Intracellular localization of GFP-Vps74 K178A, R181A 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 K178A R181A/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.
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