CIL:13452

Kre2-GFP is not efficiently maintained in the Golgi apparatus in BY4742 vps74∆ expressing Vps74 with mutations in the sulfate-binding pocket (W88A R97A). Compare with control image CIL# 13450. 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 Fig4C middle panel in J Cell Biol. 187: 967-975. 2009. Images in Fig 4C include CIL#s 13450, 13452, 13454.

Kre2-GFP在表达硫酸盐结合口袋突变体（W88A R97A）Vps74的BY4742 vps74∆菌株的高尔基体（Golgi apparatus）中无法被有效维持，需与对照图像CIL# 13450进行比对。本研究证实，Vps74与GOLPH3的硫酸盐结合口袋可介导磷脂酰肌醇4-磷酸（PtdIns4P）结合，且对其功能发挥必不可少。将在液体培养基中培养的细胞置于生长培养基中封片，使用搭载于倒置显微镜（IX-70；奥林巴斯（Olympus））的DeltaVision工作站（Applied Precision），搭配100× 数值孔径（NA）1.4的油浸物镜，以0.4 μm的z轴步长采集三维图像堆栈。成像于23℃下通过12位CCD相机（CoolSnap HQ；Photometrics）完成，并采用迭代约束算法（Agard, 1984）与实测点扩散函数进行反卷积处理。本示例为发表于《Journal of Cell Biology》2009年第187卷967-975页的图4C中间面板所展示的z堆栈近似中心处的单幅图像。图4C包含的图像编号为CIL# 13450、13452、13454。