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. 2020 Aug 3;9:e58246. doi: 10.7554/eLife.58246

Figure 6. The C-terminus of Mup1 harbors a transplantable, starvation-responsive acidic degron.

(A) Left: scheme of Mup1 topology with N- and C-terminal ubiquitination sites and acidic patches targeted by Art1-Rsp5 and Art2-Rsp5, respectively, and the C-terminal phosphorylation sites of Mup1 that promote its starvation-induced endocytosis. Ubiquitinated lysines (K) shown in green and phosphorylated serines (S) and threonines (T) in red with numbers corresponding to amino acid positions in the Mup1 sequence. Right: Hxt3 as an Art4-Rsp5 dependent cargo during nitrogen starvation. (B) Live-cell fluorescence microscopy analysis of Mup1-GFP endocytosis in cells expressing MUP1-GFP (wild type (WT)) or MUP1 D549R,D551R,E554R,D555R-GFP. Cells were treated with 20 µg/ml L-methionine (+ Met) for 1.5 hr or starved (- N) for 6 hr after 24 hr exponential growth. (C) SDS PAGE and western blot analysis with the indicated antibodies of immunoprecipitated Mup1-GFP from cells expressing MUP1-GFP (WT) or MUP1 D549R,D551R,E554R,D555R-GFP starved for the indicated times after 24 hr of exponential growth. Equal amounts of immunoprecipitated Mup1-GFP were loaded to compare the extent of ubiquitination. (D) Amino acid sequence alignment of the C-terminal acidic patches of Mup1, Can1, Lyp1, Tat2 and Ina1. The boxes indicate acidic residues (red) and phosphorylation sites (blue), which are required for Art2-dependent starvation-induced endocytosis. Red letters illustrate further acidic residues. (E) Live-cell fluorescence microscopy analysis of wild type (WT), art4∆ and art2∆ art4∆ cells expressing HXT3-GFP (top), HXT3-MUP1-C-GFP (second row), HXT3-MUP1-C K567,572R-GFP (third row) or HXT3-MUP1-C D549R,D551R,E554R,D555R-GFP (bottom). Cells were starved (- N) for 6 hr after 24 hr exponential growth. Scale bars = 5 µm. See also Figure 6—figure supplements 1 and 2.

Figure 6.

Figure 6—figure supplement 1. The role of acidic patches in AATs for starvation-induced endocytosis.

Figure 6—figure supplement 1.

(A-C) Live-cell fluorescence microscopy analysis of the indicated strains treated with 20 µg/ml L-methionine (+ Met) for 1.5 hr or starved (- N) for 6 hr after 24 hr exponential growth. Scale bars = 5 µm.
Figure 6—figure supplement 2. Art4-dependent starvation-induced endocytosis of Hxt3.

Figure 6—figure supplement 2.

(A) Live-cell fluorescence microscopy analysis of the indicated strains starved (- N) for 6 hr after 24 hr exponential growth. (B) SDS PAGE and western blot analysis with the indicated antibodies of whole cell protein extracts from wild type (WT) and art4∆ cells expressing HXT3-GFP. Cells were starved (- N) for 6 hr after 24 hr exponential growth. Coomassie staining of the membrane serves as loading control. (C) Densitometric quantification of B). Displayed is the distribution of the GFP signal into full length Hxt3-GFP and clipped GFP fragment. Mean % of total GFP signal in each lane ± standard deviation from n = 3 independent experiments. (D) Wild type (WT), art4∆, art2∆ art4∆ and art2∆ cells expressing HXT3-MUP1-C-GFP were treated as in B). (E) Densitometric quantification of C). Displayed is the distribution of the GFP signal into full-length Hxt3-Mup1-C-GFP and clipped GFP fragment. Mean % of total GFP signal in each lane ± standard deviation from n = 3 independent experiments. Scale bars = 5 µm.