CIL:13451

Intracellular localization of GFP-Vps74 W88A, R97A mutant in BY4742 vps74∆. GFP-Vps74 is localized to Golgi compartments and the cytosol (CIL# 13449) but loses localization 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/vps74∆ 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.

GFP（Green Fluorescent Protein，绿色荧光蛋白）标记的Vps74 W88A、R97A突变体在BY4742 vps74基因缺失菌株中的细胞内定位实验。GFP-Vps74原本定位于高尔基体（Golgi）区室与细胞质基质，对应细胞图像库（Cell Image Library, CIL）编号#13449，但当其硫酸盐结合口袋内的两个氨基酸发生突变后，会丧失该定位特性。本研究证实，Vps74与GOLPH3的硫酸盐结合口袋可介导与磷脂酰肌醇4-磷酸（PtdIns4P）的结合，且该结构域对其功能发挥至关重要。将在液体培养基中培养的细胞置于生长培养基中封片，基于奥林巴斯Olympus IX-70型倒置显微镜，在Applied Precision公司的DeltaVision工作站上以0.4 μm的z轴步距采集三维图像堆栈，成像物镜为100× 数值孔径（Numerical Aperture, NA）1.4的油浸物镜。图像采集于23℃环境，使用Photometrics公司CoolSnap HQ型12位电荷耦合器件相机（Charge-Coupled Device camera, CCD）完成，并通过迭代约束算法（Agard, 1984）与实测点扩散函数进行反卷积处理。本研究展示的z轴图像堆栈近似中心处的一张图像，见于《Journal of Cell Biology》2009年第187卷第967-975页的图4A中W88A R97A/vps74∆实验组面板。图4A中的图像包含以下细胞图像库编号：CIL# 13449、13451、13453、13455、13456、13457、24816、24817、24818、24819、24820、24821。