Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Apr 22;42(2):160-71.
doi: 10.1016/j.molcel.2011.02.035.

Yeast SREBP Cleavage Activation Requires the Golgi Dsc E3 Ligase Complex

Affiliations
Free PMC article

Yeast SREBP Cleavage Activation Requires the Golgi Dsc E3 Ligase Complex

Emerson V Stewart et al. Mol Cell. .
Free PMC article

Abstract

Mammalian lipid homeostasis requires proteolytic activation of membrane-bound sterol regulatory element binding protein (SREBP) transcription factors through sequential action of the Golgi Site-1 and Site-2 proteases. Here we report that while SREBP function is conserved in fungi, fission yeast employs a different mechanism for SREBP cleavage. Using genetics and biochemistry, we identified four genes defective for SREBP cleavage, dsc1-4, encoding components of a transmembrane Golgi E3 ligase complex with structural homology to the Hrd1 E3 ligase complex involved in endoplasmic reticulum-associated degradation. The Dsc complex binds SREBP and cleavage requires components of the ubiquitin-proteasome pathway: the E2-conjugating enzyme Ubc4, the Dsc1 RING E3 ligase, and the proteasome. dsc mutants display conserved aggravating genetic interactions with components of the multivesicular body pathway in fission yeast and budding yeast, which lacks SREBP. Together, these data suggest that the Golgi Dsc E3 ligase complex functions in a post-ER pathway for protein degradation.

Figures

Figure 1
Figure 1. Sre1 cytosolic cleavage requires dsc genes
(A) Western blot probed with anti-Sre1 IgG of lysates from wild-type (lane 3) and sre1Δ cells expressing truncated versions of Sre1 (aa 1–440, aa 1–430, or aa 1–420, lanes 1, 2, 4 and 5). (B) Diagram of Sre1 where black boxes represent predicted transmembrane domains. The protein is inserted into the membrane in a hairpin fashion, placing both the N- and C-termini in the cytosol. Arrow denotes cleavage site. (C) Wild-type and mutant yeast (200 cells) containing no plasmid, a plasmid expressing Sre1N (aa 1 –440), or empty vector were grown on rich medium or rich medium containing cobalt chloride (CoCl2) in the presence of oxygen or on rich medium in the absence of oxygen. (D) Western blot probed with anti-Sre1 IgG of lysates from wild-type and indicated mutants grown for 6h in the presence or absence of oxygen. P and N denote Sre1 precursor and cleaved nuclear forms, respectively. See also Figure S1.
Figure 2
Figure 2. E-MAP analysis reveals a conserved function for the Dsc proteins
(A) Diagrams of Dsc proteins where black boxes represent predicted transmembrane domains. Predicted signal sequences and conserved domains are indicated. (B) Histograms of correlation coefficients calculated by comparing the genetic interactions of 471 query mutants mated against a library of 2,277 nonessential deletions. Histograms for dsc1Δ, dsc2Δ, dsc3Δ, dsc4Δ, sre1Δ and sre2Δ are shown and relevant genes are indicated. See also Figures S2 and S3.
Figure 3
Figure 3. Dsc proteins form a complex
(A) Digitonin solubilized extracts from anp1-GFP and dsc2-Myc anp1-GFP cells were prepared, and proteins associated with Dsc2-Myc were immunopurified with anti-myc IgG-9E10 monoclonal antibody. Equal amounts of total (lanes 1 and 2) and unbound fractions (lanes 3 and 4) along with 10× bound fractions (lanes 5 and 6) were immunoblotted using anti-Dsc1 IgG, anti-Dsc3 serum, anti-Dsc4 serum, rabbit anti-Myc IgG, anti-GFP and anti-Hmg1 IgG. (B) Eluate from tandem affinity purification using dsc1-TAP cells was subjected to velocity centrifugation on a 15%–40% sucrose gradient. Fractions were analyzed by immunoblot using anti-Dsc1 IgG, anti-Dsc2 serum, anti-Dsc3 serum, and anti-Dsc4 serum. (C) Digitonin solubilized extracts from anp1-GFP, dsc2-Myc anp1-GFP, and anp1-GFP dsc2-Myc dsc3Δ cells were prepared, and proteins associated with Dsc2-Myc were immunopurified using anti-myc IgG-9E10 monoclonal antibody. Equal amounts of total (lanes 1, 2 and 3) and unbound fractions (lanes 4, 5 and 6) along with 10× bound fractions (lanes 7, 8 and 9) were immunoblotted using anti-Dsc1 IgG, anti-Dsc4 serum, anti-Dsc3 serum, and rabbit anti-Myc IgG. For (A) and (C), + denotes wild-type allele.
Figure 4
Figure 4. Sre2 cleavage requires dsc genes
(A) Diagram of Sre2 where black boxes represent predicted transmembrane domains. (B) Western blot probed with anti-Sre2 serum of lysates from wild-type and indicated deletion mutant cells. P and N denote Sre2-GFP precursor and cleaved nuclear forms, respectively. (C) Wild-type and indicated deletion mutant cells containing a plasmid expressing GFP-sre2 (pAH230) were grown in minimal medium lacking leucine and thiamine for 20h to induce expression after which cells were labeled with DAPI to stain DNA and imaged by fluorescence microscopy.
Figure 5
Figure 5. Dsc complex resides in the Golgi
(A) dsc2–6xmGFP anp1-mCherry cells were cultured in the presence of oxygen and imaged by confocal fluorescence microscopy. (B) Western blot probed with anti-Sre1 IgG of lysates from wild-type and indicated mutant cells grown in the presence of oxygen and either brefeldin A (100 µg/ml) or vehicle (2% EtOH) for 2h at 30°C. (C) Western blot probed with anti-Sre1 IgG of lysates from wild-type and indicated mutant cells grown in the presence of oxygen and either brefeldin A (100 µg/ml) or vehicle (2% EtOH) for 2h at 30°C. (D) Western blot probed with anti-Sre1 IgG of lysates from wild-type and indicated mutant cells grown for a time course at 36°C in the presence of oxygen. In each figure, P denotes Sre1 precursor. See also Figures S4 and S5.
Figure 6
Figure 6. Sre1 cleavage requires components of the ubiquitin-proteasome pathway
(A) Western blot probed with anti-Sre1 IgG of lysates (top panel) from wild-type and indicated mutants grown for 0.5h at 30°C in anaerobic conditions. Microsomes (bottom panel) were isolated from the cell cultures and immunoblotted using anti-Dsc1 IgG. (B) Western blot probed with anti-Sre1 or anti-Sre2 IgG of lysates (top panels) from sre2-GFP (WT), sre2-GFP dsc1Δ, and sre2-GFP sre1Δ cells carrying an integrated plasmid expressing either empty vector, dsc1+ or dsc1-H668A grown for 0.5h at 30°C in anaerobic conditions. Microsomes (bottom panel) were isolated from the cell cultures, treated with peptide N-glycosidase F, and immunoblotted using anti-Dsc1 IgG. (C) Wild-type and ubc4-P61S cells were grown at the non-permissive temperature (36°C) for 1h and then grown in anaerobic conditions at 36°C for the indicated times. Cell lysates were subjected to immunoblot analysis using anti-Sre1 IgG. (D) Wild-type and mts3-1 cells were grown for 3h at the non-permissive temperature (36°C) to inactivate Mts3 and then shifted to anaerobic conditions at 36°C for the indicated times prior to cell harvest. Cell lysates were immunoblotted using anti-Sre1 IgG. P and N denote Sre1 precursor and cleaved nuclear forms, respectively. (E) dsc1–1, dsc2-Myc, dsc2-Myc dsc1–1 and dsc2-Myc dsc1–1 sre2Δ cells were treated with the reversible, cell-permeable crosslinker DSP (2 mM) for 30 min and detergent solubilized extracts were prepared as described in Experimental Procedures. Proteins associated with Dsc2-Myc were immunopurified using anti-myc IgG-9E10 monoclonal antibody. Unbound (lanes 1– 4) and 75× bound fractions (lanes 5–8) were immunoblotted using anti-Sre2 serum, anti-Dsc1 IgG, and rabbit anti-Myc IgG. + denotes wild-type allele.

Similar articles

See all similar articles

Cited by 38 articles

See all "Cited by" articles

Publication types

MeSH terms

Substances

Feedback