1. Biochemistry and Chemical Biology
  2. Cell Biology
Download icon

Sorting of a multi-subunit ubiquitin ligase complex in the endolysosome system

  1. Xi Yang
  2. Felichi Mae Arines
  3. Weichao Zhang
  4. Ming Li  Is a corresponding author
  1. University of Michigan, United States
Research Article
Cite this article as: eLife 2018;7:e33116 doi: 10.7554/eLife.33116
7 figures, 1 table and 2 additional files

Figures

Targeting pathways utilized by the Dsc complex.

(A) A cartoon model showing two conserved pathways (VPS and AP3) for transporting proteins from Golgi to vacuole. Some VPS cargoes can be recycled from the endosome to Golgi by the retromer complex before reaching the vacuole. EE: early endosome, MVB: multivesicular body. (B) Localization of Ubx3-nG and Vph1-mCh in WT, vps27∆, pep12∆, and apl6∆ strain cells. Scale bar: 2 µm.

https://doi.org/10.7554/eLife.33116.003
Figure 2 with 1 supplement
Identification of two new Dsc components.

(A) A cartoon model showing known Dsc components in budding yeast. (B) A silver staining gel showing that Ypr109W and Yir014W co-immunoprecipitate with Ubx3-Flag, together with other known Dsc components. HC, heavy chain. LC, light chain. (C) 014W-Flag can selectively pull down known Dsc components. (D) 014W-Flag can selectively pull down with known Dsc components. (E) Cartoon representations of 014W and 109W. Both proteins are predicted to be tetra-spanning membrane proteins. (F) 014W-HA and 109W-Flag do not pull down each other. St: Starting material. Ub: Unbound. El: Elution.

https://doi.org/10.7554/eLife.33116.004
Figure 2—figure supplement 1
Identification of two new Dsc subunits by mass spectrometry.

(A) Mass spectrometry peptide coverage for Yir014W. The peptides identified by mass spectrometry are highlighted in red. The underlined sequences are the predicted transmembrane helices. (B) Mass spectrometry peptide coverage for Ypr109W. The peptides identified by mass spectrometry are highlighted in red. The underlined sequences are the predicted transmembrane helices. (C) Sequence similarity between Yir014W and Ypr109W. The alignment was performed with Clustal Omega (Sievers et al., 2011).

https://doi.org/10.7554/eLife.33116.005
109W and 014W have distinct subcellular localizations.

(A) 014W-nG co-localizes with FM4-64 labelled vacuole membrane, whereas 109W-nG localizes to the intracellular punctae. (B) Quantification of the number of 109 W-nG punctae. (C) Left, 109W-nG partially co-localizes with Mars-Sec7 labelled trans-Golgi (white arrows). Right, quantification of the co-localization. Error bar represents the Standard Error of the Mean (SEM). (D) Left, 109W-nG partially co-localizes with FM4-64 labelled endosomes (white arrows). Right, quantification of the co-localization. Error bar represents the SEM. (E) Co-localization between 109W and Ubx3 using rapamycin induced co-localization (RICo) assay. Left: A cartoon diagram showing the principle of the assay. Right: FRB-mCherry localization before and after rapamycin treatment. White arrows indicate the co-localization. Scale bar: 2 µm.

https://doi.org/10.7554/eLife.33116.006
Figure 3—source data 1

The source data for the quantification of 109W-nG punctae in Figure 3B.

https://doi.org/10.7554/eLife.33116.007
Figure 3—source data 2

The source data for the quantification of co-localization in Figure 3C-D.

https://doi.org/10.7554/eLife.33116.008
Figure 4 with 1 supplement
Vld1 competes with Gld1 to determine the Dsc subcellular localizations.

(A) Localization of Ubx3-nG after either VLD1 or GLD1 deletion. Note a small amount of punctae are also observed outside the vacuole in gld1∆ cells. (B) The small Ubx3-nG punctae in the gld1∆ cells co-localize with ER marker, DsRed-HDEL, whereas the punctae in vld1∆ cells do not co-localize with ER marker. (C) A competition assay to show elevating the Vld1 expression level in vld1∆ cells gradually diverts Ubx3-nG from punctae to vacuole membrane. Scale bar: 2 µm.

https://doi.org/10.7554/eLife.33116.009
Figure 4—figure supplement 1
Gld1 competes with Vld1 to determine the subcellular localization of the Dsc complex.

(A) A competition assay to show the localization of Ubx3-nG in strains with different GLD1 expression levels. The vacuoles were labeled with FM4-64. Vec: empty vector. (B) Immunoprecipitation experiments to show the interactions between Ubx3-Flag and other Dsc components in WT, Vld1-HA overexpression, and Gld1-GFP overexpression strains. Note for the Input, 1OD600 cells were loaded for each sample except Vld1-HA, lane 2 and Gld1-GFP OE, lane 3. Only 0.1 OD600 cells were loaded for these two samples due to the overexpression. Scale bar: 2 µm.

https://doi.org/10.7554/eLife.33116.010
The Golgi/endosome and vacuole subcomplexes are functionally independent.

(A) Subcellular localizations of GFP-Yif1 before (0 hr) and after (4 hr) amino acid starvation. (B) Western blot analysis of the GFP-Yif1 starvation assay. 1 OD600 cells were loaded in each lane. (C) Subcellular localizations of Cot1-GFP before (0 hr) and after (8 hr) Zn2+ withdrawal. (D) Western blot analysis of the Cot1-GFP degradation assay. Same volume of cells was loaded, with 0.25 OD600 cells loaded at 0 hr. Scale bar: 2 µm.

https://doi.org/10.7554/eLife.33116.011
Figure 6 with 2 supplements
Two core complexes that determine the subcellular localizations.

(A) Deletion analysis for Vld1-nG and Gld1-nG localizations. (B) Deletion analysis for Ubx3-nG localization. (C) Co-localization between the Ubx3-nG punctae and ER marker DsRed-HDEL in either dsc2∆ or vld1gld1∆ strains. (D) Double deletion of TUL1 and DSC3 did not change the localization patterns of Vld1-nG, Gld1-nG, or Ubx3-nG. (E) Western blot analysis of the Dsc complex assembly after deleting indicated Dsc components. St: Starting material, El: Elution. (F) A model to summarize the core complex concept. White dashed lines indicate the periphery of yeast cells. Scale bar: 2 µm.

https://doi.org/10.7554/eLife.33116.012
Figure 6—figure supplement 1
Ubx3, Vld1 and Gld1 still support the degradation of Cot1-GFP and GFP-Yif1 after neonGreen tagging.

(A) Western blot analysis of the Cot1-GFP degradation after Zn2+ withdrawal in WT, Ubx3-nG, and Vld1-nG strains. Same volume of cells was loaded for each group, with 0.25 OD600 cells loaded at 0 hr. (B) Western blot analysis of the GFP-Yif1 degradation after amino acid starvation in WT, Ubx3-nG, and Gld1-nG strains. Same volume of cells was loaded, with 0.5 OD600 cells loaded at 0 hr.

https://doi.org/10.7554/eLife.33116.013
Figure 6—figure supplement 2
Both Vld1-nG and Gld1-nG are trapped in the ER after deleting either DSC2 or UBX3.

(A) Co-localization of Vld1-nG punctae with the ER marker DsRed-HDEL in either dsc2∆ or ubx3∆ strains. (B) Co-localization of Gld1-nG punctae with the ER marker DsRed-HDEL in either dsc2∆ or ubx3∆ strains. (C) Localization of Ubx3-nG in vld1∆ gld1∆ cells expressing either an empty vector or spDsc4. The vacuoles were labeled with fluorescent dye FM4-64. Scale bar: 2 µm.

https://doi.org/10.7554/eLife.33116.014
Vld1 and Gld1 subcomplexes travel through the AP3 and VPS pathways, respectively.

(A) Subcellular localizations of Vld1-nG and Vph1-mCh in WT, pep12∆, vps27∆, and apl6∆ cells. (B) Vld1 contains a conserved acidic di-leucine motif at its C-terminus. (C) Subcellular localizations of vld1∆6AA-nG (last 6 amino acids of Vld1 deleted) in WT cell and pep12∆ cells. (D) A single E233 to A mutation caused the trafficking defects of vld1E233A-nG in both WT and pep12∆ cells. (E) Subcellular localizations of Gld1-nG and Vph1-mCh in WT, vps27∆, and pep12∆ cells. (F) Subcellular localizations of Gld1-nG in WT and vps35∆ cells. (G) A model summarizing the key findings of this study. White dashed lines indicate the periphery of yeast cells. Scale bar: 2 µm.

https://doi.org/10.7554/eLife.33116.015

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Strain, strain background (54 yeast strains)please find in Supplementary file 1
Transfected construct (11 yeast constructs)please find in Supplementary file 1
Antibodyrabbit anti-G6PDHSigma-AldrichA9521
Antibodymouse anti-GFPSanta Cruz Biotechnology, Inc.SC9996
Antibodymouse anti-FlagSigma-AldrichF7425
Antibodyrabbit anti-HALife technologies715500
Antibodymouse anti-HASigma-Aldrich12CA5
Antibodymouse anti-Vph1Invitrogen10D7
Antibodyrabbit anti-GFPTorrey Pines BiolabsTP401
AntibodyM2 anti-FLAG resinSigma-AldrichA2426
Antibodyanti-HA resinSigma-AldrichE6779
Peptide3xFlag peptideSigma-AldrichF3290
Chemical compoundRapamycinLC LaboratoriesASW-135
Chemical compoundDoxycyclineFisher BioReagents162209
Chemical compoundzymolyaseAmsbio120493–1
Chemical compoundFM4-64InvitrogenT3166
Chemical compoundProtease Inhibitor CocktailRoche21169500

Additional files

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)