Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Nov 2;35(21):2332-2349.
doi: 10.15252/embj.201693923. Epub 2016 Sep 21.

A Golgi Rhomboid Protease Rbd2 Recruits Cdc48 to Cleave Yeast SREBP

Affiliations
Free PMC article

A Golgi Rhomboid Protease Rbd2 Recruits Cdc48 to Cleave Yeast SREBP

Jiwon Hwang et al. EMBO J. .
Free PMC article

Abstract

Hypoxic growth of fungi requires sterol regulatory element-binding protein (SREBP) transcription factors, and human opportunistic fungal pathogens require SREBP activation for virulence. Proteolytic release of fission yeast SREBPs from the membrane in response to low oxygen requires the Golgi membrane-anchored Dsc E3 ligase complex. Using genetic interaction arrays, we identified Rbd2 as a rhomboid family protease required for SREBP proteolytic processing. Rbd2 is an active, Golgi-localized protease that cleaves the transmembrane segment of the TatA rhomboid model substrate. Epistasis analysis revealed that the Dsc E3 ligase acts on SREBP prior to cleavage by Rbd2. Using APEX2 proximity biotinylation, we demonstrated that Rbd2 binds the AAA-ATPase Cdc48 through a C-terminal SHP box. Interestingly, SREBP cleavage required Rbd2 binding of Cdc48, consistent with Cdc48 acting to recruit ubiquitinylated substrates. In support of this claim, overexpressing a Cdc48-binding mutant of Rbd2 bypassed the Cdc48 requirement for SREBP cleavage, demonstrating that Cdc48 likely plays a role in SREBP recognition. In the absence of functional Rbd2, SREBP precursor is degraded by the proteasome, indicating that Rbd2 activity controls the balance between SREBP activation and degradation.

Keywords: SREBP; Cdc48/p97; intramembrane proteolysis; rhomboid; ubiquitin.

Figures

Figure 1
Figure 1. Rbd2 is a Golgi‐resident rhomboid protease

Plot of correlation coefficients generated from comparison of the genetic profiles from sre2Δ and SPCC790.03Δ (rbd2Δ) to all other profiles from the genetic interaction array. Note the high correlations among dsc1‐4, sre2, and rbd2.

Protein structural predictions for Schizosaccharomyces pombe Rbd2 (blue) and E. coli GlpG (red). Protein structures were predicted using the Phyre2 server (http://www.sbg.bio.ic.ac.uk/phyre2). Rbd2 model structure was aligned to GlpG using PyMOL.

Protein sequence alignment of E. coli GlpG, human RHBDL4, S. pombe Rbd2, and S. cerevisiae Rbd2. Alignments were generated using Clustal Omega server (http://www.ebi.ac.uk/Tools/msa/clustalo). Predicted transmembrane domains (TM) and shared sequence motifs were used for alignment. Positions of functionally important, conserved rhomboid amino acid residues (including predicted catalytic dyad serine and histidine) are shaded in red. Residues boxed in green were identified as required for Sre1 activation from a genetic selection and were analyzed in Fig 2E and F. The starting residue numbers of the indicated sequences within their full‐length proteins are given. Asterisks mark residues identical in all sequences, and colons denote conservative substitutions.

Topology model for S. pombe Rbd2 with the conserved six‐pass TM. Both the N‐ and C‐termini face the cytoplasm. The protease active site serine and histidine form a catalytic dyad between TM4 and TM6. A SHP box, predicted to bind Cdc48, is positioned at the Rbd2 C‐terminus.

Cells carrying a plasmid expressing rbd2‐6xmCherry and either of two Golgi proteins dsc2‐6xGFP (dsc2‐6xGFP rbd2‐6xmCherry) or Sec72‐6xGFP (Sec72‐6xGFP rbd2‐6xmCherry) were imaged by confocal fluorescence microscopy.

Figure 2
Figure 2. SREBP activation requires rbd2

Wild‐type (WT), rbd2Δ, or sre1Δ yeast (200 cells) containing either empty vector (EV) or a plasmid expressing sre1N (Sre1 amino acids 1–440) were grown on rich medium in the presence or absence of cobalt chloride (CoCl2).

Western blot was probed with anti‐Sre1 IgG of phosphatase‐treated, whole‐cell lysates from WT and the indicated mutants grown for the indicated time in the absence of oxygen. Dsc5 serves as a loading control and was detected by chemiluminescence. P and N denote Sre1 precursor and cleaved nuclear forms, respectively.

Indicated yeast strains expressing rbd2 or chromosomal Flag‐tagged rbd2, rbd2‐S130A, or rbd2‐H182A were analyzed for Sre1 cleavage. Western blot was performed using anti‐Sre1 or anti‐Flag IgG of phosphatase‐treated, whole‐cell lysates from cells grown for 3 h in the presence or absence of oxygen.

Indicated yeast strains expressing rbd2 or chromosomal Flag‐tagged rbd2, rbd2‐S130A, or rbd2‐H182A were analyzed for Sre2 cleavage by Western blot probed with anti‐Sre2 serum or anti‐Flag IgG of phosphatase‐treated, whole‐cell lysates from cells grown in the presence of oxygen.

Sre1 cleavage assay was performed as in (C). G128V, A127D, and A186T mutant strains were obtained from our previous mutagenesis screening (Stewart et al, 2012). Dsc5 serves as a loading control.

Sre2 cleavage assay for indicated yeast strains was performed as in (D).

A model substrate of Sre2 containing amino acids 423–793 (Sre2MS) with an N‐terminal 3xFlag epitope tag was expressed on a plasmid under the control of a CaMV promoter in sre2Δ, dsc1Δsre2Δ, or rbd2Δsre2Δ yeast strains. Whole‐cell lysates from indicated yeast strains were analyzed by Western blot with anti‐Flag antibody. Three independent isolates (A–C) are shown for each strain. Empty vector (EV) transformation serves as a negative control.

Figure 3
Figure 3. Uncleaved SREBP is degraded in a proteasome‐dependent and Dsc E3 ligase‐dependent manner in the absence of rbd2

Wild‐type (WT) and the indicated mutant strains were grown for 3 h at the non‐permissive temperature (36°C) to inactivate Mts3. Phosphatase‐treated, whole‐cell lysates were analyzed by Western blot with anti‐Sre2 serum and imaged using chemiluminescence. P and N denote Sre2 precursor and cleaved nuclear forms, respectively.

WT, rbd2Δ, and sre2Δ cells were treated with bortezomib (Bz) for 3 h, and phosphatase‐treated, whole‐cell lysates were analyzed by Western blot with anti‐Sre2 serum.

WT, dsc1Δ, and rbd2Δ cells carrying either empty vector (EV) or a plasmid expressing 3xFlag‐Sre2MS (MS) were treated with bortezomib (Bz) for 3 h. Whole‐cell lysates were analyzed by Western blot with anti‐Flag antibody.

Whole‐cell lysates from the indicated strains were analyzed by Western blot with anti‐Sre2 serum.

Catalytically dead Dsc1 E3 ligase (dsc1‐C634A) mutant and other indicated yeast strains were analyzed by Western blot probed with anti‐Sre2 serum and imaged using chemiluminescence.

Figure EV1
Figure EV1. Sre1 and Sre2 precursor degradation requires dsc1

Western blot of phosphatase‐treated, whole‐cell lysates from indicated strains was probed with anti‐Sre2 IgG. P and N denote Sre2 precursor and cleaved forms, respectively.

Western blot was probed with anti‐Sre1 IgG of phosphatase‐treated, whole‐cell lysates from wild‐type cells and the indicated mutants grown for 3 h in the presence or absence of oxygen.

Figure 4
Figure 4. Rbd2 is an active rhomboid protease

Human HEK293T cells were transfected with plasmids expressing different 3xHA‐tagged rhomboid proteases: Schizosaccharomyces pombe rbd2, S. pombe rbd2 recoded for human expression, human RHBDL2, D. melanogaster Rho1, and P. falciparum ROM4. Whole‐cell lysates were analyzed by Western blot to detect the HA‐tagged rhomboid. Asterisks indicate loading control bands detected with antibody ab179726 (Abcam).

Human HEK293T cells were transfected with plasmids expressing the indicated rhomboid substrates and either recoded S. pombe rbd2, D. melanogaster Rho1, or P. falciparum ROM4. Conditioned media and cell lysates were analyzed by Western blot. P and C denote precursor and cleaved products, respectively.

Human HEK293T cells were transfected with plasmids expressing the GFP‐TatA‐Flag substrate and the indicated rhomboid proteases. Whole‐cell lysates were analyzed by Western blot. P, CN, CC denote precursor, N‐terminal cleaved, and C‐terminal cleaved products, respectively.

Human HEK293T cells were transfected with plasmids expressing the indicated GFP‐TatA‐Flag substrates and in the absence or presence of recoded S. pombe rbd2. Whole‐cell lysates were analyzed by Western blot.

Human HEK293T cells were transfected with plasmids expressing the indicated GFP‐TatA‐Flag substrates and different rhomboid proteases. Whole‐cell lysates were analyzed by Western blot.

Human HEK293T cells were transfected with plasmids expressing the GFP‐TatA (A8L)‐Flag and recoded S. pombe rbd2. C‐terminal cleavage products were purified using anti‐Flag antibodies, and purified protein was analyzed by mass spectrometry.

Figure 5
Figure 5. Rbd2 cleavage requires Lys 743

Diagram of Sre2MS substrate (aa 423–793). The enlarged model shows predicted positions of TM1 and TM2 denoting the 26 amino acids tested for mutagenesis by colored squares. Numbers indicate the number of amino acid residues. Mutants showing a block in cleavage due to defects in Dsc1 function are indicated in orange, Rbd2 function in pink, a combination of defects in Dsc1 and Rbd2 in blue (intermediate), and residues showing normal cleavage are indicated in gray.

Whole‐cell lysates from WT, rbd2Δ, and dsc1Δ cells carrying a plasmid expressing 3xFlag‐Sre2MS WT or indicated mutants were analyzed by Western blot with anti‐Flag antibody. P and N denote Sre2MS precursor and cleaved forms, respectively.

Model outlining the predicted fate of Sre2MS in different yeast strains. U denotes either mono‐ubiquitin or poly‐ubiquitin.

Whole‐cell lysates from WT, rbd2Δ, and dsc1Δ cells carrying a plasmid expressing 3xFlag‐Sre2MS WT or indicated mutants were analyzed by Western blot with anti‐Flag antibody. P and N denote Sre2MS precursor and cleaved forms, respectively. Asterisks indicate non‐specific, loading control bands, which were used to normalize the intensities of P and N in (F). Two independent isolates, A and B, are shown for each strain.

Intensities of individual P and N bands from Western blots were quantified using LI‐COR software. Relative intensity of P and N to corresponding non‐specific band (asterisk) in each lane was normalized to that of P level in isolate A of WT strain for each mutagenesis. The ratio of N to P or relative P level was presented in bar graphs.

The average ratio of N to P level for different Sre2MS mutants in wild‐type cells (Appendix Fig S2) was plotted. Cleavage was scored as normal if the ratio was > 2.

For Sre2MS mutants showing defects in cleavage, the average fold‐increase in P level in dsc1Δ cells compared to WT cells from two isolates (Appendix Fig S2) was plotted. Mutants were grouped into three phenotypic classes (intermediate, dsc1Δ, and rbd2∆) according to observed precursor accumulation. Precursor accumulation was defined as dsc1Δ if < 1.5 (orange), rbd2Δ if > 3 (pink), and intermediate if in between (blue).

Figure 6
Figure 6. Rbd2 binds Cdc48 through a C‐terminal SHP box

Alignment of SHP box sequences from human UFD1, human p47, S. cerevisiae Dfm1, and Schizosaccharomyces pombe Rbd2 C‐terminal SHP box (aa 242–251). Asterisks denote residues identical in all sequences, colons mark conservative substitutions, and dots mark semi‐conservative substitutions.

Recombinant proteins GST‐fused Rbd2 C‐terminus (Rbd2200–251), Dsc5 UBX (Dsc5323–425), Dsc2 UBA (Dsc2298–372), and GST‐HA‐V5 control were bound to GST magnetic beads and incubated with S. pombe cytosol fraction from wild‐type cells. Equivalent amounts of unbound and bound fractions were probed for anti‐Cdc48, anti‐ubiquitin, and anti‐GST IgG.

GST pull‐down assay was performed as in (B) using truncated forms of Rbd2 C‐terminus; Rbd2200–251, Rbd2200–225, Rbd2225–251, and Rbd2200–240. Unbound (1×) and bound (5×) fractions were analyzed by Western blotting

Single residue mutants of conserved glycine residues in Rbd2200–251 (G244R and G246R) were tested for Cdc48 binding as in (C).

Data information: Western blots (B–D) were images using chemiluminescence.
Figure 7
Figure 7. APEX2 system detects Rbd2–Cdc48 binding in cells

Detergent‐solubilized yeast extracts from cells containing chromosomal Flag‐tagged rbd2 or rbd2‐G246R in rbd2Δ background were prepared, and proteins associated with Rbd2‐Flag were immunopurified using anti‐Flag magnetic beads. Equal amount of total proteins (lanes 1–3) along with 25× bound fractions (lanes 4–6) were analyzed by Western blot with anti‐Cdc48 serum and anti‐Flag IgG.

Illustration outlining APEX2‐catalyzed biotin‐phenol labeling. An Rbd2‐Flag‐APEX fusion protein oxidizes biotin‐phenol to biotin‐phenoxyl radical that covalently labels binding partners.

Biotin‐phenol labeling was performed for 1 min with H2O2 treatment as described in Materials and Methods. WT cells or rbd2Δ cells carrying rbd2‐Flag‐APEX2 (rbd2‐F‐AX) or rbd2‐G246R‐Flag‐APEX2 (G246R‐F‐AX) plasmid were lysed after biotin‐labeling reaction, and proteins were denatured by heating the cells in lysis buffer containing 1% SDS. Biotinylated proteins were then enriched using streptavidin magnetic beads. Lysates and 50×‐enriched eluates were analyzed by Western blot with IRDye 800CW Streptavidin, anti‐Cdc48 serum, or anti‐Flag IgG.

Figure EV2
Figure EV2. Rbd2 catalytic dead mutant binds Cdc48

WT, rbd2Δ, or two isolates (A and B) of yeast expressing rbd2‐Flag‐APEX2 under control of the constitutive adh1 + promoter in rbd2Δ background were assayed for Sre1 cleavage. Western blot was probed with anti‐Sre1 IgG of whole‐cell lysates from the indicated strains grown for 3 h in the presence or absence of oxygen. P and N denote Sre1 precursor and cleaved forms, respectively. Note that whole‐cell lysates were not treated with phosphatase, and multiple bands of Sre1N represent phosphorylated forms.

rbd2Δ cells expressing rbd2‐Flag‐APEX2, rbd2‐G246R‐Flag‐APEX2, or rbd2‐S130A‐Flag‐APEX2 from a plasmid were lysed after biotin‐labeling reaction, and proteins were denatured by heating the cells in a lysis buffer containing 1% SDS. Biotinylated proteins were then enriched using streptavidin magnetic beads. Lysates and 20×‐enriched eluates were analyzed by Western blot with IRDye 800CW Streptavidin, anti‐Cdc48 serum, and anti‐Flag IgG.

Figure 8
Figure 8. SREBP activation requires Cdc48 binding to Rbd2

Yeast strains (200 cells) containing chromosomal Flag‐tagged rbd2, rbd2‐G244R, rbd2‐G246R or rbd2‐S130A in rbd2Δ background, and sre1Δ yeast were grown on rich medium in the absence or presence of cobalt chloride (CoCl2).

Western blot of phosphatase‐treated, whole‐cell lysates from strains in (A) grown for 3 h in the presence or absence of oxygen was probed with anti‐Sre1 or anti‐Flag IgG. P and N denote Sre1 precursor and cleaved nuclear forms, respectively.

Sre2 cleavage was assayed by Western blot using anti‐Sre2 serum or anti‐Flag IgG of phosphatase‐treated, whole‐cell lysates from the indicated strains. N denotes Sre2 cleaved nuclear form.

Whole‐cell lysates from indicated yeast strains carrying a plasmid expressing 3xFlag‐Sre2MS were analyzed by Western blot with anti‐Flag antibody. Two independent isolates, A and B, are shown for each strain. Asterisk denotes non‐specific band.

Diagram of four Rbd2 chimeras fused to the SHP box from Schizosaccharomyces pombe Der1‐like protein (SPBC365.08c). The SHP boxes of Rbd2 and Der1‐like protein are shaded red and green, respectively. Asterisks denote point mutation in the SHP box.

Yeast strains containing Flag‐tagged rbd2‐SHP, rbd2‐SHP*, rbd2‐G246R‐SHP, and rbd2‐G246R‐SHP* were generated in rbd2Δ background by chromosomal integration of the rbd2 fusion proteins depicted in (E). Indicated yeast strains were then assayed for Sre1 cleavage as in (B).

Indicated strains were assayed for Sre2 cleavage as in (C).

Wild type, sre2Δ, or strains containing chromosomal Flag‐tagged rbd2, rbd2‐G246R, or rbd2‐S130S in rbd2Δ background (lane 7–12) were treated with bortezomib (Bz) for 2 h, and phosphatase‐treated, whole‐cell lysates were analyzed by Western blot with anti‐Sre2 serum or anti‐Flag IgG.

Figure EV3
Figure EV3. Fusion of UBX domain to Rbd2‐G246R partially rescues Sre1 cleavage
Yeast strains containing Flag‐tagged rbd2‐UBX and rbd2‐G246R‐UBX (diagrammed) were generated in rbd2Δ background by chromosomal integration. Whole‐cell lysates from indicated strains expressing Flag‐tagged Rbd2 variants grown for 3 h in the presence or absence of oxygen were analyzed by Western blotting with anti‐Sre1 or anti‐Flag IgG.
Figure 9
Figure 9. Overexpression of Rbd2 bypasses requirement for Cdc48 binding to Rbd2

Western blot of phosphatase‐treated, whole‐cell lysates from yeast strains carrying rbd2‐Flag‐APEX2 (rbd2‐F), rbd2‐G246R‐Flag‐APEX2 (G246R), or rbd2‐S130A‐Flag‐APEX2 (S130A) plasmid in rbd2Δ background and sre1Δ yeast was probed with anti‐Sre1 or anti‐Flag IgG. P and N denote Sre1 precursor and cleaved nuclear forms, respectively.

Indicated yeast strains (200 cells) containing chromosomal Flag‐tagged rbd2, rbd2‐G246R, or rbd2‐S130A in rbd2Δ background (lane 3–5) or yeast strains (200 cells) carrying rbd2‐Flag‐APEX2, rbd2‐G246R‐Flag‐APEX2, or rbd2‐S130A‐Flag‐APEX2 plasmid in rbd2Δ background (lane 6–8) were grown on rich medium in the absence or presence of cobalt chloride (CoCl2).

Comparison of Rbd2 protein levels from overexpression system to endogenous expression. Western blot of whole‐cell lysates from yeast strains containing chromosomal Flag‐tagged rbd2, rbd2‐G246R, or rbd2‐S130A in rbd2Δ background and yeast strains carrying rbd2‐Flag‐APEX2, rbd2‐G246R‐Flag‐APEX2, or rbd2‐S130A‐Flag‐APEX2 plasmid in rbd2Δ background was probed with anti‐Flag IgG. For quantification, the intensity of Flag band in each lane was normalized to the non‐specific band (asterisk).

rbd2Δ cells expressing indicated plasmids of GFP‐Anp1 or GFP‐Sre2MS together with rbd2‐Flag‐APEX2, rbd2‐S130A‐Flag‐APEX2, or rbd2‐S130A/G246R‐Flag‐APEX2 were treated with Bz at a concentration of 1 mM for 2 h prior to biotin‐labeling reaction. After labeling termination, cells were lysed and denatured as described in Materials and Methods. Biotinylated proteins were then enriched using streptavidin magnetic beads. Lysates (1× input) and 100×‐enriched eluates were analyzed by Western blot with IRDye 800CW Streptavidin, anti‐Flag IgG, anti‐Cdc48 serum, and anti‐GFP IgG. Intensity of GFP band in enriched sample was normalized to that in the corresponding input sample, then normalized to the Flag intensity in enriched sample. Relative intensities of biotinylated GFP level are shown in bar graph. Error bars denote standard deviation from three biological replicates.

Figure EV4
Figure EV4. Sre1 cleavage does not require signal peptide peptidase ypf1
Western blot of phosphatase‐treated, whole‐cell lysates from wild‐type cells and the indicated mutants grown for 3 h in the presence or absence of oxygen was probed with anti‐Sre1 IgG. P and N denote Sre1 precursor and cleaved forms, respectively.
Figure EV5
Figure EV5. Aspergillus fumigatus RbdB contains a conserved SHP box

Alignment of SHP box sequences from Schizosaccharomyces pombe Rbd2 C‐terminal SHP box (aa 242–251) and A. fumigatus RbdB C‐terminal SHP box (aa 263–272). Asterisks denote identical residues, colon marks conservative substitution, and dots mark semi‐conservative substitutions

Recombinant proteins GST‐fused S. pombe Rbd2 C‐terminus (aa 200–251), A. fumigatus RbdB C‐terminus (aa 211–272), S. cerevisiae Rbd2 C‐terminus (aa 192–262), and GST were bound to GST magnetic beads and incubated with S. pombe cytosol fraction from wild‐type cells. 5× bound fractions were probed for anti‐Cdc48 and anti‐GST IgG. The S. pombe cytosol fraction was used for 1× input (In.) loading.

Figure 10
Figure 10. Model for yeast SREBP cleavage
Under low sterol and oxygen conditions, Sre1 exits the ER and travels to the Golgi. Golgi‐localized Sre1 is ubiquitinylated by E2 Ubc4 and Dsc E3 ligase. Ubiquitinylated Sre1 is recruited by Cdc48 and cleaved by Rbd2. The intermediate cleaved product is further processed by a second, unknown protease to generate Sre1N that traffics to the nucleus to activate transcription. When Rbd2 is inactive (dashed box), ubiquitinylated Sre1 is subjected to proteasomal degradation. SRE, sterol regulatory element; U, either mono‐ubiquitin or poly‐ubiquitin.

Similar articles

See all similar articles

Cited by 11 articles

See all "Cited by" articles

Publication types

MeSH terms

Substances

LinkOut - more resources

Feedback