Identification of ERGIC-53 as an intracellular transport receptor of alpha1-antitrypsin

Abstract

Secretory proteins are exported from the endoplasmic reticulum (ER) by bulk flow and/or receptor-mediated transport. Our understanding of this process is limited because of the low number of identified transport receptors and cognate cargo proteins. In mammalian cells, the lectin ER Golgi intermediate compartment 53-kD protein (ERGIC-53) represents the best characterized cargo receptor. It assists ER export of a subset of glycoproteins including coagulation factors V and VIII and cathepsin C and Z. Here, we report a novel screening strategy to identify protein interactions in the lumen of the secretory pathway using a yellow fluorescent protein-based protein fragment complementation assay. By screening a human liver complementary DNA library, we identify alpha1-antitrypsin (alpha1-AT) as previously unrecognized cargo of ERGIC-53 and show that cargo capture is carbohydrate- and conformation-dependent. ERGIC-53 knockdown and knockout cells display a specific secretion defect of alpha1-AT that is corrected by reintroducing ERGIC-53. The results reveal ERGIC-53 to be an intracellular transport receptor of alpha1-AT and provide direct evidence for active receptor-mediated ER export of a soluble secretory protein in higher eukaryotes.

Figure 1.

Strategy of the ERGIC-53 cargo hunt based on YFP PCA. (A) Schematic representation of the YFP PCA approach to screen for ERGIC-53 cargo proteins in the lumen of the secretory pathway. YFP PCA is based on the reconstitution of functional YFP from two nonfluorescent fragments that are brought into close proximity by two interacting proteins. YFP2 was fused to the ERGIC-53 bait protein, whereas YFP1 was fused to a human liver cDNA library to express YFP1-tagged prey proteins. (B) The YFP2–ERGIC-53 bait protein was expressed from the kanamycin-resistant pCMV vector, whereas the cDNA-YFP1 library was constructed in the ampicillin-resistant pcDNA3 vector. (C) Flow chart of the screening strategy.

Figure 2.

Screening of the cDNA-YFP1 library for ERGIC-53 interaction partners. (A) COS-1 cells were transfected with the indicated bait and prey constructs and analyzed by FACS as described in Materials and methods. Coexpression of YFP2–ERGIC-53 and the cDNA-YFP1 library resulted in the specific detection of 1.63% YFP positive (YFP⁺) cells. In a nonsaturating screen, ∼500 fluorescent cells were collected by FACS and total DNA was extracted and transformed into bacteria, which resulted in the recovery of several hundred prey clones. (B) 48 prey clones were randomly selected and plasmids were isolated and individually assayed by YFP PCA with YFP2–ERGIC-53 in COS-1 cells. Fluorometric analysis revealed that prey plasmids 17, 32, 33, and 44 (indicted by red boxes) were positive and reconstitute fluorescent YFP when expressed with YFP2–ERGIC-53. Bars represent fluorometric values of a single screening experiment. The threshold for a positive hit was set to 250 arbitrary fluorescence units, which corresponds to a ∼1.5-fold induction in YFP fluorescence in comparison to untransfected cells. (C) Sequence analyses identified α1-AT as a cDNA insert in all four positive prey plasmids.

Figure 3.

ERGIC-53 captures α1-AT in a carbohydrate- and conformation-dependent manner. The indicated YFP PCA constructs were expressed in HeLa cells for 24 (A) and 48 h (B). 20 μM Lactacystin (lact) and 200 μM kifunensine (kif) were applied where indicated for 24 h. YFP complementation was analyzed by fluorometric analysis of cell suspensions in microtiter plates and expression levels of the different constructs were probed by Western blotting (WB) using polyclonal antibodies against ERGIC-53 and α1-AT. Endogenous ERGIC-53 functions as an input control. Bars represent mean ± SD (n = 3).

Figure 4.

α1-AT secretion is impaired in ERGIC-53 knockdown cells. HepG2 cells were transiently transfected for 96 h with control and ERGIC-53–specific siRNA duplexes. (A) Western blotting using monoclonal anti–ERGIC-53 and anti–CLIMP-63 and polyclonal anti–α1-AT antibodies. ERGIC-53 is efficiently silenced, which results in intracellular accumulation of α1-AT. CLIMP-63, an ER-resident membrane protein, functions as an input control. (B) [³⁵S]methionine pulse chase of endogenous α1-AT and albumin. HepG2 cells were labeled for 15 min with [³⁵S]methionine and chased for the indicated times, and α1-AT and albumin were recovered by immunoprecipitation from cell lysates (intracellular) and conditioned medium (extracellular) using anti–α1-AT and anti-albumin antibodies, respectively. (C) The amount of intracellular and extracellular α1-AT and albumin was quantified by densitometric scanning of the band intensities and the secreted fraction of total protein [extracellular/(intracellular + extracellular)] was determined for each time point. Bars represent mean ± SD (n = 3). Results analyzed by paired t test: NS, P > 0.05; **, P < 0.01.

Figure 5.

ERGIC-53 is an intracellular transport receptor of α1-AT. α1-AT was transfected into wild-type (+/+) and ERGIC-53 knockout (−/−) MEFs and expressed for 24 h. (A) [³⁵S]methionine pulse chase of α1-AT and endogenous fibronectin. MEFs were labeled with [³⁵S]methionine for 15 min and chased for the indicated times, and α1-AT and fibronectin were recovered by immunoprecipitation from cell lysates (intracellular) and conditioned medium (extracellular) using anti–α1-AT and anti-fibronectin antibodies, respectively. (B) The secreted fraction of α1-AT and fibronectin was quantified as described in Fig. 4 C. (C) α1-AT was coexpressed with human ERGIC-53 in ERGIC-53 knockout MEFs (MEF −/− plus ERGIC-53). Western blotting was performed using polyclonal antibodies against ERGIC-53, α1-AT, and CLIMP-63. (D) [³⁵S]methionine pulse chase of α1-AT. (E) The secreted fraction of α1-AT after a 60-min chase was quantified by densitometric scanning and calculated as in Fig. 4 [extracellular/(intracellular + extracellular)]. Bars represent mean ± SD (n = 3). Results analyzed by paired t test: NS, P > 0.05; *, P < 0.05.