Quality control and substrate-dependent downregulation of the nutrient transporter Fur4

Abstract

Upon exposure to stress conditions, unfolded cell-surface nutrient transporters are rapidly internalized and degraded via the multivesicular body (MVB) pathway. Similarly, high concentrations of nutrients result in the downregulation of the corresponding transporters. Our studies using the yeast transporter Fur4 revealed that substrate-induced downregulation and quality control utilize a common mechanism. This mechanism is based on a conformation-sensing domain, termed LID (loop interaction domain), that regulates site-specific ubiquitination (also known as degron). Conformational alterations in the transporter induced by unfolding or substrate binding are transmitted to the LID, rendering the degron accessible for ubiquitination by Rsp5. As a consequence, the transporter is rapidly degraded. We propose that the LID-degron system is a conserved, chaperone-independent mechanism responsible for conformation-induced downregulation of many cell-surface transporters under physiological and pathological conditions.

Figure 1

Structure of the transporters Fur4 and Mhp1. (A) Schematic representation of Fur4 which contains 12 transmembrane domains. The N-terminally localized degron of Fur4 is composed of the ubiquitination site (Ub) and the PEST-like sequence, which is followed by the LID (Loop Interaction Domain). Deletion of the first 60 amino acids removes the degron (∆N60). (B) Amino acid sequence alignment of the N-terminal regions of Fur4, Mhp1 and other homologous yeast transporters. Amino acids in Fur4 that have been mutated to alanine are labeled in red. The asparagine 115 of Fur4 is labeled in purple. (C) Side- and bottom-view of the Mhp1 structure (based on crystal structure 2JLN). The LID is labeled in red and the cytoplasmic loops are indicated (L2–3, L6–7, L8–9, L10–11).

Figure 2

Stress-induced downregulation of Fur4 is dependent on the N-terminal degron. (A) Fluorescence microscopy of yeast expressing wild-type and different mutant forms of Fur4-GFP, before and after treatment with uracil, peroxide or heat-shock. (B) Intracellular uracil concentration in cells expressing Fur4^∆N60-GFP. Cells were either not treated or treated with peroxide for 20min and uracil concentration was determined before and after addition of 5µg/ml uracil to the medium. (C) Growth at 37°C of fur4∆ strains containing plasmids that express either wild-type or mutant forms of FUR4-GFP. (D) Growth of fur4∆ strains expressing wild-type or different mutant forms of Fur4-GFP in liquid medium (YNB) at 30°C in the presence or absence of 1M sorbitol. The graph represents the average growth of three cultures. (E) Downregulation of wild-type and N115H mutant of Fur4-GFP after a 10min heat-shock.

Figure 3

Extracellular and intracellular substrate initiates downregulation of Fur4. (A) Downregulation of wild-type (WT) and mutant (K272A; K38,41R) Fur4-GFP in the presence of uracil or leflunomide. The fluorescence microscopy pictures are inverted and thus black indicates the localization of GFP. Dashed lines outline cells with no discernable plasma membrane signal. (B) Optical density (OD 600nm) of yeast cultures grown over-night in the presence or absence of leflunomide. Yeast used for the experiment were deleted for Fur4 and transformed either with empty vector (-) or plasmids expressing either wild-type or the K272A mutant of Fur4-GFP. The results show the average growth of three cultures. (C) Schematic of the uracil and cytosine metabolism of yeast. (D) Uracil- and cytosine-induced downregulation of wild-type and K272A mutant of Fur4-GFP expressed in either wild-type, ∆cdd1-2µFCY1 or 2µFUR1 strains. (E) Quantification of the fluorescence microscopy shown in D (50 cells were quantified for each experiment). The graph shows the percentile of cells with a particular range of internal-to-total GFP signal (0.0–0.2, 0.2–0.4, etc.).

Figure 4

The LID regulates Fur4 degradation. (A) Downregulation of wild-type and LID mutants of Fur4-GFP after treatment with uracil, high temperature or leflunomide. (B) Deletion of the N-terminal 90 or 110 amino acids of Fur4-GFP resulted in ER retention and degradation of the protein. (C) Quantification of the leflunomide treatment shown in A. Approximately 30 cells were analyzed for the presence or absence of plasma membrane localized Fur4-GFP. (D) Heat-shock and substrate-induced downregulation of wild-type Fur4-GFP and M96BPA mutant, before and after UV treatment. (E) Quantification of the analysis shown in D (N=50). (F) Localization of different N-terminal mutants of Fur4-GFP before and after heat-shock or leflunomide exposure.

Figure 5

Quality control of Mup1 depends on Rsp5 but does not require Art1. Fluorescence microscopy of different Mup1-GFP expressing yeast strains before and after treatment with methionine, peroxide or heat-shock.

Figure 6

Model of substrate- and stress-induced Fur4 downregulation mediated by the LID-degron system.