Protein Folding in the Endoplasmic Reticulum

Calnexin (CNX) and calreticulin (CRT) represent a novel class of molecular chaperones resident in the ER (72-74). Calnexin (also termed p88 and IP90) was described by three different groups as a protein that was transiendy associated in the ER with class I major 

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Braakman, I., Helenius, J., and Helenius, A. (1992). Manipulating disulfide bond formation and protein folding in the endoplasmic reticulum. EMBO J. 11, 1717—1722. 

Zhang, K. and Kaufman, R.J. (2006a). Protein folding in the endoplasmic reticulum and the unfolded protein response. Handb. Exp. Pharmacol. 172, 69-91. 

Tu BP, Weissman JS. The FAD- and 02-dependent reaction cycle of Erol-mediated oxidative protein folding in the endoplasmic reticulum. Mol. Cell. 2002 10 983-994. 

Knittler MR, Dirks S, Haas IG (1995) Molecular chaperones involved in protein degradation in the endoplasmic reticulum quantitative interaction of the heat shock cognate protein BiP with partially folded immunoglobulin light chains that are degraded in the endoplasmic reticulum. Proc Natl Acad Sd USA 92 1764-1768... 

It is unknown how and when the receptor chemokine complexes form when the two proteins are coexpressed in Sf9 cells. One possibility is that the chemokine molecules are secreted in the cell culture medium and from there, bind to the receptors that are expressed and trafficked to the plasma membrane. Alternatively, the proteins may bind one another at the time of folding in the endoplasmic reticulum and be trafficked to the cell surface together as a complex. In either case, the interaction interface is in an oxidizing environment which promotes disulfide bond formation for those... 

IP3 mobilizes Ca2+ from intra- or extracellular stores. The interior of a cell is kept very low in Ca2+ ions, at a concentration less than 10-9 M., while the outside [Ca2+] is about 10-3 M. This million-fold concentration gradient is the result of cellular calcium-dependent ATPase protein. Ca-ATPase uses up to a third of the ATP synthesized by a cell to maintain the concentration gradient. The stores of Ca2+ available for use inside the cell are found primarily in the endoplasmic reticulum. A large store of Ca2+ exists in the mitochondrial matrix, but this seems to be a final dumping ground —in other words, calcium ions in the mitochondria don t come into the cytoplasm. 

At least two major slow processes occur in the folding of disulfide-containing proteins the cis-trans isomerizations of Xaa-Pro peptide bonds and the formation of the correct disulfide bonds. The latter is catalyzed by protein disulfide-isomerase (PDl). This enzyme occurs at high concentration in the endoplasmic reticulum (Hawkins et al., 1991) and there is good experimental evidence that PDl is required for the de novo folding of nascent secretory proteins (Bulleid and Freedman, 1988). Cyclophilins have recently also been localized in the ER (Hasel et al., 1991) and in other compartments of the secretory pathway (Caroni et al., 1991). Their biological function is not known. 

Clearly, the action of prolyl isomerases is not restricted to the slow folding of polypeptide chains with intact disulfides, but they also accelerate the oxidative folding of reduced proteins, which resemble more closely the nascent polypeptide chains as they occur in the endoplasmic reticulum. The simultaneous presence of PPI markedly enhances the efficiency of PDI as a catalyst of disulfide bond formation. Both enzymes act according to their specificity and catalyze the isomerization of prolyl peptide bonds and the formation of disulfide bonds, respectively, in the folding protein chains. It remains to be demonstrated that a similar concerted action of the two enzymes can take place in the course of de novo synthesis and folding of proteins in the cell. 

A retarding effect of CsA on cellular folding, mediated possibly by the inhibition of PPI activity, was observed for two large proteins, collagen and transferrin. Both are secreted proteins that mature to their native oligomeric structure in the endoplasmic reticulum. 

Inositol trisphosphate opens a calcium transport channel in the membrane of the endoplasmic reticulum. This leads to an influx of calcium from storage in the endoplasmic reticulum and a 10-fold increase in the cytosolic concentration of calcium ions. Calmodulin is a small calcium binding protein found in all cells. Its affinity for calcium is such that, at the resting concentration of calcium in the cytosol (of the order of 0.1 /xmol per L), little or none is bound to calmodulin. When the cytosolic concentration of calcium rises to about 1 /xmol per L, as occurs in response to opening of the endoplasmic reticulum calcium transport channel, calmodulin binds 4 mol of calcium per mol of protein. When this occurs, calmodulin undergoes a conformational change, and calcium-calmodulin binds to, and activates, cytosolic protein kinases, which in turn phosphorylate target enzymes. 

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