Christopher S. Wood, Karl R. Schmitz, Nicholas J. Bessman, Thanuja Gangi Setty, Kathryn M. Ferguson, Christopher G. Burd (2011) CIL:24817, Saccharomyces cerevisiae S288c. CIL. Dataset

Intracellular localization of GFP-Vps74 W88A, R97A mutant in BY4742 sac1∆ vps74∆ mutant. SAC1 encodes an integral membrane phosphoinositide phosphatase and in the sac1∆ vps74∆ mutant, GFP-Vps74 localizes, in addition to the Golgi apparatus, to nuclear ER and cortical ER and/or PM (CIL# 24816). This localization of GFP-Vps74 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 W88A R97A/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 sac1∆ vps74∆ 突变体中，GFP-Vps74 W88A、R97A 突变体的细胞内定位，除了高尔基体外，还定位于核质内质网、皮质内质网及/或质膜（CIL# 24816）。当硫酸结合口袋中的两个氨基酸发生突变时，GFP-Vps74 的这种定位即消失。该研究证实，Vps74 和 GOLPH3 的硫酸结合口袋介导 PtdIns4P 结合，对功能至关重要。在液体培养基中培养的细胞，经生长培养基固定后，在 DeltaVision 工作站（Applied Precision）上，基于倒置显微镜（IX-70；Olympus）并使用 100× NA 1.4 油浸镜头，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，并采用 Agard（1984）提出的迭代约束算法及其测量的点扩散函数进行去卷积。图 4A 中 W88A R97A/sac1∆ 面板中展示的图像来自 z 堆栈的大致中心，见《细胞生物学》杂志第 187 卷第 967-975 页，2009 年。图 4A 中的图像包括 CIL#s 13449、13451、13453、13455、13456、13457、24816、24817、24818、24819、24820、24821。