Fig. 6. Overexpression of Sla1(118-511) region disrupts fluid phase uptake of lucifer yellow and subsequent trafficking to the vacuole. Cells expressing GST alone (KAY439), GST-Sla1(118-511SH3#3) (KAY508), GST-Sla1(118-511SH3#3)* (KAY657) or GST-Sla1 (KAY439) were grown overnight in synthetic media containing 2% glucose, then inoculated into media with 2% galactose to induce expression of the various fusion proteins respectively. (A) Cells expressing GST alone displayed normal lucifer yellow uptake before and after induction and exhibited a strong fluorescence within the vacuole. (B) In cells induced to overexpress GST-Sla1p (118-511SH3#3), lucifer yellow uptake was disrupted. These cells showed little vacuolar staining compared to those expressing GST alone and possessed more abundant brightly stained dots outside the vacuole. Lucifer yellow staining at the plasma membrane of these cells also appeared brighter than in the control cells. (C) A mutant form of Sla1 (118-511SH3#3)* was expressed that cannot bind to another strong interactor of the 118-511 domain (Ysc84p). The same defect in uptake and subsequent trafficking was seen with this mutants with the Sla1(118-511SH3#3) mutant, indicating that the defect is not because of the domain blocking normal Ysc84 function. (D) Overexpression of full-length Sla1p abrogated lucifer yellow uptake but did not result in accumulation of stained endosomes. Bar, 10 µM.