Kre2-GFP is not efficiently maintained in the Golgi apparatus in BY4742 vps74∆ expressing Vps74 with mutations in the sulfate-binding pocket (K178A R181A). 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 bottom panel in J Cell Biol. 187: 967-975. 2009. Images in Fig 4C include CIL#s 13450, 13452, 13454.
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