Organizational diversity among distinct glycoprotein endoplasmic reticulum-associated degradation programs

Abstract

Protein folding and quality control in the early secretory pathway function as posttranslational checkpoints in eukaryote gene expression. Herein, an aberrant form of the hepatic secretory protein alpha1-antitrypsin was stably expressed in a human embryonic kidney cell line to elucidate the mechanisms by which glycoprotein endoplasmic reticulum-associated degradation (GERAD) is administered in cells from higher eukaryotes. After biosynthesis, genetic variant PI Z underwent alternative phases of secretion and degradation, the latter of which was mediated by the proteasome. Degradation required release from calnexin- and asparagine-linked oligosaccharide modification by endoplasmic reticulum mannosidase I, the latter of which occurred as PI Z was bound to the molecular chaperone grp78/BiP. That a distinct GERAD program operates in human embryonic kidney cells was supported by the extent of PI Z secretion, apparent lack of polymerization, inability of calnexin to participate in the degradation process, and sequestration of the glycoprotein folding sensor UDP-glucose:glycoprotein glucosyltransferase in the Golgi complex. Because UDP-glucose:glycoprotein glucosyltransferase sustains calnexin binding, its altered distribution is consistent with a GERAD program that hinders the reentry of substrates into the calnexin cycle, allowing grp78/BiP to partner with a lectin, other than calnexin, in the recognition of a two-component GERAD signal to facilitate substrate recruitment. How the processing of a mutant protein, rather than the mutation itself, can contribute to disease pathogenesis, is discussed.

Figure 1

Secretion, degradation, and asparagine-linked oligosaccharide processing for synthesized variant PI Z in HEK/Z1. (A) Fluorographic detection after SDS-PAGE of variant PI Z (Z) immunoprecipitated from the NP-40-soluble (s) and insoluble (i) cell lysates (c) and medium (m) after a 15-min pulse of HEK/Z1 with [³⁵S]methionine, and chase for up to 7 h. The discrete mobility shift (*) reflects asparagine-linked oligosaccharide modification during intracellular retention. (B) Results from the pulse-chase experiment are depicted as the percentage of radiolabeled variant PI Z remaining in the cells (open circles), the percentage secreted into the medium (closed circles), and the percentage degraded (shaded circles) at each time point.

Figure 2

Proteasome-mediated degradation of PI Z in HEK/Z1. Fluorographic detection after SDS-PAGE of variant PI Z (Z) immunoprecipitated from the cell lysate (cells) and medium (med.) from HEK/Z1 after a 15-min pulse with [³⁵S]methionine and a 5-h chase with (±) or without (−) media supplemented with 0.025 mM lactacystin (Lct). The discrete mobility shift (*) reflects asparagine-linked oligosaccharide modification during intracellular retention.

Figure 3

Newly synthesized variant PI Z undergoes sequential physical interaction with calnexin and grp78/BiP. (A) Calnexin and KDEL immunoblots of variant PI Z (PI Z) immunoprecipitates (Immppt.) generated under steady-state conditions from HEK/Z1 (lane 2) and from the total cell extract (Extract) (lane1), the latter of which was used merely to show the relative migration of the endogenous immunoreactive protein. The immunological detection of calnexin (Cxn), grp78/BiP and grp94 is shown. (B) SDS-PAGE and fluorographic detection of immunoprecipitated variant PI Z (Z) released from a calnexin (Cxn → Z) or grp78/BiP (Grp78/BiP → Z) immunoprecipitate after a 15-min pulse of HEK/Z1 with [³⁵S]methionine and chase.

Figure 4

Proposed order of events that coincide with the secretion, or intracellular retention and degradation of newly synthesized variant PI Z in HEK/Z1. A model is depicted in which partial deglucosylation by glucosidases I and II (step 1) leads to the assembly of newly synthesized and unfolded AAT (a) with calnexin (Cxn) (step 2) before conformational maturation (A) (step 3) and secretion. The remaining nonnative population of molecules eventually fails to bind calnexin and assemble with grp78/BiP (BiP) (step 4) before degradation by the proteasome, which requires asparagine-linked oligosacharide modification by ER mannosidase I (step 5) and recognition by a lectin (step 6). In the absence of proteasomal degradation, a significant fraction of nonnative molecules can attain conformational maturation (dashed arrow), and are secreted. The steps inhibited with castanospermine (Cst) or kifunensine (Kif) are shown, as are the predicted number of glucose (G) and mannose (M) units.

Figure 5

Kifunensine-sensitive degradation of variant PI Z in HEK/Z1. (A) Structural organization of the 14-unit asparagine-linked oligosaccharide precursor consisting of glucose (open squares), mannose (closed ovals), and N-acetylglucosamine (closed squares), attached to the Asn-x-Ser/Thr concensus sequence, is shown. The sites of hydrolysis by glucosidase I (Glc'ase I), glucosidase II (Glc'ase II), ER mannosidase I (ER ManI), and ER mannosidase II (ER Man II) are depicted, as is the glucose transferred by UDP-UGT. The two hydrolytic sites for glucosidase II are shown (a and b), and the combinations of sugars that constitute the Man8B isomer are depicted with a plus (±). (B) Fluorographic detection after SDS-PAGE of variant PI Z (Z) immunoprecipitated from the cell lysates (cells) and medium (med.) from HEK/Z1 after a 15-min pulse with [³⁵S]methionine and 5-h chase with (±) or without (−) media supplemented with 0.1 mM kifunensine (Kif). The discrete mobility shift (*) reflects asparagine-linked oligosaccharide modification during intracellular retention. Also, the band intensities in lanes 4 and 5 are misleading, possibly resulting from changes in band density caused by altered oligosaccharide modification and mobility. (C) Calnexin and KDEL immunoblots of variant PI Z (PI Z) immunoprecipitates (Immppt.) generated under steady-state conditions from HEK/Z1 under control conditions (lane 2) or treated with deoxymannojirimycin (+Dmj) (lane 3), as well as from a HEK/Z1 cell extract (Extract) (lane 1), the latter of which was used merely to show the relative migration of the endogenous immunoreactive protein. The immunological detection of calnexin (Cxn) and grp78/BiP is shown by the arrows.

Figure 6

Intracellular distribution of UGT by indirect immunofluorescence microscopy (see MATERIALS AND METHODS). Hepa1a (H1A) cells (A–C) and HEK293 (HEK) cells (D–F) were grown on coverslips, fixed in methanol and incubated with antibodies against UDP-UGT, calnexin (CNX), or Golgi mannosidase II (GMII), before incubation with species-specific fluorescein isothiocyanate-conjugated fluorescent secondary antibodies. In each case, the fluorescence pattern was indicative of >95% of cells. Brefeldin A (BFA) was added to H1A cells (B) and HEK cells (E) 1.5 h before methanol fixation at a final concentration of 2 μg/ml.

Figure 7

Intracellular distribution of molecular chaperones by indirect immunofluorescence microscopy (see MATERIALS AND METHODS). Hepa1a (H1A) cells (A–C) and HEK293 (HEK) cells (D–F) were grown on coverslips, fixed in methanol, and incubated with antibodies against calreticulin (CRT), calnexin (CRT), or grp78/BiP (BiP) (see MATERIALS AND METHODS) before incubation with species-specific fluorescein isothiocyanate-conjugated fluorescent secondary antibodies. In each case, the fluorescence pattern was indicative of >95% of the cells.