Specific targeting of Rgs1-mC to sub cellular vesicular structures is mediated by the N-terminal DEP-DEP domain.

<p>Subcellular localization and immuno-blot confirmation of protein expression. (<b>A</b>) Verification of expression of the mCherry tagged protein and individual domain fragments by western blot analysis and microscopy. Whole cell extracts were made from the <i>rgs1</i>Δ, Rgs1-mC, N-Rgs1-mC and C-Rgs1-mC strains cultured in CM. The protein samples were subjected to SDS-PAGE and analyzed by western blot using antisera against DEP-DEP domain (α-DEP) and mCherry (α-RFP). Specific immuno reactive bands corresponding to the full-length protein (Rgs1-mC), N-terminal DEP-DEP domain and C-terminal RGS domain were detected in each case. The <i>rgs1</i>Δ lysate served as a control. The white asterisk indicates a probable post-translationally modified form of Rgs1. Molecular mass standards (kDa) are indicted on the right. Intracellular localization of the individual domain fragments and the full-length Rgs1 protein. Conidia were harvested from the C-Rgs1-mC, N-Rgs1-mC and Rgs1-mC strains and microscopic observations were made at the indicated time points. DIC and wide field images were captured using the requisite filter sets. The arrowhead points to the punctate vesicles prevalent in the Rgs1-mC and N-Rgs1-mC strains at 0 h. Scale bar = 10 µm. (B) The DEP-A and DEP-B domain contribute to punctate/vesicular localization of Rgs1. Conidia from the indicated mCherry tagged strains were visualized using a wide field microscope and images captured at the indicated time point. The arrowheads in each image indicate the region of the conidium magnified and depicted as inset. Scale bar = 10 µm. (<b>C</b>) Vacuolar localization of the catalytic core domain (C-Rgs1-mC). Representative images from a colocalization experiment between the C-Rgs1-mC strain with a vacuole specific dye, CMAC. The merged panel suggests a predominant trafficking of the core RGS domain to the vacuole. Scale bar = 10 µm.