Christopher S. Wood, Karl R. Schmitz, Nicholas J. Bessman, Thanuja Gangi Setty, Kathryn M. Ferguson, Christopher G. Burd (2011) CIL:24821, Saccharomyces cerevisiae S288c. CIL. Dataset

Intracellular localization of GFP-GOLPH3 R171A, R174A mutant in BY4742 sac1∆ vps74∆ mutant. 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 (CIL# 24819). This localization of GFP-GOLPH3 is lost when two amino acids are mutated in the sulfate-binding pocket. 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 R171A R174A/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.

本研究探讨了在BY4742 sac1∆ vps74∆突变体中，GFP-GOLPH3 R171A、R174A突变体在细胞内的定位情况。SAC1编码一种整合膜磷脂酰肌醇磷酸酯磷酸酶，在sac1∆ vps74∆突变体中，GFP-GOLPH3除了定位于高尔基体外，还定位于核内质网、皮质质网和/或质膜（CIL# 24819）。当突变体中的两个氨基酸在硫酸结合口袋发生突变时，GFP-GOLPH3的这种定位将丧失。本研究证实，Vps74和GOLPH3的硫酸结合口袋介导PtdIns4P的结合，对于其功能至关重要。培养在液体培养基中的细胞被固定在生长培养基中，并使用DeltaVision工作站（Applied Precision）上的倒置显微镜（IX-70；Olympus）及100× NA 1.4油浸镜头，以0.4-µm的z轴增量收集3D图像堆栈。图像在23°C下由12位CCD相机（CoolSnap HQ；Photometrics）捕捉，并使用迭代约束算法（Agard，1984）及测量的点扩散函数进行去卷积。J Cell Biol. 2009年，第187卷，第967-975页的图4A中，展示了来自z堆栈大致中心的图像，其中包含CIL#s 13449、13451、13453、13455、13456、13457、24816、24817、24818、24819、24820、24821。