Nh

Such isomerizations sometimes are the rate-limiting step in the folding of protein domains. Many peptidyl-prolyl iso-merases can catalyze the rotation of exposed peptidyl-prolyl bonds indiscriminately in numerous proteins, but some have very specific protein substrates.

Many important secretory and membrane proteins synthesized on the ER are built of two or more polypeptide sub-units. In all cases, the assembly of subunits constituting these

Oligosaccharyl transferase

Dolichol oligosaccharide

Cytosol

ER lumen

Oligosaccharyl transferase

Dolichol oligosaccharide

Calnexin

Calreticulin

Membrane-spanning Luminal a helix a helix

Membrane-spanning Luminal a helix a helix

Completed HA0 monomer

HA0 trimer

Calreticulin

Completed HA0 monomer

▲ FIGURE 16-21 Folding and assembly of hemagglutinin (HA0) trimer in the ER. Transient binding of the chaperone BiR (step |1a|) to the nascent chain and of two lectins, calnexin and calreticulin, to certain oligosaccharide chains (step 1b) promotes proper folding of adjacent segments. A total of seven /V-linked oligosaccharide chains are added to the luminal portion of the nascent chain during cotranslational translocation, and RDI catalyzes the formation of six disulfide bonds per monomer. Completed HA0 monomers are anchored in the membrane by a single membrane-spanning a helix with their N-terminus in the lumen (step|2|). Interaction of three HA0 chains with one another, initially via their transmembrane a helices, apparently triggers formation of a long stem containing one a helix from the luminal part of each HA0 polypeptide. Finally, interactions between the three globular heads occur, generating a stable HA0 trimer (step|3|). [See U. Tatu et al., 1995, EMBO J. 14:1340, and D. Hebert et al., 1997, J. Cell Biol. 139:613.]

multisubunit (multimeric) proteins occurs in the ER. An important class of multimeric secreted proteins is the immunoglobulins, which contain two heavy (H) and two light (L) chains, all linked by intrachain disulfide bonds. Hemag-glutinin (HA) is another multimeric protein that provides a good illustration of folding and subunit assembly (see Figure 16-21). This trimeric protein forms the spikes that protrude from the surface of an influenza virus particle. HA is formed within the ER of an infected host cell from three copies of a precursor protein termed HA0, which has a single membrane-spanning a helix. In the Golgi complex, each of the three HA0 proteins is cleaved to form two polypeptides, HA 1 and HA2; thus each HA molecule that eventually resides on the viral surface contains three copies of HA1 and three of HA2 (see Figure 3-7). The trimer is stabilized by interactions between the large exoplasmic domains of the constituent polypeptides, which extend into the ER lumen; after HA is transported to the cell surface, these domains extend into the extracellular space. Interactions between the smaller cytosolic and membrane-spanning portions of the HA sub-units also help stabilize the trimeric protein.

The time course of HA0 folding and assembly in vivo can be determined by pulse-labeling experiments. In a typical experiment, virus-infected cells are pulse-labeled with a radioactive amino acid; at various times during the subsequent chase period, membranes are solubilized by detergent and exposed to monoclonal antibodies specific for either HA0 monomer or trimer. Immediately after the pulse, the monomer-specific antibody is able to immunoprecipitate all the radioactive HA0 protein. During the chase period, increasing proportions of the total radioactive HA0 protein instead react with the trimer-specific monoclonal antibody. Such experiments have shown that each newly made HA0 polypeptide requires approximately 10 minutes to fold and be incorporated into a trimer in living cells.

Improperly Folded Proteins in the ER Induce Expression of Protein-Folding Catalysts

Wild-type proteins that are synthesized on the rough ER cannot exit this compartment until they achieve their completely folded conformation. Likewise, almost any mutation that prevents proper folding of a protein in the ER also blocks movement of the polypeptide from the ER lumen or membrane to the Golgi complex. The mechanisms for retaining unfolded or incompletely folded proteins within the ER probably increase the overall efficiency of folding by keeping intermediate forms in proximity to folding catalysts, which are most abundant in the ER. Improperly folded proteins retained within the ER generally are found permanently bound

Hac1

transcription factor

Spliced Hac1 mRNA

Translation

Endonuclease-cut Hac1 mRNA^^ ^

Unspliced Hac1 mRNA

Ire1

monomer

Cytosol ER

lumen

Endonuclease-cut Hac1 mRNA^^ ^

Unspliced Hac1 mRNA

Ire1

monomer

Cytosol ER

lumen

Unfolded proteins

Unfolded proteins with BiP bound

Unfolded proteins

Unfolded proteins with BiP bound to the ER chaperones BiP and calnexin. Thus these luminal folding catalysts perform two related functions: assisting in the folding of normal proteins by preventing their aggregation and binding to irreversibly misfolded proteins.

Both mammalian cells and yeasts respond to the presence of unfolded proteins in the rough ER by increasing transcription of several genes encoding ER chaperones and other folding catalysts. A key participant in this unfolded-protein response is Ire1, an ER membrane protein that exists as both a monomer and a dimer. The dimeric form, but not the monomeric form, promotes formation of Hac1, a transcription factor in yeast that activates expression of the genes induced in the unfolded-protein response. As depicted in Figure 16-22, binding of BiP to the luminal domain of monomeric Ire1 prevents formation of the Ire1 dimer. Thus the quantity of free BiP in the ER lumen probably determines the relative proportion of monomeric and dimeric Ire1. Accumulation of unfolded proteins within the ER lumen sequesters BiP molecules, making them unavailable for binding to Ire1. As a result the level of dimeric Ire1 increases, leading to an increase in the level of Hac1 and production of proteins that assist in protein folding.

Mammalian cells contain an additional regulatory pathway that operates in response to unfolded proteins in the ER. In this pathway, accumulation of unfolded proteins in the ER triggers proteolysis of ATF6, a transmembrane protein in the ER membrane. The cytosolic domain of ATF6 released by proteolysis then moves to the nucleus, where it stimulates transcription of the genes encoding ER chaperones. Activa-

FIGURE 16-22 The unfolded-protein response. Ire1, a transmembrane protein in the ER membrane, has a binding site for BiP on its luminal domain; the cytosolic domain contains a specific RNA endonuclease. Step 1: Accumulating unfolded proteins in the ER lumen bind BiP molecules, releasing them from monomeric Ire1. Dimerization of Ire1 then activates its endonuclease activity. Steps 2,13: The unspliced mRNA precursor encoding the transcription factor Hac1 is cleaved by dimeric Ire1, and the two exons are joined to form functional Hac1 mRNA. Current evidence indicates that this processing occurs in the cytosol, although pre-mRNA processing generally occurs in the nucleus. Step 4: Hac1 is translated into Hac1 protein, which then moves back into the nucleus and activates transcription of genes encoding several protein-folding catalysts. [See U. Ruegsegger et al., 2001, Cell 107:103; A. Bertolotti et al., 2000, Nature Cell Biol. 2:326; and C. Sidrauski and P Walter, 1997, Cell 90:1031.]

tion of a transcription factor by such regulated intramembrane proteolysis also occurs in the Notch signaling pathway (see Figure 14-29). We will encounter another example of this phenomenon, the cholesterol-responsive transcription factor SREBP, in Chapter 18.

The hereditary form of emphysema illustrates the detrimental effects that can result from misfolding of proteins in the ER. This disease is caused by a point mutation in a1-antitrypsin, which normally is secreted by hepatocytes and macrophages. The wild-type protein binds to and inhibits trypsin and also the blood protease elastase. In the absence of a1-antitrypsin, elastase degrades the fine tissue in the lung that participates in the absorption of oxygen, eventually producing the symptoms of emphysema. Although the mutant a1-antitrypsin is synthesized in the rough ER, it does not fold properly, forming an almost crystalline aggregate that is not exported from the ER. In hepatocytes, the secretion of other proteins also becomes impaired, as the rough ER is filled with aggregated a1-antitrypsin. I

Unassembled or Misfolded Proteins in the ER Are Often Transported to the Cytosol for Degradation

Misfolded secretory and membrane proteins, as well as the unassembled subunits of multimeric proteins, often are degraded within an hour or two after their synthesis in the rough ER. For many years, researchers thought that prote-olytic enzymes within the ER catalyzed degradation of mis-folded or unassembled polypeptides, but such proteases were never found. More recent studies have shown that misfolded membrane and secretory proteins are transported from the ER lumen, "backwards" through the translocon, into the cytosol, where they are degraded by the ubiquitin-mediated proteolytic pathway (see Figure 3-13).

Ubiquitinylating enzymes localized to the cytosolic face of the ER add ubiquitin to misfolded ER proteins as they exit the ER. This reaction, which is coupled to hydrolysis of ATP, may provide some of the energy required to drag these proteins back to the cytosol. The resulting polyubiquitinylated polypeptides are quickly degraded in proteasomes. Exactly how misfolded soluble and membrane proteins in the ER are recognized and targeted to the translocon for export to the cytosol is not known.

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