Quality Control in the Secretory Pathway: The Role of Calreticulin, Calnexin and BiP in the Retention of Glycoproteins with C-Terminal Truncations

Abstract

Unlike properly folded and assembled proteins, most misfolded and incompletely assembled proteins are retained in the endoplasmic reticulum of mammalian cells and degraded without transport to the Golgi complex. To analyze the mechanisms underlying this unique sorting process and its fidelity, the fate of C-terminally truncated fragments of influenza hemagglutinin was determined. An assortment of different fragments was generated by adding puromycin at low concentrations to influenza virus-infected tissue culture cells. Of the fragments generated, <2% was secreted, indicating that the system for detecting defects in newly synthesized proteins is quite stringent. The majority of secreted species corresponded to folding domains within the viral spike glycoprotein. The retained fragments acquired a partially folded structure with intrachain disulfide bonds and conformation-dependent antigenic epitopes. They associated with two lectin-like endoplasmic reticulum chaperones (calnexin and calreticulin) but not BiP/GRP78. Inhibition of the association with calnexin and calreticulin by the addition of castanospermine significantly increased fragment secretion. However, it also caused association with BiP/GRP78. These results indicated that the association with calnexin and calreticulin was involved in retaining the fragments. They also suggested that BiP/GRP78 could serve as a backup for calnexin and calreticulin in retaining the fragments. In summary, the results showed that the quality control system in the secretory pathway was efficient and sensitive to folding defects, and that it involved multiple interactions with endoplasmic reticulum chaperones.

Cell Surface Expression of α1D-Adrenergic Receptors Is Controlled by Heterodimerization with α1B-Adrenergic Receptors

“…Some GPCRs contain ER retention motifs that must be removed or masked before they can be exported, including the CB1 N terminus (68) and GABA B -R1 C terminus (34). Other GPCRs may be targeted by ER retention proteins such as HSP79 analogue BiP/GRP78 (69) or the lectin-like proteins calnexin and calreticulin (70,71), preventing surface expression. We show here that ␣ 1B -ARs promote ␣ 1D -AR surface expression, presumably by direct heterodimerization.…”

The cytoplasmic tail of the influenza C virus glycoprotein HEF negatively affects transport to the cell surface.

“…A conformational change of glycoproteins prior to leaving the ER is a common feature of many proteins destined to travel to other intracellular organelles or to the cell surface (Zhang et al, 1997). In the case of HEF, the conformational change appears to be especially pronounced, because it results in a dramatic decrease of the electrophoretic mobility under non-reducing conditions (Herrler et al, 1979).…”

Monitoring chaperone engagement of substrates in the endoplasmic reticulum of live cells

“…For the chaperone probe, we selected Crt for several reasons: the availability of a knockout cell line (19), chemical inhibitors of its function (20), well characterized biochemical properties (21)(22)(23)(24)(25)(26), and structural information (24,(27)(28)(29). Based on this information, we generated a GFP-tagged Crt (Crt-GFP) (Fig.…”