The KDEL trafficking receptor exploits pH to tune the strength of an unusual short hydrogen bond

The endoplasmic reticulum (ER) is the main site of protein synthesis in eukaryotic cells and requires a high concentration of luminal chaperones to function. During protein synthesis, ER luminal chaperones are swept along the secretory pathway and must be retrieved to maintain cell viability. ER protein retrieval is achieved by the KDEL receptor, which recognises a C-terminal Lys-Asp-Glu-Leu (KDEL) sequence. Recognition of ER proteins by the KDEL receptor is pH dependent, with binding occurring under acidic conditions in the Golgi and release under conditions of higher pH in the ER. Recent crystal structures of the KDEL receptor in the apo and peptide bound state suggested that peptide binding drives the formation of a short-hydrogen bond that locks the KDEL sequence in the receptor and activates the receptor for COPI binding in the cytoplasm. Using quantum mechanical calculations we demonstrate that the strength of this short hydrogen bond is reinforced following protonation of a nearby histidine, providing a conceptual link between receptor protonation and KDEL peptide binding. Protonation also controls the water networks adjacent to the peptide binding site, leading to a conformational change that ultimately allows the receptor-complex to be recognized by the COPI system.

The KDEL Trafficking Receptor Exploits pH to Tune the Strength of an Unusual Short Hydrogen Bond

The endoplasmic reticulum (ER) is the main site of protein synthesis in eukaryotic cells and requires a high concentration of luminal chaperones to function. During protein synthesis, ER luminal chaperones are swept along the secretory pathway and must be retrieved to maintain cell viability. ER protein retrieval is achieved by the KDEL receptor, which recognises a C-terminal Lys-Asp-Glu-Leu (KDEL) sequence. Recognition of ER proteins by the KDEL receptor is pH dependent, with binding occurring under acidic conditions in the Golgi and release under conditions of higher pH in the ER. Recent crystal structures of the KDEL receptor in the apo and peptide bound state suggested that peptide binding drives the formation of a short-hydrogen bond that locks the KDEL sequence in the receptor and activates the receptor for COPI binding in the cytoplasm. Using quantum mechanical calculations we demonstrate that the strength of this short hydrogen bond is reinforced following protonation of a nearby histidine, linking receptor protonation to high affinity peptide binding. Protonation also controls the wetting of a cavity adjacent to the peptide binding site, leading to a conformational change that ultimately allows the complex to be recognized by the COPI system.

Pathogenic Effects of Impaired Retrieval between the Endoplasmic Reticulum and Golgi Complex

Cellular activities, such as growth and secretion, are dependent on correct protein folding and intracellular protein transport. Injury, like ischemia, malnutrition, and invasion of toxic substances, affect the folding environment in the endoplasmic reticulum (ER). The ER senses this information, following which cells adapt their response to varied situations through the unfolded protein response. Activation of the KDEL receptor, resulting from the secretion from the ER of chaperones containing the KDEL sequence, plays an important role in this adaptation. The KDEL receptor was initially shown to be necessary for the retention of KDEL sequence-containing proteins in the ER. However, it has become clear that the activated KDEL receptor also regulates bidirectional transport between the ER and the Golgi complex, as well as from the Golgi to the secretory pathway. In addition, it has been suggested that the signal for KDEL receptor activation may also affect several other cellular activities. In this review, we discuss KDEL receptor-mediated bidirectional transport and signaling and describe disease models and human diseases related to KDEL receptor dysfunction.

Retrotranslocation of the Chaperone Calreticulin from the Endoplasmic Reticulum Lumen to the Cytosol

ABSTRACT Polypeptide folding and quality control in the endoplasmic reticulum (ER) are mediated by protein chaperones, including calreticulin (CRT). ER localization of CRT is specified by two types of targeting signals, an N-terminal hydrophobic signal sequence that directs insertion into the ER and a C-terminal KDEL sequence that is responsible for retention in the ER. CRT has been implicated in a number of cytoplasmic and nuclear processes, suggesting that there may be a pathway for generating cytosolic CRT. Here we show that CRT is fully inserted into the ER, undergoes processing by signal peptidase, and subsequently undergoes retrotranslocation to the cytoplasm. A transcription-based reporter assay revealed an important role for the C-terminal Ca2+ binding domain in CRT retrotranslocation. Neither ubiquitylation nor proteasome activity was necessary for retrotranslocation, which indicates that the pathway is different from that used by unfolded proteins targeted for destruction. Forced expression of cytosolic CRT is sufficient to rescue a cell adhesion defect observed in mouse embryo fibroblasts from crt −/− mice. The ability of CRT to retrotranslocate from the ER lumen to the cytosol explains how CRT can change compartments and modulate cell adhesion, transcription, and translation.

Transport of an external Lys-Asp-Glu-Leu (KDEL) protein from the plasma membrane to the endoplasmic reticulum: studies with cholera toxin in Vero cells.

The A2 chain of cholera toxin (CTX) contains a COOH-terminal Lys-Asp-Glu-Leu (KDEL) sequence. We have, therefore, analyzed by immunofluorescence and by subcellular fractionation in Vero cells whether CTX can used to demonstrate a retrograde transport of KDEL proteins from the Golgi to the ER. Immunofluorescence studies reveal that after a pulse treatment with CTX, the CTX-A and B subunits (CTX-A and CTX-B) reach Golgi-like structures after 15-20 min (maximum after 30 min). Between 30 and 90 min, CTX-A (but not CTX-B) appear in the intermediate compartment and in the ER, whereas the CTX-B are translocated to the lysosomes. Subcellular fractionation studies confirm these results: after CTX uptake for 15 min, CTX-A is associated only with endosomal and Golgi compartments. After 30 min, a small amount of CTX-A appears in the ER in a trypsin-resistant form, and after 60 min, a significant amount appears. CTX-A seems to be transported mainly in its oxidized form (CTX-A1-S-S-CTX-A2) from the Golgi to the ER, where it becomes slowly reduced to form free CTX A1 and CTX-A2, as indicated by experiments in which cells were homogenized 30 and 90 min after the onset of CTX uptake in the presence of N-ethylmaleimide. Nocodazol applied after accumulation of CTX in Golgi inhibits the appearance of CTX-A in the ER and delays the increase of 3',5'cAMP, indicating the participation of microtubules in the retrograde Golgi-ER transport.