GEEC Endocytosis in Drosophila Figure 7. A conserved role for GBF1 in fluid phase uptake in Drosophila and mammalian cells. (A) Normalized uptake of Fdex and TfR was measured as before in S2R+ cells treated with dsRNAs corresponding to various predicted Arf GAPs and GEFs from the Drosophila genome (corresponding CG numbers are: arfgap1 CG6838; arfgap2 CG16728; gap20 CG4937; gap40 CG8243; gap69 CG4237; garz CG8487; schizo CG32434; arfgef1 CG7578). Notably depletion of the Arf GEF garz was singled out because it affected Fdex but not Tf internalization, while other other depletions either had no major effect or affected both pathways significantly. Values are represented as mean6s.e.m. derived from 2 independent experiments with n.200 cells in each condition. Asterix denote P,0.05 by Student’s t-test. (B) Cartoon representation of domain structures in human GBF1 (hGBF1)and Drosophila garz (dgarz) proteins. The glutamic acid to lysine substitution mutation within the catalytic Sec7 domain is marked as E794K. A dot plot comparison of hGBF1 and dgarz primary sequences shows broad conservation throughout the length of the proteins, when a stringent moving window of 6 matches every 10 amino acids is applied. (C) Quantitative uptake of Fdex and TfR in S2R+ cells depleted of garz (left panel) or CHO cells overexpressing non-functional GBF1 (GFPGBF1E794K right panel). In the left panel, representative images of S2R+ cells (outlined) treated with control or garz dsRNA are shown in 3 channels, depicting uptake/binding with Fdex, TfR and Okt respectively. The bar graph below the panel shows the quantitation of Fdex and TfR uptake normalized to control values. Values are represented as mean6s.e.m. derived from 2 independent experiments with n.200 cells in each condition. Asterix denote P,0.05 by Student’s t-test. In the right panel, representative images of CHO cells pulsed with Rdex or TfR and overexpressing GFPGBF1E794K (transfected cells are visible in the GFP channel) are shown. Bars in C = 20 mm. doi:10.1371/journal.pone.0006768.g007 PLoS ONE | www.plosone.org 11 August 2009 | Volume 4 | Issue 8 | e6768