Figure 1.

Functional characterization of truncated Fur transporters. (A) C-terminally-truncated Fur transporters show modified apparent substrate specificities. Growth test of strains expressing, from gpdA_p promoter, nontruncated (WT) and truncated Fur-GFP transporters. The test is performed at 37° on MM media containing as sole nitrogen source nitrate (control), UA, or ALL, or on nitrate media containing a nucleobase toxic analog (5FU, 5FC, or 5FUd). The growth on UA is recorded at both 37 and 25°. WT stands for a standard A. nidulans wild-type strain. Δ7 is a mutant strain carrying total deletions in all seven major transporters for nucleobases, nucleosides, and ALL (uapAΔ uapCΔ azgAΔ furDΔ furAΔ fcyBΔ cntAΔ). The Δ7 strain has an intact endogenous furE gene transporter, but this is very little expressed under standard conditions and thus does not contribute to detectable transport of its potential substrates (UA, URA, or ALL). All other strains are single-copy isogenic transformants of Δ7 expressing the indicated nontruncated or truncated Fur transporter. K252F is a missense mutation increasing the activity of FurE (Krypotou et al. 2015). (B) C-terminally-truncated Fur transporters do not undergo endocytosis. Subcellular localization of nontruncated (WT) and truncated GFP-tagged Fur transporters, expressed from the gpdA_p promoter, as analyzed by in vivo inverted epifluorescence microscopy. Samples are grown for 18 hr at 25° under control (MM with nitrate as N source), or substrate-elicited (ALL or URA) or ammonium-elicited endocytic conditions (MM with nitrate as N source plus addition of substrate or ammonium for the last 2 hr of the culture). Endocytic turnover is identified by the appearance of cytoplasmic structures corresponding to endosomes and vacuoles and the progressive diminution of the peripheral fluorescent signal (Gournas et al. 2010; Krypotou et al. 2015). Notice that different Fur transporters have distinct sensitivities to endocytosis (FurE > FurD, FurA), but truncation of the C-terminus stabilizes the Fur-GFP chimeras in all cases. (C) C-terminally-truncated Fur transporters show dramatically reduced turnover. Western blot analysis of total protein extracts of strains expressing WT and truncated Fur-GFP versions, using anti-GFP (upper panel) or anti-actin (control, lower panel) antibody. Growth conditions are as in (B). Free GFP levels reflect vacuolar degradation of Fur-GFP proteins. (D and E) C-terminally-truncated Fur transporters show modified transport kinetics. (D) Time course of [³H]-URA uptake by truncated (FurD-ΔC and FurE-ΔC) and nontruncated (FurD and FurE) transporters. SD is depicted with error bars. (E) K_i/m values (micromolar) for truncated and nontruncated FurD and FurE transporters determined using [³H]-URA uptake competition. Results are averages of three measurements for each concentration point. SD was < 20%. 5FC, 5-fluorocytosine; 5FU, 5-fluorouracil; 5FUd, 5-fluorouridine; ALL, allantoin; MM, minimal media; n.m., nonmeasurable; URA, uracil; UA, uric acid; WT, wild-type.