Subunit Architecture of the Golgi Dsc E3 Ligase Required for Sterol Regulatory Element-binding Protein (SREBP) Cleavage in Fission Yeast

Abstract

Background: Fungal sterol regulatory element-binding protein cleavage requires the Golgi Dsc E3 ligase complex of undefined structure. Results: Biochemical and bioinformatic analyses provide insight into Dsc E3 ligase architecture, indicating that Dsc2 is a ubiquitin-binding rhomboid pseudoprotease. Conclusion:The five Dsc E3 ligase subunits form subcomplexes and display defined connectivity. Significance: Multisubunit E3 ligase complexes in the ER and Golgi exhibit conserved subunit organization.

Endoplasmic Reticulum Exit of Golgi-resident Defective for SREBP Cleavage (Dsc) E3 Ligase Complex Requires Its Activity

“…In the absence of Dsc2, Dsc3, or Dsc4, Dsc1 migrated with an intermediate mobility as compared with the mature and deglycosylated forms (Fig. 3A, lane 2) (8). Unexpectedly, we observed that Dsc1 mutants lacking E3 ligase activity also displayed incomplete glycosylation (Fig.…”

“…Interestingly, complete glycosylation of Dsc1 requires Dsc2, Dsc3, and Dsc4 (8). In the absence of Dsc2, Dsc3, or Dsc4, Dsc1 migrated with an intermediate mobility as compared with the mature and deglycosylated forms (Fig.…”

“…We speculated previously that the Dsc1 glycosylation defect in cells lacking Dsc subunits was due to either improper complex assembly or Dsc1 mislocalization (8). To assess Dsc E3 ligase complex assembly in strains lacking E3 ligase activity, we performed co-immunoprecipitation studies with digitonin-solubilized membranes from wild-type and RING domain mutant cells.…”

“…The Dsc E3 ligase complex has a hierarchical organization where Dsc2-Dsc3-Dsc4 define the core, and Dsc1 and Dsc5 are peripheral subunits (Fig. 1A) (8). To assay Dsc E3 ligase localization, we determined the localization of the core subunit Dsc2 in mutant strains using live cell fluorescence confocal microscopy.…”

“…Co-immunoprecipitation-Co-immunoprecipitation assays were performed as described previously (8). Briefly, cells (6 ϫ 10 8 ) from exponentially growing wild-type or mutant strains were collected and lysed using glass beads in digitonin lysis buffer (50 mM HEPES, pH 6.8, 1% (w/v) digitonin, 50 mM KOAc, 2 mM Mg(OAc) 2 , 1 mM CaCl 2 , 200 mM sorbitol, 1 mM NaF, 0.3 mM Na 3 VO 2 , supplemented with 1ϫ protease inhibitors).…”