced branching from existing tips in response to stress, and blocked the induction of actin filaments that would otherwise arise from Wsh3/Tea4 overproduction.
PP1全酶由少量催化亚基以及一系列调节亚基、靶向亚基组成。粟酒裂殖酵母基因组编码两种高度相关的催化亚基，即Dis2和Sds21。任一蛋白质的基因都可单独缺失，然而，两者同时缺失是致死的。我们将增强型绿色荧光蛋白（EGFP）编码序列融合到内源性sds21(+)和dis2(+)基因的5'端。Dis2.NEGFP积聚在细胞核中，与着丝粒、细胞尖端的焦点和内吞小泡相关。这种肌动蛋白依赖性内吞作用发生在细胞核和生长尖端之间，并朝着生长尖端极化。当存在dis2(+)时，Sds21.NEGFP主要是一种核蛋白，在核仁中大量富集。当dis2(+)缺失时，Sds21.NEGFP水平增加，然后在着丝粒、内吞小泡和细胞尖端清晰检测到Sds21.NEGFP。Dis2.NEGFP通过formin结合应激途径支架Wsh3（也称为Tea4）被招募到细胞尖端。Wsh3/Tea4在未受干扰的细胞周期中调节极化尖端生长，并在渗透应激后控制极化生长。在Wsh3/Tea4中突变PP1招募RVXF基序会阻断PP1结合，改变细胞周期调节的生长以诱导分支，在应激时从现有尖端诱导分支，并阻断否则会因Wsh3/Tea4过量产生而产生的肌动蛋白丝的诱导。
