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. 2019 Oct 28;17(10):e3000512. doi: 10.1371/journal.pbio.3000512

Fig 2. The endocytosis of the low-affinity thiamine transporters Nrt1 and Thi72 is also mediated by Art2.

Fig 2

(A) Strains expressing NRT1-GFP (top panel) and THI72-GFP (bottom panel) under THI7 endogenous promoter were grown in thiamine-free medium up to early-log phase and harvested after 120-min incubation with thiamine (“Th.”) at a concentration of 100 μM for total protein extractions. Extracts were immunoblotted with anti-GFP and anti-Pma1 as a loading control. Values are quantification of band intensity. (B) Localization of Nrt1-GFP or Thi72-GFP in WT, art2Δ, art9Δ, rsp5, and art2Δart9Δ strains after addition of an excess of thiamine into culture grown in thiamine-free medium. The vacuolar membrane is stained with FM4–64. Scale bar represents 5 μm. Quantification shows the ratio of internal-over-PM fluorescence intensity as described in Materials and methods section (n > 50 cells). (****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05) The numerical data are included in S2 Data. GFP, green fluorescent protein; Nrt1, nicotinamide riboside transporter 1; ns, nonsignificant; PM, plasma membrane; Pma1, plasma membrane ATPase 1; WT, wild type.