Protein Modifications Folding and Quality Control in the ER

Membrane and soluble secretory proteins synthesized on the rough ER undergo four principal modifications before they reach their final destinations: (1) addition and processing of carbohydrates (glycosylation) in the ER and Golgi, (2) formation of disulfide bonds in the ER, (3) proper folding of polypeptide chains and assembly of multisubunit proteins in the ER, and (4) specific proteolytic cleavages in the ER, Golgi, and secretory vesicles.

One or more carbohydrate chains are added to the vast majority of proteins that are synthesized on the rough ER; indeed, glycosylation is the principal chemical modification to most of these proteins. Carbohydrate chains in glycopro-teins may be attached to the hydroxyl group in serine and threonine residues or to the amide nitrogen of asparagine. These are referred to as O-linked and N-linked oligosaccha-rides, respectively. O-linked oligosaccharides, such as those found in collagen and glycophorin, often contain only one to four sugar residues. The more common N-linked oligosaccharides are larger and more complex, containing several branches in mammalian cells. In this section we focus on N-linked oligosaccharides, whose initial synthesis occurs in the ER. After the initial glycosylation of a protein in the ER, the oligosaccharide chain is modified in the ER and commonly in the Golgi, as well.

Disulfide bond formation, protein folding, and assembly of multimeric proteins, which take place exclusively in the rough ER, also are discussed in this section. Only properly folded and assembled proteins are transported from the rough ER to the Golgi complex and ultimately to the cell surface or other final destination. Unfolded, misfolded, or partly folded and assembled proteins are selectively retained in the rough ER. We consider several features of such "quality control" in the latter part of this section.

As discussed previously, N-terminal ER signal sequences are cleaved from secretory proteins and type I membrane proteins in the ER. Some proteins also undergo other specific proteolytic cleavages in the Golgi complex or forming secretory vesicles. We cover these cleavages, as well as carbohydrate modifications that occur primarily or exclusively in the Golgi complex, in the next chapter.

A Preformed /V-Linked Oligosaccharide Is Added to Many Proteins in the Rough ER

Biosynthesis of all N-linked oligosaccharides begins in the rough ER with addition of a preformed oligosaccharide precursor containing 14 residues (Figure 16-16). The structure of this precursor is the same in plants, animals, and single-celled eukaryotes—a branched oligosaccharide, containing three glucose (Glc), nine mannose (Man), and two N-acetylglucosamine (GlcNAc) molecules, which can be written as Glc3Man9(GlcNAc)2. This branched carbohydrate structure is modified in the ER and Golgi compartments, but 5 of the 14 residues are conserved in the structures of all N-linked oligosaccharides on secretory and membrane proteins.

The precursor oligosaccharide is linked by a pyrophos-phoryl residue to dolichol, a long-chain polyisoprenoid lipid that is firmly embedded in the ER membrane and acts as a carrier for the oligosaccharide. The dolichol pyrophospho-ryl oligosaccharide is formed on the ER membrane in a complex set of reactions catalyzed by enzymes attached to the cytosolic or luminal faces of the rough ER membrane

GlcNAc = N-Acetylglucosamine Man = Mannose Glc = Glucose ^^^ = Conserved = Variable



â–˛ FIGURE 16-16 Common 14-residue precursor of W-linked oligosaccharides that is added to nascent proteins in the rough ER. Subsequent removal and in some cases addition of specific sugar residues occur in the ER and Golgi complex. The core region, composed of five residues highlighted in purple, is retained in all N-linked oligosaccharides. The precursor can be linked only to asparagine (Asn) residues that are separated by one amino acid (X) from a serine (Ser) or threonine (Thr) on the carboxyl side.

(Figure 16-17). The final dolichol pyrophosphoryl oligosac-charide is oriented so that the oligosaccharide portion faces the ER lumen.

The entire 14-residue precursor is transferred from the dolichol carrier to an asparagine residue on a nascent polypeptide as it emerges into the ER lumen (Figure 16-18, step 1). Only asparagine residues in the tripeptide sequences Asn-X-Ser and Asn-X-Thr (where X is any amino acid except proline) are substrates for oligosaccharyl transferase, the enzyme that catalyzes this reaction. Two of the three sub-units of this enzyme are ER membrane proteins whose cytosol-facing domains bind to the ribosome, localizing a third subunit of the transferase, the catalytic subunit, near the growing polypeptide chain in the ER lumen. Not all Asn-X-Ser/Thr sequences become glycosylated; for instance, rapid folding of a segment of a protein containing an Asn-X-Ser/Thr sequence may prevent transfer of the oligosaccharide precursor to it.

Immediately after the entire precursor, Glc3Man9(Glc-NAc) 2, is transferred to a nascent polypeptide, three different enzymes remove all three glucose residues and one particular mannose residue (Figure 16-18, steps 2-4). The three glucose residues, which are the last residues added during synthesis of the precursor on the dolichol carrier, appear to act as a sig nal that the oligosaccharide is complete and ready to be transferred to a protein.

Oligosaccharide Side Chains May Promote Folding and Stability of Glycoproteins

The oligosaccharides attached to glycoproteins serve various functions. For example, some proteins require A-linked oligosaccharides in order to fold properly in the ER. This function has been demonstrated in studies with the antibiotic tunicamycin, which blocks the first step in formation of the dolichol-linked precursor of A-linked oligosaccharides (see Figure 16-17). In the presence of tunicamycin, for instance, the hemagglutinin precursor polypeptide (HA0) is synthesized, but it cannot fold properly and form a normal trimer; in this case, the protein remains, misfolded, in the rough ER. Moreover, mutation in the HA sequence of just one asparagine that normally is glycosylated to a glutamine residue, thereby preventing addition of an A-linked oligosac-charide to that site, causes the protein to accumulate in the ER in an unfolded state.

In addition to promoting proper folding, A-linked oligosaccharides also confer stability on many secreted gly-

Blocked by tunicamycin


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