Christopher S. Wood, Karl R. Schmitz, Nicholas J. Bessman, Thanuja Gangi Setty, Kathryn M. Ferguson, Christopher G. Burd (2011) CIL:24819, Saccharomyces cerevisiae S288c. CIL. Dataset

Intracellular localization of GFP-GOLPH3 in BY4742 vps74∆ sac1∆. SAC1 encodes an integral membrane phosphoinositide phosphatase and in the sac1∆ vps74∆ mutant, GFP-GOLPH3 localizes, in addition to the Golgi apparatus, to nuclear ER and cortical ER and/or PM. Control image for images showing ectopic expression of GFP-GOLPH3 with mutations in the sulfate-binding pocket (CIL#s 24820, 24821). This study demonstrates that the sulfate-binding pocket of Vps74 and GOLPH3 mediates PtdIns4P-binding and is essential for function. Cells grown in liquid medium were mounted in growth medium and 3D image stacks were collected at 0.4-µm z increments on a DeltaVision workstation (Applied Precision) based on an inverted microscope (IX-70; Olympus) using a 100× NA 1.4 oil immersion lens. Images were captured at 23C with a 12-bit CCD camera (CoolSnap HQ; Photometrics) and deconvolved using the iterative-constrained algorithm (Agard, 1984) and the measured point spread function. One image from the approximate center of z stack is shown in Fig4A GFP-GOLPH3 wild-type/sac1∆ panel in J Cell Biol. 187: 967-975. 2009. Images in Fig 4A include CIL#s 13449, 13451, 13453, 13455, 13456, 13457, 24816, 24817, 24818, 24819, 24820, 24821.

本研究揭示了在 BY4742 vps74∆ sac1∆ 突变体中，GFP-GOLPH3 的细胞内定位，除了高尔基体之外，还分布于细胞核、内质网和细胞质膜。SAC1 编码一种整合膜磷脂酰肌醇磷酸酶，在 sac1∆ vps74∆ 突变体中，GFP-GOLPH3 的这种定位现象尤为显著。对照图像展示了具有硫酸结合口袋突变（CIL#s 24820, 24821）的 GFP-GOLPH3 异位表达情况。研究证实，Vps74 和 GOLPH3 的硫酸结合口袋介导了 PtdIns4P 的结合，对于其功能至关重要。培养于液体培养基中的细胞被固定于生长培养基中，并在 DeltaVision 工作站（Applied Precision）上，基于倒置显微镜（IX-70; Olympus）和 100× NA 1.4 油浸物镜，以 0.4-µm 的 z 增量收集 3D 图像堆栈。图像在 23°C 下，使用 12 位 CCD 相机（CoolSnap HQ; Photometrics）捕获，并利用迭代约束算法（Agard, 1984）和测量的点扩散函数进行去卷积处理。图 4A 中展示的来自 z 堆栈中心的图像之一，可见于 J Cell Biol. 187: 967-975. 2009 的 GFP-GOLPH3 野生型/sac1∆ 组面板。图 4A 包含 CIL#s 13449, 13451, 13453, 13455, 13456, 13457, 24816, 24817, 24818, 24819, 24820, 24821 的图像。