Calcium as a crucial cofactor for low density lipoprotein receptor folding in the endoplasmic reticulum

Abstract

The family of low density lipoprotein (LDL) receptors mediate uptake of a plethora of ligands from the circulation and couple this to signaling, thereby performing a crucial role in physiological processes including embryonic development, cancer development, homeostasis of lipoproteins, viral infection, and neuronal plasticity. Structural integrity of individual ectodomain modules in these receptors depends on calcium, and we showed before that the LDL receptor folds its modules late after synthesis via intermediates with abundant non-native disulfide bonds and structure. Using a radioactive pulse-chase approach, we here show that for proper LDL receptor folding, calcium had to be present from the very early start of folding, which suggests at least some native, essential coordination of calcium ions at the still largely non-native folding phase. As long as the protein was in the endoplasmic reticulum (ER), its folding was reversible, which changed only upon both proper incorporation of calcium and exit from the ER. Coevolution of protein folding with the high calcium concentration in the ER may be the basis for the need for this cation throughout the folding process even though calcium is only stably integrated in native repeats at a later stage.

FIGURE 1.

Effect of ER calcium depletion on LDL receptor folding and transport to the Golgi complex. HeLa cells overexpressing wild-type LDL receptor were pulse-labeled for 5 min and chased for the indicated periods in the presence of thapsigargin, A23187, or DMSO (Control). Under the conditions used (see “Experimental Procedures”), thapsigargin and A23187 both deplete ER calcium levels, albeit with different mechanisms and with different effect on cytosolic calcium concentration. a, samples were precipitated with polyclonal antiserum 121, which recognizes all forms of the LDL receptor, whether folded, misfolded, reduced, or denatured, and independent of subcellular location. Immunoprecipitates then were subjected to nonreducing SDS-PAGE. ER represents newly synthesized ER localized LDL receptor molecules, and G represents O-glycosylated “mature” LDL receptor molecules that have reached the Golgi complex or beyond. The gray reference lines help comparison of mobilities. b, the same samples as in a, now reduced. c, as a, except that lysates were immunoprecipitated with monoclonal antibody C7, which recognizes the most N-terminal repeat of the LDL receptor, but only when properly folded and containing its disulfide bonds and bound calcium. d, samples as in a of cells treated with A23187, DMSO (Control), or thapsigargin were analyzed next to each. NR, nonreducing SDS-PAGE; R, reducing SDS-PAGE.

FIGURE 2.

Specificity of ER calcium depletion on LDL receptor folding. a, HeLa cells overexpressing wild-type LDL receptor were pulse-labeled for 4 min and chased for 0 or 2 h in the presence of thapsigargin or DMSO (Control). The LDL receptor was precipitated in parallel with polyclonal antiserum 121 and monoclonal antibodies C7, 6B2, 7H2, 5G2, and 6E2, against properly folded and disulfide-bonded ligand-binding repeats LR1, LR1, LR3, LR5, and LR7, respectively). The samples were analyzed by reducing SDS-PAGE. Ab, antibody. b, influenza virus infected CHO15B cells, expressing as control HA, were pulse-labeled for 2 min and chased for the indicated periods in the presence of DMSO (Control) or thapsigargin. The samples were immunoprecipitated with a polyclonal antibody that recognizes all conformations of HA and were subjected to nonreducing (NR) and reducing (R) SDS-PAGE. IT1 and IT2 are the two HA folding intermediates with incomplete sets of disulfide bonds that fold into NT, which is the native monomer with native epitopes and its six native disulfide bonds. ER represents the ER forms of HA. Upon transport to the Golgi complex, the N-linked glycans of HA are trimmed substantially in these cells, which increases electrophoretic mobility. G represents HA molecules that have reached the Golgi complex and beyond. NP is the viral nucleoprotein, which functions as marker and loading control. c, HeLa cells overexpressing the LDL receptor P678L mutant were pulse-labeled for 5 min and chased for 2 h in the presence of thapsigargin. The samples were immunoprecipitated with polyclonal antibody 121 and subjected to reducing SDS-PAGE.

FIGURE 3.

Cartoon representing disulfide bond formation during folding of the LDL receptor in the presence (Control) and absence of calcium. Control cartoon reprinted with permission (28, 34).

FIGURE 4.

DTT resistance of disulfide bonds during LDL receptor folding. HeLa cells overexpressing the LDL receptor were pulse-labeled for 5 min and chased for 0 or 1 h. The cells then were cooled immediately on ice (ch+DTT 0′) or chased for an additional 5 min in the presence of 10 mm DTT (ch+DTT 5′). The media for starvation, pulse, and chase all contained solvent (DMSO; control) (a) or 100 nm thapsigargin (b). Detergent cell lysates were immunoprecipitated in parallel with polyclonal antiserum 121 and monoclonal antibody C7. The samples were analyzed by reducing (Red, +) and nonreducing (Red, −) SDS-PAGE.

FIGURE 5.

Resistance of the LDL receptor to calcium depletion during folding. HeLa cells overexpressing the LDL receptor were pulse-labeled for 5 min, chased for 1 h in complete chase medium (ch+Ca²⁺), and then depleted of calcium in an additional 1-h chase (ch-Ca²⁺) in thapsigargin (lanes 4 and 9) or in calcium-free medium containing A23187 (lanes 5 and 10). Detergent cell lysates were immunoprecipitated in parallel with polyclonal antiserum 121 (lanes 1–5) and monoclonal antibody C7 (lanes 6–10). The control samples were pulse-labeled and chased for the same time intervals in complete medium containing calcium and DMSO (lanes 1–3 and 6–8). The samples were subjected to nonreducing (a) and reducing (b) SDS-PAGE.

FIGURE 6.

Rescue from early calcium depletion. HeLa cells were pulse-labeled for 5 min (p±Ca²⁺) and chased for 15 min or 2 h in calcium-free medium containing A23187 (ch - Ca²⁺). For the rescued samples (ch+Ca²⁺) the chase medium was replaced by calcium-containing complete chase medium without A23187. Control samples were pulse-labeled for 5 min (p + Ca²⁺) and chased for 1 h (ch+Ca²⁺) in complete medium containing DMSO. a, samples precipitated in parallel with polyclonal antiserum 121 (lanes 1–7) or monoclonal antibody C7 (lanes 8–14) were subjected to nonreducing SDS-PAGE. b, the same samples as in a, now reduced.