Figure 2.

PtdIns 3–kinase activity in mutants lacking a component of Vps30p complexes. TN125 (wild type; lane 1), AKY15 (Δvps30; lane 2), AKY13 (Δapg14; lane 3), AKY114 (Δvps38; lane 4), AKY109 (Δvps34; lane 5), and AKY115 (Δvps15; lane 6) cells were grown in YPD medium at 28°C. Total lysates were incubated with soybean PtdIns, [γ-³²P]ATP, and 60 μM cold ATP in buffer L (20 mM Hepes-NaOH, pH 7.5, 10 mM MgCl₂) for 5 min at 25°C. Lipids were extracted using chloroform–methanol, and samples were separated on Silica gel 60 TLC plates (Merck) with the borate system (Walsh et al. 1991) and detected by autoradiography using BAS2000.