Inverting the Topology of a Transmembrane Protein by Regulating the Translocation of the First Transmembrane Helix

Abstract

TM4SF20 (transmembrane 4 L6 family 20) is a polytopic membrane protein that inhibits proteolytic processing of CREB3L1 (cAMP response element-binding protein 3-like 1), a membrane-bound transcription factor that blocks cell division and activates collagen synthesis. Here we report that ceramide stimulates CREB3L1 cleavage by inverting the orientation of TM4SF20 in membranes. In the absence of ceramide, the N terminus of the first transmembrane helix of TM4SF20 is inserted into the endoplasmic reticulum (ER) lumen. This translocation requires TRAM2 (translocating chain-associated membrane protein 2), a membrane protein containing a putative ceramide-interacting domain. In the presence of ceramide, the N terminus of the first transmembrane domain of TM4SF20 is exposed to cytosol. Consequently, the membrane topology of TM4SF20 is inverted, and this form of TM4SF20 stimulates CREB3L1 cleavage. In the presence of ceramide, translocation of TM4SF20 is TRAM2-independent. We designate this mechanism-causing regulated inversion of the membrane topology as "regulated alternative translocation."

Figure 1. Ceramide induces appearance of TM4SF20(B)

(A) Proposed model illustrating ceramide-induced RAT of TM4SF20. The N-terminal loop and the 1st transmembrane helix are highlighted in blue and yellow, respectively. The two polar residues G22 and N26 in the first transmembrane helix are marked in red. The three N-linked glycosylation sites in loop 3 are indicated. N80 in loop 2 is not glycosylated because of its close proximity to the transmembrane helix. The A and B forms of TM4SF20 are designated as TM4SF20(A) and TM4SF20(B), respectively. (B and C) On day 0, A549 cells were seeded at 4×10⁵ cells per 60-mm dish. On day 1, cells were treated with or without 6 μM C₆-ceramide for 4 h. (B) Cells were harvested and separated into nuclear and membrane fractions, and analyzed by immunoblot analysis with indicated antibodies. Immunoblot analysis with antibodies against calnexin and lysine-specific demethylase 1 (LSD1) served as loading controls for membrane and nuclear fractions, respectively. (C) The amount of TM4SF20 mRNA was quantified through RT-QPCR with the value in untreated cells set to 1. Results are reported as mean ± S.E.M. of three independent experiments. (D) On day 0, A549/pTM4SF20 cells were seeded at 4×10⁵ cells per 60-mm dish. On day 1, cells were treated with 6 μM C₆-ceramide for the indicated time. Cells were then harvested for the analysis of RIP of CREB3L1 as described in B. Immunoblot with anti-Myc was used to detect the stably transfected TM4SF20 tagged with the Myc epitope at the C-terminus of the protein. (E) On day 0, A549/pTM4SF20 cells were seeded at 4×10⁵ cells per 60-mm dish. On day 1, cells were treated with 0.24 units/ml of sphingomyelinase for the indicated time. Cells were then harvested for immunoblot analysis as described in D. Asterisk denotes a band derived from TM4SF20 the appearance of which is not always reproducible (e.g. Figure 1D) and is not always induced by ceramide treatment (e.g. Figure 3A). This band is thus not further pursued in this study. (F) On day 0, A549/pTM4SF20 cells were seeded at 4×10⁵ cells per 60-mm dish. On day 1, cells were treated with 500 nM doxorubicin for 24 h, or 6 μM C₆-ceramide for 8 h followed by immunoblot analysis with the indicated antibodies. See also Figure S1

Figure 2. Ceramide alters the membrane topology of TM4SF20

(A) The amino acid sequence and hydropathy plot of TM4SF20. The putative membrane-spanning sequence is underlined. Potential N-linked glycosylation sites are highlighted in red. The residue-specific hydropathy index was calculated over a window of 18 residues by the method of Kyte and Doolittle. (B) On day 0, A549/pTM4SF20 cells were seeded at 4×10⁵ cells per 60-mm dish. On day 1, after incubation with 6 μM C₆-ceramide for 8 h, cells were harvested and cell lysate were incubated in the absence and presence of the indicated endoglycosidase, subjected to SDS/PAGE followed by immunoblot analysis. (C) On day 0, A549 cells were seeded at 4×10⁵ cells per 60-mm dish. On day 1, cells were transfected with 0.1 μg of wild type or the mutant pCMV-TM4SF20-Myc as indicated. On day 2, cells were treated with 6 μM C₆-ceramide for 8 h. Cell lysates were incubated in the absence or presence of PNGase F followed by immunoblot analysis. (D) A549/pTM4SF20 cells were seeded and treated as described in C. Membrane vesicles were subjected to protease protection assay as described in Experimental Procedure followed by immunoblot analysis with anti-Myc detecting the epitope tagged at the C-terminus of TM4SF20. See also Figure S2

Figure 3. Ceramide induces RAT of TM4SF20

(A) On day 0, A549 cells were seeded at 4×10⁵ cells per 60-mm dish. On day 1, cells were transfected with 0.1 μg of plasmid encoding TM4SF20 tagged with the Myc epitope at C- (TM4SF20-Myc) or N-terminus of the protein (Myc-TM4SF20). On day 2, cells were treated with or without 6 μM C₆-ceramide for 8 h. Cell lysates were subjected to immunoblot analysis with anti-Myc to detect TM4SF20. (B and C) On day 0, A549 cells were seeded at 1×10⁵ cells per 60-mm dish. On day 1, the cells were transfected with a control siRNA (C) or that targeting SPCS3 (S). On day 3, the cells were transfected with 0.1 μg of plasmids encoding N or C-terminally tagged TM4SF20 and SPC3 containing synonymous mutations at the region targeted by the siRNA as described in A. On day 4, some cells were treated with 6 μM C₆-ceramide for 8 h as indicated (B) while others were not treated with the lipid (C). Cell lysates were subjected to immunoblot analysis with the indicated antibody. (D) On day 0, A549 cells were seeded at 5×10⁵ cells per 60-mm dish. On Day 1, cells were transfected as described in A with 0.1 μg of indicated plasmids encoding C-terminally Myc-tagged TM4SF20 with or without the Flag epitope inserted N-terminal to the first transmembrane helix. After incubation for 8 h, cells were treated with 5 μM C₆-ceramide. On day 2, after 16 h of the treatment, cells were harvested and lysates of the cells were subject to immunoprecipitation with anti-Flag. Supernatant and pellet fractions of the precipitation were loaded at 1:1 ratio and analyzed by immunoblot analysis with anti-Myc. (E) A549 cells were seeded, transfected with the indicated plasmids encoding the proline scanning mutants of TM4SF20, and treated with C₆-ceramide as described in (D). Cell lysates were subjected to immunoblot analysis with anti-Myc to detect TM4SF20. (F) A549 cells were seeded on day 0 and transfected on day 1 as described in (A) with pCMV-TM4SF20-Myc(C47, 77, 78, 101, 116, 126, 151, 217, 219S) in which cysteine residues are only presented in peptide N-terminal to the putative signal peptidase cleavage site. After 6 h, the cells were treated with 5 μM C₆-ceramide as indicated for 16 h. On day 2, cysteines residues from the cell lysate were labeled with maleimide-biotin (MBP) as indicated. Following isolation of TM4SF20 through immunoprecipitation with anti-Myc, the presence of cysteine residues in TM4SF20 was detected by immunoblot with streptavidin-HRP as described in Experimental Procedure. Asterisk denotes light chain of anti-Myc. (G) A549 cells were seeded on day 0 and transfected on day 1 as described in (A) with Myc-TM4SF20. On day 2, cells were treated with 6 μM C₆-ceramide for 8 h. Membrane vesicles were subjected to protease protection assay as described in Figure 2D. (H) On day 0, A549/pTM4SF20 cells were seeded at 4×10⁵ cells per 60-mm dish. On day 1, cells were treated with 50 μM cycloheximide or 4 μM C₆-ceramide as indicated for 5 h. Cell lysates were subjected to immunoblot analysis with anti-Myc to detect TM4SF20. See also Figure S3

Figure 4. The first transmembrane helix of TM4SF20 is critical for RAT of the protein

(A) A549 cells were seeded, transfected with a plasmid encoding either wildtype AP or mutant AP in which the signal peptide is replaced by the N-terminal sequence of TM4SF20, and analyzed by immunoblot analysis following PNGase F treatment as described in Figure 2C. (B–D) A549 cells were seeded, treated, transfected with a plasmid encoding TM4SF20-Myc in which the N-terminal sequence was replaced by the signal peptide of prolactin (B) and that contains the indicated point mutation (C and D), and analyzed as described in Figure 3A. (E) A model predicting ceramide-induced RAT of TM4SF20-AP fusion protein. The putative signal sequence and the first transmembrane helix of TM4SF20 are highlighted in blue and yellow, respectively. (F and G) A549 cells were set up at 4×10⁵ cells per 60-mm dish. On day 1 they were transfected with 0.5 μg of the plasmid encoding the fusion protein. On day 2 the cells were treated with 3 μM C₆-ceramide for 8 h as indicated. (F) Cell lysates were subjected to PNGaseF treatment followed by immunoblot analysis with anti-Myc to detect the transfected fusion protein. (G) Membrane vesicles of ceramide-treated cells were subjected to protease protection assay as described in Figure 2D.

Figure 5. Ceramide induces RAT of TM4SF20 through TRAM2

(A–D) On day 0, A549/pTM4SF20 cells were seeded at 1×10⁵ cells per 60-mm dish. On day 1, the cells were transfected with indicated siRNAs. On day 3, some of the cells were harvested for quantification of indicated mRNA through RT-QPCR with the value in cells transfected with the control siRNA set to 1 (A and C). Some of the cells were harvested for quantification of sphingolipids (E). The rest of the cells were treated with 6 μM C₆-ceramide as indicated for 8 h. Cell lysates were subjected to immunoblot analysis of TM4SF20 with anti-Myc (B and D). (A, C and E) Results are reported as mean ± S.E.M. of three independent experiments.

Figure 6. TM4SF20(B) activates CREB3L1 cleavage

(A) A549 cells were seeded on day 0 at 1.5×10⁶ cells per 100-mm dish. On day 1, cells were infected with lentivirus encoding the indicated proteins. On day 2, 24 h later, the cells were switched into fresh medium containing 2 μg/ml puromycin for selection of the virus-infected cells. On day 4, cells were treated with or without 6 μM C₆-ceramide for 8 h. Cells were then harvested and analyzed as described in Figure 1D. (B) A549/pTM4SF20 cells were set up and treated as described in Figure 5A. Cells were also fractionated into nucleus and membranes for analysis of RIP of CREB3L1 as described in Figure 1B.