A new class of endoplasmic reticulum export signal PhiXPhiXPhi for transmembrane proteins and its selective interaction with Sec24C

Abstract

Protein export from the endoplasmic reticulum (ER) depends on the interaction between a signal motif on the cargo and a cargo recognition site on the coatomer protein complex II. A hydrophobic sequence in the N terminus of the bovine anion exchanger 1 (AE1) anion exchanger facilitated the ER export of human AE1Δ11, an ER-retained AE1 mutant, through interaction with a specific Sec24 isoform. The cell surface expression and N-glycan processing of various substitution mutants or chimeras of human and bovine AE1 proteins and their Δ11 mutants in HEK293 cells were examined. The N-terminal sequence (V/L/F)X(I/L)X(M/L), (26)VSIPM(30) in bovine AE1, which is comparable with ΦXΦXΦ, acted as the ER export signal for AE1 and AE1Δ11 (Φ is a hydrophobic amino acid, and X is any amino acid). The AE1-Ly49E chimeric protein possessing the ΦXΦXΦ motif exhibited effective cell surface expression and N-glycan maturation via the coatomer protein complex II pathway, whereas a chimera lacking this motif was retained in the ER. A synthetic polypeptide containing the N terminus of bovine AE1 bound the Sec23A-Sec24C complex through a selective interaction with Sec24C. Co-transfection of Sec24C-AAA, in which the residues (895)LIL(897) (the binding site for another ER export signal motif IXM on Sec24C and Sec24D) were mutated to (895)AAA(897), specifically increased ER retention of the AE1-Ly49E chimera. These findings demonstrate that the ΦXΦXΦ sequence functions as a novel signal motif for the ER export of cargo proteins through an exclusive interaction with Sec24C.

Keywords: AE1; Anion Transport; COPII; ER Export; Endoplasmic Reticulum (ER); Erythrocyte; Intracellular Trafficking; Membrane Proteins; Molecular Cell Biology; Sec24.

FIGURE 1.

Expression of human and bovine AE1 and their C-terminally truncated mutants in transfected HEK293 cells. A, schematic illustration of erythroid AE1 based on the crystallographic structure of the cytoplasmic domain (36) and the topology model of the transmembrane domain (45). The amino acid sequences of the N- and C-terminal regions of human and bovine AE1 are aligned, and residues highlighted in blue are identical between humans and cattle. The N-terminal sequences of canine, equine, and murine AE1 are also shown. The N-terminal sequence varies between the species. The functions of these amino acid sequences in ER export were analyzed (Figs. 4–7), and asterisks indicate the hydrophobic residues that correspond to the ΦXΦXΦ motif. B, HEK293 cells were transfected with EGFP-tagged hAE1 or bAE1 and their C-terminally truncated mutants. The cells were fixed after 48 h, and EGFP and the ER marker calnexin were visualized. A merged image of EGFP- (green), calnexin- (red), and DAPI-stained nuclei (blue) is shown. The indicated areas in the merged images are magnified, and EGFP fluorescence at the plasma membrane is indicated by arrowheads. Bars, 10 μm. C, at 48 h after transfection, cell surface proteins were labeled with sulfo-NHS-SS-biotin. After solubilization, biotin-labeled proteins were separated from intracellular proteins on NeutrAvidin beads. AE1 proteins in total (Total), intracellular (Intracellular), and cell surface (Surface) fractions were detected by immunoblotting with an anti-GFP antibody. The amount of cell surface fraction loaded is 10-fold higher than that of the other fractions. Migrating positions of size markers are shown in kDa.

FIGURE 2.

Intracellular localization of a series of bAE1-hAE1 chimeric proteins and their Δ11 mutants. A, a series of bAE1 and hAE1 chimeric proteins are schematically illustrated with the numbers of the amino acid residues shown. The overall structures of erythroid and kidney AE1 are presented along with a representative illustration of the hAE1/b[1–403] mutant. CYT, TM, and Ct indicate the cytoplasmic domain, the transmembrane domain, and the short C-terminal cytoplasmic tail, respectively. The chimeras hAE1/b[1–403], hAE1/b[1–75], hAE1/b[1–17], hAE1/b[17–75], hAE1/b[17–55], hAE1/b[17–37], hAE1/b[23–37], hAE1/b[25–29], and hAE1/b[33–35] have a hAE1 backbone and contain amino acid residues 1–403, 1–75, 1–17, 17–75, 17–55, 17–37, 23–37, 25–29, and 33–35 from bAE1, respectively. Conversely, the chimeras bAE1/h[1–385], bAE1/h[18–39], and bAE1/h[26–30] contain the N-terminal amino acid residues 1–385, 18–39, and 26–30 of hAE1, respectively, in the corresponding regions of bAE1. The Δ11 mutants of these chimeras, in which the 11 C-terminal amino acid residues were deleted, were also prepared. hAE1 has a single N-glycosylation site on the fourth extracellular loop in the TM, whereas bAE1 does not (29). B, various bAE1-hAE1 chimeras and their Δ11 mutants were transfected into HEK293 cells, and their intracellular localizations were determined by confocal laser microscopy. EGFP fluorescence at the plasma membrane is indicated by arrowheads. C, HEK293 cells were transfected with EGFP-hAE1/b[25–29], EGFP-bAE1/h[26–30], and their Δ11 mutants. The cells were fixed at 48 h after transfection and stained with an anti-calnexin antibody. EGFP fluorescence at the cell surface is indicated by arrowheads. EGFP- (green), calnexin- (red), and DAPI-stained nuclei (blue) are shown in the merged images. Bars, 10 μm. D, cell surface expression of hAE1/b[25–29], bAE1/h[26–30], and their Δ11 mutants was examined by cell surface biotinylation as described in the legend for Fig. 1. AE1 proteins in the total (Total), intracellular (Intracellular), and cell surface (Surface) fractions were detected by immunoblotting using an anti-GFP antibody. The amount of cell surface fraction loaded is 10-fold higher than that of the other fractions. The migrating positions of the size markers are shown in kDa.

FIGURE 3.

Identification of the N-terminal amino acid sequence of AE1 required for its cell surface expression. A, various amino acid substitution mutants of EGFP-bAE1 and EGFP-hAE1Δ11 were transfected into HEK293 cells. Mutated amino acid residues are highlighted. B–D, the cell surface expression of these mutants, as determined by laser confocal microscopy and cell surface biotinylation studies, are summarized by + and −, indicating positive and negative (less than 5% of the total amount) cell surface expression, respectively. CYT, TM, and Ct indicate the N-terminal cytoplasmic domain, the transmembrane domain, and the short cytoplasmic C-terminal tail, respectively. bAE1 is not N-glycosylated (29), whereas hAE1 possesses a single N-glycan in the extracellular loop. B and C, HEK293 cells were transfected with various EGFP-bAE1 mutants in which one or two amino acid residues in the ²⁵SVSIPM³⁰ sequence were substituted by the indicated residue(s). After 48 h, the intracellular localizations of the mutants was analyzed by confocal laser microscopy (B) and cell surface biotinylation (C), as described in the legend of Fig. 1. In B, EGFP fluorescence at the plasma membrane is indicated by arrowheads. EGFP- (green), calnexin- (red), and DAPI-stained nuclei are shown in the merged images. Bars, 10 μm. In C, bAE1 mutants detected by immunoblotting of the total (T), intracellular (I), and cell surface (S) fractions are shown, and the migrating positions of the size markers are shown in kDa. Cell surface expression of each mutant was determined by the presence of EGFP fluorescence at the plasma membrane (B) and when the relative abundance of the mutant in the cell surface fraction comprised more than 5% of the total amount, as determined by densitometric scanning of the immunoblots (C). D and E, intracellular localization of hAE1Δ11/P27V, hAE1Δ11/S29I, and hAE1Δ11/P27V/S29I was examined by confocal laser microscopy (D) and cell surface biotinylation (E) as described in the legend to Fig. 1. EGFP fluorescence at the plasma membrane is indicated by arrowheads in D. Total (Total), intracellular (Intracellular), and cell surface (Surface) fractions were deglycosylated with endo H or peptide N-glycosidase F (PNGase). The amount of cell surface fraction loaded is 10-fold higher than that of the other fractions. Symbols indicate the migrating positions of each hAE1Δ11 mutant with mature and processed N-glycans (bars), endo H-sensitive immature N-glycans (closed arrowheads), and the deglycosylated polypeptides (open arrowheads). The migrating positions of the size markers are shown in kDa. Bars, 10 μm.

FIGURE 4.

Intracellular distribution of the AE1-Ly49E chimeric proteins. A, schematic illustration of the AE1 N terminus-Ly49E chimeric proteins (bN[1–37]Ly, hN[1–39]Ly, and hN[P27V/S29I]Ly), which contain the N-terminal cytoplasmic regions (AE1 Nt) of bAE1, hAE, or hAE1/P27V/S29I and the transmembrane and extracellular domains of Ly49E with an N-terminal EGFP tag. B–D, AE1-Ly49E mutants were transfected into HEK293 cells, and their intracellular localizations were analyzed by biotinylation (B), deglycosylation (C), and confocal laser microscopy (D) as described in the legends to Figs. 1 and 2. A representative immunoblot of three independent experiments is presented in B. T, I, and S indicate the total, intracellular, and cell surface fractions, respectively. The amount of cell surface fraction loaded is 10-fold higher than that of the other fractions. In B and C, bars, closed arrowheads, and open arrowheads indicate the migrating positions of the mutant proteins whose N-glycan is endo H-resistant, endo H-sensitive, and deglycosylated, respectively. The migrating positions of the size markers are shown in kDa. In D, EGFP fluorescence (green) was visualized in HEK293 cells expressing bN[1–37]Ly, hN[1–39]Ly, or hN[P27V/S29I]Ly, and the ER, ER exit sites, ERGIC, and cis-Golgi were stained with anti-calnexin, anti-Sec23A, anti-ERGIC53, and anti-GM130 antibodies, respectively (red). Representative images are shown. The indicated areas of the region of interest (ROI) in the merged images are magnified (Magnified). Bars, 10 μm. E, Pearson's coefficients of co-localization between AE1-Ly49E chimeric proteins and organelle markers were analyzed. Co-localization was analyzed for each marker in the ROIs of 24 cells using LSM5 PASCAL co-localization software. The data are shown as the means ± S.D. (n = 24).

FIGURE 5.

Effect of Sar1A expression on the N-glycan processing of AE1-Ly49E reporter proteins. A, HEK293 cells were co-transfected with a AE1-Ly49E mutant (bN[1–37]Ly, hN[1–39]Ly, or hN[P29V/S29I]Ly) and either an empty vector (Mock), C-terminally Myc-tagged Sar1A (Sar1-myc), or its activated form (Sar1 H79G). After 48 h of incubation, cell lysates were prepared, and AE1-Ly49E mutants were detected by immunoblotting with an anti-GFP antibody. A representative immunoblot from three independent experiments is presented. Bars and arrowheads indicate the migrating positions of AE1-Ly49E mutants with mature and immature N-glycans, respectively. The migrating positions of the size markers are shown in kDa. B, the abundance of the AE1-Ly49E mutants bearing mature N-glycans relative to the total amount was quantitated by densitometric scanning of the immunoblots. The data are expressed as the means ± S.D. (n = 3). *, p < 0.05; **, p < 0.005.

FIGURE 6.

Roles of the N-terminal sequences of erythroid AE1 from several species in the N-glycan processing and cell surface expression of the protein. A, schematic illustration of EGFP-tagged AE1Δ11 mutants of canine, equine, and murine erythroid AE1. Equine and murine erythroid AE1 contain the N-terminal sequences ¹¹IEVIV¹⁵ and ³⁵LTIPV³⁹, which conform to the ΦXΦXΦ motif, respectively, whereas canine erythroid AE1 does not contain this motif as shown in Fig. 1A. B, EGFP-AE1Δ11 mutants described above were transfected into HEK293 cells, which were then fixed and stained for calnexin. Equine and murine AE1Δ11 were abundant at the plasma membrane (arrowheads), whereas canine AE1Δ11 exhibited a similar pattern to calnexin. EGFP- (green), calnexin- (red), and DAPI-stained nuclei (blue) are shown in the merged images. C, the partial sequences of the N-terminal regions of canine, equine, and murine erythroid AE1 (AE1 Nt) were substituted for the N-terminal cytoplasmic region of Ly49E to create the AE1-Ly49E chimeric proteins cN[1–46]Ly, eqN[1–41]Ly, and mN[1–49]Ly, respectively. D and E, HEK293 cells were transfected with cN[1–46]Ly, eqN[1–41]Ly, and mN[1–49]Ly and incubated for 48 h. The intracellular localization of the mutants was examined by confocal laser microscopy after staining with an anti-calnexin antibody (D), and N-glycan processing was analyzed by deglycosylation as described in the legends to Figs. 2 and 3 (E). In D, the plasma membrane expression of eqN[1–41]Ly and mN[1–49]Ly is indicated by arrowheads. Bars, 10 μm. In E, total cell lysates from the transfected cells were mock treated, endo H-treated, or peptide N-glycosidase F (PNGase)-treated and then immunoblotted with an anti-EGFP antibody. The migrating positions of each AE1-Ly49E mutant with mature and processed N-glycans (bars), endo H-sensitive immature N-glycans (closed arrowheads), and the deglycosylated polypeptides (open arrowheads) are indicated. The migrating positions of the size markers are shown in kDa.

FIGURE 7.

Selective interaction between the ΦXΦXΦ motif and Sec24C. A, HEK293 cell lysate (lane L) was incubated with bacterially expressed bN[1–37]-Halo or hN[1–39]-Halo protein that was immobilized on HaloLink resin or with the resin alone (Control). Bound proteins (lane B) were separated by SDS-PAGE on a 5–20% gradient gel followed by staining with Coomassie Brilliant Blue (CBB). The 120- and 85-kDa polypeptides that specifically bound to bN[1–37] were subjected to MS/MS analysis and were identified as Sec24C and Sec23A, respectively (indicated by arrowheads). Lane I contains the bait protein, and the asterisk indicates the bait protein that leaked from the resin. Immunoblotting (IB) with an anti-Sec23A antibody detected a band specifically in the fraction that bound the bN[1–37]-Halo-coupled resin and in the total cell lysate, which migrated at a size comparable with that of the 85-kDa protein (lower panel). Proteins in each fraction were separated on an 8% gel and were immunoblotted. B, lysates from HEK293 cells expressing Myc-tagged Sec24 isoforms (Sec24A, Sec24B, Sec24C, and Sec24D) or Sec24C-AAA were incubated with bN[1–37]-Halo-coupled resin as described above. Sec24 proteins in cell lysates (L), unbound (U), and bound (B) fractions were detected by immunoblotting with an anti-Myc antibody (indicated by arrowheads). C and D, the effect of Sec24C-AAA on N-glycan processing of AE1-Ly49E chimeric proteins was examined. HEK293 cells were co-transfected with an AE1-Ly49E mutant (bN[1–37]Ly, hN[1–39]Ly, or hN[P27V/S29I]Ly) or VSV-G-EGFP and either the empty vector (Mock), Myc-tagged Sec24C (Sec24C), or its Sec24C-AAA mutant (Sec24C-AAA). After 48 h, the AE1-Ly49E and VSV-G proteins and Sec24C proteins in the cell lysates were detected by immunoblotting with anti-GFP and anti-Myc antibodies, respectively. Representative immunoblots from three independent experiments are shown in C. The migrating positions of AE1-Ly49E mutants and VSV-G with mature and immature N-glycans are indicated by a bar and an arrowhead, respectively. The asterisk indicates Sec24C proteins. The migrating positions of the size markers are shown in kDa. D, the abundance of AE1-Ly49E and VSV-G proteins with mature and immature N-glycans relative to the total amount were quantitated by densitometric scanning of the immunoblots. The data are expressed as the means ± S.D. (n = 3). *, p < 0.05; **, p < 0.005.