How, then, do calnexin and calreticulin distinguish properly folded from incompletely folded proteins? The answer lies in yet another ER enzyme, a glucosyl transferase that keeps adding a glucose to those oligosaccharides that have lost their last glucose. It adds the glucose, however, only to oligosaccharides that are attached to unfolded proteins. Thus, an unfolded protein undergoes continuous cycles of glucose trimming (by glucosidase) and glucose addition (by glucosyl transferase), maintaining an affinity for calnexin and calreticulin until it has achieved its fully folded state (Figure 12–49).
improperly folded proteins are exported from the er and Degraded in the Cytosol
Despite all the help from chaperones, many protein molecules (more than 80% for some proteins) translocated into the ER fail to achieve their properly folded or oligomeric state. Such proteins are exported from the ER back into the cytosol, where they are degraded in proteasomes (discussed in Chapter 6). In many ways, the mechanism of retrotranslocation is similar to other post-translational
Figure 12–49 The role of N-linked glycosylation in ER protein folding.
The er-membrane-bound chaperone protein calnexin binds to incompletely folded proteins containing one terminal glucose on N-linked oligosaccharides, trapping the protein in the er. removal of the terminal glucose by a glucosidase releases the protein from calnexin. a glucosyl transferase is the crucial enzyme that determines whether the protein is folded properly or not: if the protein is still incompletely folded, the enzyme transfers a new glucose from UDp-glucose to the N-linked oligosaccharide, renewing the protein’s affinity for calnexin and retaining it in the er. The cycle repeats until the protein has folded completely. Calreticulin functions similarly, except that it is a soluble er resident protein. another er chaperone, erp57 (not shown), collaborates with calnexin and calreticulin in retaining an incompletely folded protein in the er. erp57 recognizes free sulfhydryl groups, which are a sign of incomplete disulfide bond formation.
modes of translocation. For example, like translocation into mitochondria or chloroplasts, chaperone proteins are necessary to keep the polypeptide chain in an unfolded state prior to and during translocation. Similarly, a source of energy is required to provide directionality to the transport and to pull the protein into the cytosol. Finally, a translocator is necessary.
Selecting proteins from the ER for degradation is a challenging process: mis-folded proteins or unassembled protein subunits should be degraded, but folding intermediates of newly made proteins should not. Help in making this distinction comes from the N-linked oligosaccharides, which serve as timers that measure how long a protein has spent in the ER. The slow trimming of a particular man-nose on the core oligosaccharide tree by an enzyme (a mannosidase) in the ER creates a new oligosaccharide structure that ER-lumenal lectins of the retrotranslocation apparatus recognize. Proteins that fold and exit from the ER faster than the mannosidase can remove its target mannose therefore escape degradation.
In addition to the lectins in the ER that recognize the oligosaccharides, chaperones and protein disulfide isomerases (enzymes mentioned earlier that catalyze the formation and breakage of S–S bonds) associate with the proteins that must be degraded. The chaperones prevent the unfolded proteins from aggregating, and the disulfide isomerases break disulfide bonds that may have formed incorrectly, so that a linear polypeptide chain can be translocated back into the cytosol.