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

Fig 3. The Art2-dependent ubiquitylation of Thi7-GFP C terminus is required for its endocytosis.

Fig 3

(A) GFP-tagged KR-mutated THI7 was expressed in a thi7Δnrt1Δthi72Δ strain and grown in a thiamine-free medium. Serial 10-fold dilutions of yeast cells in early log phase were spotted on thiamine-free oxythiamine-containing medium. The expression of pRS416 (empty vector) and the nontagged version were used as controls. Oxythiamine was transported by all the KR Thi7 mutants (B) Localization of the different KR mutant versions of Thi7-GFP in the WT strain after thiamine treatment (final concentration: 100 μM): Thi7KR-GFP, Thi7NterKR-GFP, Thi7CterKR-GFP, and Thi7Cter6KR-GFP. (C) Top: THI7-GFP and THI7KR-GFP were expressed in end3Δ and end3Δart2Δ strains and grown in thiamine-free medium to early log phase before a 30-min incubation with 100 μM thiamine. Bottom: THI7-GFP, Thi7KR-GFP, Thi7NterKR-GFP, Thi7CterKR-GFP, or Thi7Cter6KR-GFP were expressed in art2Δ strain (complemented or not with 3xHA-ART2) and grown in thiamine-free medium to early log phase before a 30-min incubation with 100 μM thiamine. After solubilization, IP of transporters was performed using the GFP-Trap kit. Samples were immunoblotted with anti-GFP and anti-Ub. Quantification is based on the intensity of the Ub band and normalized to the intensity of the corresponding GFP signal. GFP, green fluorescent protein; IB, immunoblotting; IP, immunoprecipitation; KR, lysine-to-arginine; Ub, ubiquitin; WT, wild type.