Carbohydrate- and conformation-dependent cargo capture for ER-exit

Abstract

Some secretory proteins leave the endoplasmic reticulum (ER) by a receptor-mediated cargo capture mechanism, but the signals required for the cargo-receptor interaction are largely unknown. Here, we describe a novel targeting motif that is composed of a high-mannose type oligosaccharide intimately associated with a surface-exposed peptide beta-hairpin loop. The motif accounts for lectin ERGIC-53-assisted ER-export of the lyososomal enzyme procathepsin Z. The second oligosaccharide chain of procathepsin Z exhibits no binding activity for ERGIC-53, illustrating the selective lectin properties of ERGIC-53. Our data suggest that the conformation-based motif is only present in fully folded procathepsin Z and that its recognition by ERGIC-53 reflects a quality control mechanism that acts complementary to the primary folding machinery in the ER. A similar oligosaccharide/beta-hairpin loop structure is present in cathepsin C, another cargo of ERGIC-53, suggesting the general nature of this ER-exit signal. To our knowledge this is the first documentation of an ER-exit signal in soluble cargo in conjunction with its decoding by a transport receptor.

Figure 1.

CatZr is the Chinese hamster ortholog of procatZ. (A) Sequence alignment of prepro-catZ from different species. Sequences are available from GenBank/EMBL/DDBJ under accession numbers Q9EPP7 (hamster), Q9WUU7 (mouse), Q9R1T3 (rat), Q9UBR2 (human), NP_491023 (worm). Signal peptides are underlined; the propeptide cleavage site is marked with a triangle; peptides used for immunizing rabbits are boxed; identified catZr-peptides are shaded; glycosylation sites are bold and marked with an asterisk; the two strands of the β-hairpin loop (G₁₇₁TCNEFKECHA₁₈₁) and the S-S linkage within the loop are indicated by a black bar. (B) Cross-linking and coimmunoprecipitation of GMAA-ERGIC-53 and hamster procatZ. GMAA cells were treated with or without DSP and subjected to anti-ERGIC-53 immunoprecipitation (IP). For comparison, 1% of the total homogenate (H) is shown. GMAA-ERGIC-53 and catZ were visualized by Western blotting (WB) using anti-myc or affinity-purified anti-catZ. Note that procatZ can be cross-linked to ERGIC-53. p, procatZ; m, mature catZ. (C) Maturation of endogenous procatZ in GM and GMAA cells (fluorogram). The cells were pulsed for 15 min with [³⁵S]methionine, chased as indicated, and subjected to anti-catZ immunoprecipitation. p, procatZ; i, intermediate catZ; m, mature catZ. Note that in GMAA cells the transport of catZ is slowed. (D) Coimmunoprecipitation of human procatZ with GMAA-ERGIC-53. GMAA cells were transfected with HA-tagged wt-procatZ cDNA, cross-linked with DSP, and subjected to immunoprecipitation using anti-ERGIC-53 (IP). procatZ was visualized by anti-HA immunoblotting. Note that human procatZ can be coprecipitated with ERGIC-53 in a DSP-dependent way.

Figure 3.

Efficient binding of procatZ to ERGIC-53 requires a combined carbohydrate/peptide motif. (A) Three-dimensional model of the peptide β-hairpin loop (cyan) and the high-mannose N184-linked glycan (yellow). The peptide structure was obtained from the protein data bank (PDB ID: 1DEU). The glycan is shown in a hypothetical conformation. Selected amino acid side chains that are likely to anchor the adjacent oligosaccharide by hydrogen bonds and the disulfide linkage are shown. As depicted in single letter code, mutations to eliminate the hairpin to the level of the disufide-linkage (Δloop^*) or completely (Δloop) were engineered by introducing a DG-turn (corresponding to procatZ turns at positions 72/73, 252/253, or 282/283). (B) Secretion of Δloop and N184Q+Δloop (fluorogram). Transfected CHO cells were pulsed for 5 min, chased for different times, and analyzed by immunoprecipitation of HA-tagged procatZ from the cell lysate (c) or the medium (m). cg, complex glycosylated catZ; i, intermediate catZ; p, procatZ. (C) Quantification of initial (1 h) secretion of the glycan and Δloop mutants. The pulse-chase experiments were performed as in Figures 2A and 3B, and fluorograms were quantified by densitometric scanning. Means ± SD (n = 3). The reduction in secretion of N224Q is statistically not significantly different from wt as opposed to that of the other constructs. (D) Binding of glycan and Δloop mutants to GMAA-ERGIC-53. Sequential immunoprecipitation of GMAA-ERGIC-53 and recombinant procatZ (bottom panel) after a 10-min pulse with or without 15-min chase and DSP-treatment. The top panel shows 10% of the first (anti-ERGIC-53) immunoprecipitate. Molecular weight markers in kDa are indicated at the left margin. (E) Quantification of the binding of glycan and Δloop mutants to GMAA-ERGIC-53. The experiments were as in D. Fluorograms were quantified by densitometric scanning, normalized to ERGIC-53 (recovered in the first immunoprecipitation), and expressed as percentage of wt/15-min chase (mean ± SD, n = 3). ProcatZ mutants lacking the full carbohydrate/peptide motif exhibit weak binding that does not increase after 15-min chase.

Figure 2.

Characterization of procatZ N-glycosylation-site mutants. (A) Top: CHO cells were transfected with wt or glycosylation-site mutant procatZ cDNAs, pulsed for 5 min with [³⁵S]methionine, and chased for the indicated times. Intracellular and secreted procatZ was recovered by anti-HA immunoprecipitation (fluorogram). c, cells; m, medium; p, procatZ; i, intermediate catZ; cg; complex glycosylated catZ. Bottom: pulse-chase analysis as in top panel but using denaturing immunoprecipitation. Note that under these experimental conditions N184 + 224Q is readily detectable. (B) ProcatZ protein secreted from transfected CHO cells after a 5-min pulse and a 3-h chase was isolated by anti-HA immunoprecipitation and subjected to endoH or PNGase F digestion (fluorogram). Numbers indicate doubly-, singly-, and nonglycosylated procatZ. Wt-procatZ and N184Q are partially resistant to endoH, whereas N224Q is almost completely endoH-sensitive. (C) Cross-linking and coimmunoprecipitation of GMAA-ERGIC-53 and procatZ mutants. Anti-ERGIC-53 immunoprecipitates from GMAA-cells expressing the indicated procatZ mutants and treated with or without DSP were analyzed for the presence of procatZ by anti-HA immunoblotting. One percent of the total homogenate (H) was loaded as an expression control. Note that DSP-dependent coisolation is decreased by N184Q, but unaffected by N224Q. IP, immunoprecipitation; WB, Western blotting.

Figure 4.

The full binding motif is required for the transport inhibition induced by GMAA-ERGIC-53. Comparison of the secretion of wt and mutant procatZ after 5-min pulse and 1-h chase from GMAA and Lec1 cells. Top: representative fluorograms. Bottom: quantification of the inhibition of secretion in GMAA versus Lec 1 cells (mean ± SD, n = 3). Calculation: 100[lane4/(lane3+lane4)]/[lane2/(lane1+lane2)]. Lane numbers are in italic. Note that only in the case of wt and N224Q the delay in secretion is statistically significantly different from zero (p < 0.05, Student's t test). c, cell lysate; m, medium.

Figure 5.

Folding kinetics of procatZ mutants. (A) CHO cells were transfected with wt-procatZ cDNA, pulsed for 5 min and chased for 15 min. ProcatZ immunoprecipitates were separated by SDS-PAGE in the presence or absence of dithiothreitol. The electrophoretic mobility of nonreduced procatZ is increased, because intramolecular disulfide bonds remain. The 29-kDa molecular-weight marker is indicated. (B) Pulse (5 min)-chase time course of procatZ mutants that were subjected to nonreducing gel electrophoresis after immunoprecipitation. Note that the conversion to a sharp band indicating the folded state is efficient in wt-procatZ and Δloop, but delayed in N184Q (fluorograms).

Figure 6.

The β-hairpin loop protects the N184-glycan of procatZ from complex glycosylation. CHO cells transfected with wt-procatZ, Δloop^*, and Δloop cDNA were pulsed for 5 min and chased for 3 h. Secreted procatZ was collected from the medium by anti-HA immunoprecipitation, probed for the sensitivity to endoH and PN-Gase F, and analyzed by SDS-PAGE/fluorography. Numbers indicate doubly-, singly-, and nonglycosylated procatZ after endoH treatment. The graph plots the percentages of endoH-resistant oligosaccharides for the three procatZ mutants as determined by densitometry.

Figure 7.

Superimposition of the hairpin loop motifs of procatZ and cathepsin C. The two highlighted hepta-peptides of procatZ (red) and cathepsin C (orange) were superimposed using the Swiss PDB viewer software (available at www.expasy.ch). Shown are the peptide backbones of procatZ (white, PDB ID: 1DEU) and cathepsin C (CatC, gray, PDB ID: 1K3B), the side chains of glycosylation-site asparagines 184 and 95, and the disulfide bridge within the procatZ hairpin. Note that the overlay is restricted to the β-hairpin loops. The full sequence of the cathepsin C motif is K₈₂YKEEGSKVTTYCNET₉₇ (β-strands are underlined; numbering according to Turk et al., 2001).