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. Author manuscript; available in PMC: 2011 May 18.
Published in final edited form as: Dev Cell. 2010 May 18;18(5):828–840. doi: 10.1016/j.devcel.2010.03.016

Figure 2. Sec2p-PI4P interaction is required for Sec2p localization.

Figure 2

(A) The Sec2 sequence between a.a. 374 and 508 is necessary for PI4P binding. Positively charged patches are shown in bold. (B) GST-tagged Sec2 wild-type (WT), or Sec2 with mutations in positively charged patches 1, 2, or 3, were purified from E. Coli. Proteins were incubated with liposomes containing 10 mol% PS or PI4P. Liposomes were precipitated by centrifugation at 100,000 x g and bound proteins were detected with anti-Sec2 antibody. The intensity of the bands was quantified using Image J. (C) Localization of Sec2-3xGFP or Sec2-KA-3xGFP was examined. Cells were grown overnight at 25°C in a synthetic medium containing 2% glucose. Bars are 2 μm. Arrowheads indicate normal localization of wildtype Sec2. Arrow in bottom panel shows normal localization of Sec2-KA, but with reduced intensity. A total of approximately 80 cells were counted for each experiment. Values indicate the percentage of cells showing bud or mother-daughter neck localization of Sec2-3xGFP. Mean and S.D. of three different experiments are shown. (D) The localization of Sec2-3xGFP (left), mCherry-FAPP1-PH (middle) and the merge (right) was examined in wildtype cells. Cells were grown at 25°C in a synthetic medium containing 2% glucose and then live cells were analyzed with a spinning disk confocal microscope. Insets and arrowheads show colocalization between Sec2-3xGFP and mCherry-FAPP1-PH. Bar is 2 μm.