Membrane-anchored ubiquitin ligase complex is required for the turnover of lysosomal membrane proteins

Abstract

Cells must regulate the abundance and activity of numerous nutrient transporters in different organelle membranes to achieve nutrient homeostasis. As the recycling center and major storage organelle, lysosomes are essential for maintaining nutrient homeostasis. However, very little is known about mechanisms that govern the regulation of its membrane proteins. In this study, we demonstrated that changes of Zn(2+) levels trigger the downregulation of vacuolar Zn(2+) transporters. Low Zn(2+) levels cause the degradation of the influx transporter Cot1, whereas high Zn(2+) levels trigger the degradation of the efflux channel Zrt3. The degradation process depends on the vacuole membrane recycling and degradation pathway. Unexpectedly, we identified a RING domain-containing E3 ligase Tul1 and its interacting proteins in the Dsc complex that are important for the ubiquitination of Cot1 and partial ubiquitination of Zrt3. Our study demonstrated that the Dsc complex can function at the vacuole to regulate the composition and lifetime of vacuolar membrane proteins.

Figure 1.

Vacuolar Zn²⁺ transporters can be downregulated in response to changes in Zn²⁺ concentration. (A) A schematic diagram illustrating the topology of vacuolar Zn²⁺ transporters, including Zrc1, Cot1, and Zrt3. (B) Localization of chromosomally tagged Zrc1-GFP, Cot1-GFP, Zrt3*-GFP and Vph1-mCherry, before and 6 h after Zn²⁺ withdrawal in YNB media. Dashed lines highlight the cell surface. (C) Western blot analysis of chromosomally tagged Zrc1-GFP, Cot1-GFP, and Zrt3*-GFP over the course of 6 h of Zn²⁺ withdrawal in YNB media. The samples were blotted with an anti-GFP antibody. G6PDH was used as a loading control. Same volume of cells was loaded in each lane, with 1 OD₆₀₀ cells loaded at 0 h. (D) Quantification of 1C. All protein levels were normalized using G6PDH level. (E) Localization of Zrt3*-GFP under different Zn²⁺ treatments. Mid-log cells grown in YPD (0 h) were transferred into YNB without Zn²⁺ and incubated for 3 h (−Zn²⁺ 3 h). Then, 2 mM ZnCl₂ was added and cells were incubated for another 3 h (+Zn²⁺ 3 h). (F) Western blot analysis of Zrt3*-GFP and Vph1 levels when yeast cells were shifted from YNB without Zn²⁺ to YNB with excessive Zn²⁺ (2 mM ZnCl₂). G6PDH was used as a loading control. 1 OD₆₀₀ cells were loaded in each lane. (G) Quantification of F. All protein levels were normalized using G6PDH level. (H) Localization of tet-O7 Cot1-GFP and Vph1-mCherry before and after Zn²⁺ withdrawal in YNB media. The incubation was performed in the presence of 2 µg/ml doxycycline (Dox). Right: Line-scan analysis of the image. (I) Degradation kinetics for tet-O7 Cot1-GFP after addition of 2 µg/ml Dox and Zn²⁺ withdrawal in YNB media. G6PDH was used as a loading control. Same volume of cells were loaded, with 1 OD₆₀₀ cells loaded at 0 h. (J) Quantification of I. All protein levels were normalized using G6PDH level. Bar, 1 µm.

Figure 2.

ESCRTs, but not the autophagy machinery, are required for the degradation of Cot1-GFP. (A) Localization of tet-O7 Cot1-GFP in WT and autophagy mutants 4 h after Zn²⁺ withdrawal from YNB media and addition of 2 µg/ml Dox. (B) Degradation kinetics for tet-O7 Cot1-GFP in WT and autophagy mutants. G6PDH was used as a loading control. Same volume of cells was loaded, with 1 OD₆₀₀ cells loaded at 0 h. (C) Localization of tet-O7 Cot1-GFP 4 h after Zn²⁺ withdrawal in WT and ESCRT mutants. Arrows highlight the aberrant endosomes. (D) Degradation kinetics for tet-O7 Cot1-GFP in WT and ESCRT mutants. G6PDH was used as a loading control. Same volume of cells was loaded, with 1 OD₆₀₀ cells loaded at 0 h. Bar, 1 µm.

Figure 3.

Tul1, but not the VAcUL-1 complex, is required for Cot1-GFP degradation. (A) ssh4Δ and rsp5-1 mutants did not block the internalization of Cot1-GFP into the vacuole lumen after Zn²⁺ withdrawal from the YNB media. (B) Degradation kinetics for tet-O7 Cot1-GFP in WT, ssh4Δ and rsp5-1 mutants. G6PDH was used as a loading control. Same volume of cells was loaded, with 1 OD₆₀₀ cells loaded at 0 h. (C) The internalization of Cot1-GFP into the vacuole lumen was blocked in tul1Δ strain after Zn²⁺ withdrawal from the YNB media. (D) Degradation kinetics for tet-O7 Cot1-GFP in WT and ssh4Δ strains. G6PDH was used as a loading control. Same volume of cells was loaded, with 1 OD₆₀₀ cells loaded at 0 h. Bar, 1 µm.

Figure 4.

Both Tul1 and Rsp5 contribute to the degradation of Zrt3*-GFP. (A) The internalization of Zrt3*-GFP into the vacuole lumen was not blocked in ssh4Δ strain after the addition of 2mM ZnCl₂ to the YNB media. Cells were pretreated with YNB without Zn²⁺ for 3 h. (B) Degradation kinetics for Zrt3*-GFP in WT and ssh4Δ strains. G6PDH was used as a loading control. Same volume of cells was loaded, with 1 OD₆₀₀ cells loaded at 0 h. (C) Localization of Zrt3*-GFP in WT, tul1Δ, rsp5-1, and tul1Δ rsp5-1 mutant strains before and after addition of 2mM ZnCl₂ to YNB media. Cells were pretreated with YNB without Zn²⁺ for 3 h at 26°C. Then, the culture was shifted to 37°C for 30 min before the addition of ZnCl₂ and the incubation was continued for another 3 h at 37°C. (D) Degradation kinetics for Zrt3*-GFP in WT, tul1Δ, rsp5-1, and tul1Δ rsp5-1 mutant strains. The cells were pretreated with YNB media without Zn²⁺ for 3 h at 26°C. Then, the culture was shifted to 37°C for 30 min before the addition of ZnCl₂ to 2 mM and the incubation was continued for another 3 h at 37°C. G6PDH was used as a loading control. Same volume of cells was loaded, with 1 OD₆₀₀ cells loaded at 0 h. Bar, 1 µm.

Figure 5.

The Dsc complex and Cdc48 are important for Cot1-GFP degradation. (A) Localization of tet-O7 Cot1-GFP after Zn²⁺ withdrawal in WT, deletion mutants for the Dsc complex and the cdc48^E315K allele. (B) Degradation kinetics for tet-O7 Cot1-GFP in WT, dsc2Δ, dsc3Δ, and ubx3Δ mutant strains. G6PDH was used as a loading control. Same volume of cells was loaded, with 1 OD₆₀₀ cells loaded at 0 h. (C) Degradation kinetics for tet-O7 Cot1-GFP in WT and cdc48^E315K mutant strains. G6PDH was used as a loading control. Same volume of cells was loaded; with 1 OD₆₀₀ cells loaded at 0 h. (D) Localization of chromosomally tagged Cot1-GFP before and after Zn²⁺ withdrawal in dsc deletion mutants overexpressing Tul1. (E) Degradation kinetics of chromosomally tagged Cot1-GFP in dsc deletion mutants overexpressing Tul1. G6PDH was used as a loading control. 1 OD₆₀₀ of cells were loaded in each lane. Bar, 1 µm.

Figure 6.

Overexpression of Tul1 causes a constitutive degradation of a subset of vacuolar membrane proteins. (A) Localization of chromosomally tagged Ypq2-GFP, Fth1-GFP, and Vph1-GFP in cells overexpressing Ssh4, Tul1, or the empty vector. (B) Western blot analysis of protein levels for chromosomally tagged Ypq2-GFP, Fth1-GFP, and Vph1-GFP in cells overexpressing Ssh4 (S-OE), Tu1(T-OE), or the empty vector (Vec). G6PDH was used as a loading control. 1 OD₆₀₀ of cells were loaded for Ypq2 and Fth1 samples, 0.4 OD₆₀₀ of cells were loaded for Vph1 samples owing to the high expression level of Vph1-GFP. (C) Localization of chromosomally tagged Ypq1-GFP, Zrt3*-GFP, Zrc1-GFP, and Cot1-GFP in cells overexpressing Ssh4, Tul1, or the empty vector. (D) Western blot analysis of protein levels for chromosomally tagged Ypq1-GFP, Zrt3*-GFP, Zrc1-GFP, and Cot1-GFP in cells overexpressing Ssh4, Tul1, or only the empty vector. G6PDH was used as a loading control. 1 OD₆₀₀ of cells were loaded for each lane. (E) Localization of chromosomally tagged Ycf1-GFP, Vba4-GFP, and Ypl162c-GFP in cells overexpressing Ssh4, Tu1, or the empty vector. (F) Western blot analysis of protein levels for chromosomally tagged Ycf1-GFP, Vba4-GFP, and Ypl162c-GFP in cells overexpressing Ssh4, Tul1, or the empty vector. G6PDH was used as a loading control. 1 OD₆₀₀ cells were loaded for each lane. Bar, 1 µm.

Figure 7.

A significant fraction of the Dsc complex localizes to the vacuole membrane. (A) Left: Schematics illustrating the position where GFP (or Phluorin) is inserted. Tul1 contains an N-terminal luminal domain with multiple glycosylation sites, seven transmembrane helices, and a C-terminal RING domain. Right: Localization of Tul1-GFP, Tul1-pH-Ring, and Cot1-Mars 6 h after Zn²⁺ withdrawal from the YNB media. Both Tul1-GFP and Tul1-pH-Ring were overexpressed under the tet-O7 promoter, and Cot1-Mars was chromosomally tagged. White arrows highlight the punctae outside the vacuole. (B) Cot1-Mars was constitutively internalized into the vacuole lumen in cells overexpressing Tul1-pH-Ring, whereas Cot1-Mars stayed on the vacuole membrane in cells transformed with an empty vector (yellow arrows). Tul1-pH-Ring was overexpressed under the tet-O7 promoter, and Cot1-Mars was chromosomally tagged. (C) Localization of chromosomally tagged Ubx3-GFP and Cot1-Mars before and 6 h after removing Zn²⁺ from the YNB media. White lines highlight the position for line-scanning analysis, which was shown on the right. Bar, 1 µm.

Figure 8.

Biochemical evidence for the vacuolar localization of the Dsc complex and a model for the regulation of vacuolar membrane proteins. (A) Subcellular fractionation of WT yeast cells. The whole-cell lysate (T) was separated into P13, P100, and S100 fractions by differential centrifugation and probed with the indicated antibodies. (B) Purified vacuole membrane fraction was compared with the whole-cell lysate (total) by Western blot analysis. Samples that contain an equal amount of Vph1 were loaded in each lane. Approximately 60% of Ubx3 was estimated to localize to the vacuole membrane. (C) Immunoprecipitation experiments from the WT vacuole membrane fraction using preimmune, Dsc2, and Dsc3 antibodies. The immunoprecipitation reaction was probed with the indicated antibodies. (D) Immunoprecipitation experiment from the UBX3-FLAG vacuole membrane fraction using the M2 Flag antibody. The immunoprecipitation reaction was probed with indicated antibodies. (E) Silver staining analysis of the eluates from Fig. 7 D. (F) Two distinct E3 ligase systems converge on the vacuole membrane to regulate different vacuolar membrane transporters via the vReD pathway.