Figure 3. Ypp1 functions in the PtdIns4P signaling pathway to positively regulate Stt4 kinase activity. (A) The relative growth capabilities of wild type, stt4-4, and ypp1-7 at 26 (top left) and 37°C (bottom left). The relative growth capabil- ity of ypp1-7 with overexpression of YPP1 from a 2 plasmid, plasmid alone (pRS426), or overexpressed STT4 at 37°C (top right). Similar growth proﬁle of stt4-4 at 37°C in the presence of vector alone or in the presence of overexpressed YPP1 (bottom right). (B) Whole cell lysate from teto-YPP1-HA cells grown in the presence or absence of doxycycline were ana- lyzed by Western blot with an anti-HA antibody. The relative abundance of Ypp1-HA under the indicated conditions (top) and the relative abundance of a control protein, G6PDH, from the same lysate (bottom) are shown. (C) Phosphoinositide lev- els in teto-YPP1 cells with and without doxycycline and control R1158 in the presence of doxycycline. Data are presented as means and SD (error bars) of three independent experiments. is similar to wild-type levels. Our data imply that Ypp1’s im- pact on PtdIns4P levels comes primarily through Stt4; how- ever, we are unable to completely rule out the possibility that it affects PtdIns4P levels maintained by the other essential PtdIns 4-kinase, Pik1. We addressed this concern by visualizing the pools of PtdIns4P at the Golgi and PM in various ypp1 mutant strains. The GFP-tagged tandem Osh2 PH domain has been shown to effectively localize to the two distinct populations of PtdIns4P in the cell, the Pik1-generated Golgi pool and the Stt4-generated PM pool (Roy and Levine, 2004). A disruption in either Pik1 or Stt4 activity redistributes GFP-2xPHOsh2 from its wild-type lo- calization (Fig. 4 C, top). In the stt4ts mutant at the restrictive temperature, the PM pool of PtdIns4P disappears, whereas under similar conditions in the pik1ts mutant the Golgi pool of PtdIns4P disappears. We again used the yTHC teto-YPP1 yeast strain to study the localization of GFP-2xPHOsh2 upon Ypp1 depletion. In the absence of doxycycline, when Ypp1 is present near normal levels, the localization of GFP-2xPHOsh2 was similar to wild type (Fig. 4 C). After 20 h of doxycycline treatment, however, in which the Ypp1 protein has been de- pleted, the GFP-2xPHOsh2 is absent from the PM. This implies that, without Ypp1, PtdIns4P is no longer generated at the PM. Similar results were seen in sac1 ypp1 (unpublished data) confirming the loss of PtdIns4P at the PM in the absence of Ypp1. An Stt4–Ypp1 complex coordinates PIK patch signaling at the PM We next analyzed the organization of the PM Stt4 PIK patch. We asked if Ypp1 recruits Stt4 to the PM and/or structurally or- ganizes Stt4 to regulate PtdIns4P metabolism. We detected very low amounts of GFP-Stt4 with either Western blot or fluores- cence microscopy in the sac1 ypp1 strain compared with wild type or sac1 . Therefore, we hypothesized that Ypp1 may be required for the stability of Stt4. We determined that attenuation of Ypp1 levels in the teto-YPP1 GFP-STT4 strain caused a cor- responding decrease of GFP-Stt4 (Fig. 5 A). Within 9 h, GFP- Stt4 decreased to <10% its initial levels and is barely visible by 16 h. Therefore, we found it very difficult to assess Stt4 distri- bution in the absence of Ypp1. To avoid this problem, we monitored GFP-Stt4 localiza- tion at various time points after the addition of doxycycline to teto-YPP1 GFP-STT4 yeast. This allowed us to study the redis- tribution of Stt4 as the Ypp1 protein was steadily diminished. After 9 h of doxycycline-induced Ypp1 depletion, GFP-Stt4 YPP1 AND EFR3 MEDIATE PIK PATCH ORGANIZATION • Baird et al. 1065