Key Concepts Of Section 163

Protein Modifications, Folding, and Quality Control in the ER

■ All A-linked oligosaccharides, which are bound to as-paragine residues, contain a core of three mannose and two A -acetylglucosamine residues and usually have several branches (see Figure 16-16).

■ O-linked oligosaccharides, which are bound to serine or threonine residues, are generally short, often containing only one to four sugar residues.

■ Formation of all A-linked oligosaccharides begins with assembly of a ubiquitous 14-residue high-mannose precursor on dolichol, a lipid in the membrane of the rough ER (see Figure 16-17). After this preformed oligosaccha-ride is transferred to specific asparagine residues of nascent polypeptide chains in the ER lumen, three glucose residues and one mannose residue are removed (see Figure 16-18).

■ Oligosaccharide side chains may assist in the proper folding of glycoproteins, help protect the mature proteins from proteolysis, participate in cell-cell adhesion, and function as antigens.

■ Disulfide bonds are added to many secretory proteins and the exoplasmic domain of membrane proteins in the ER. Protein disulfide isomerase (PDI), present in the ER lumen, catalyzes both the formation and the rearrangement of disulfide bonds (see Figure 16-19).

■ The chaperone BiP, the lectins calnexin and calreticulin, and peptidyl-prolyl isomerases work together to assure proper folding of newly made secretory and membrane proteins in the ER. The subunits of multimeric proteins also assemble in the ER.

■ Only properly folded proteins and assembled subunits are transported from the rough ER to the Golgi complex in vesicles.

■ The accumulation of abnormally folded proteins and unassembled subunits in the ER can induce increased expression of ER protein-folding catalysts via the unfolded-protein response (see Figure 16-22).

■ Unassembled or misfolded proteins in the ER often are transported back through the translocon to the cytosol, where they are degraded in the ubiquitin/proteasome pathway.

The cell wall surrounding gram-negative bacteria comprises an inner membrane, which is the main permeability barrier for the cytoplasm, a periplasmic space containing various proteins and a layer of peptidoglycan, and an outer membrane, which is permeable to small molecules but not proteins. The peptidoglycan layer gives the cell wall its strength, while the periplasmic proteins function in sensing and importing extracellular molecules and in assembling and maintaining the structural integrity of the cell wall. These proteins (like all bacterial proteins) are synthesized on cytosolic ribo-somes and then are translocated in the unfolded state across the inner membrane (also called the cytoplasmic membrane). Most proteins translocated across the inner membrane remain associated with the bacterial cell, either as a membrane protein inserted into the outer or inner membrane or trapped within the periplasmic space. Some bacterial species also possess specialized translocation systems that enable proteins to move through both membranes of the cell wall into the extracellular space. In this section, we describe both types of protein secretion in gram-negative bacteria.

Cytosolic SecA ATPase Pushes Bacterial Polypeptides Through Translocons into the Periplasmic Space

The mechanism for translocating bacterial proteins across the inner membrane shares several key features with the translocation of proteins into the ER of eukaryotic cells. First, translocated proteins usually contain an N-terminal hydrophobic signal sequence, which is cleaved by a signal peptidase. Second, bacterial proteins pass through the inner membrane in a channel, or translocon, composed of proteins that are structurally similar to the eukaryotic Sec61 complex. Third, bacterial cells express two proteins, Ffh and its receptor (FtsY), that are homologs of the SRP and SRP receptor, respectively. In bacteria, however, these latter proteins appear to function mainly in the insertion of hydrophobic membrane proteins into the inner membrane. Indeed, all bacterial proteins that are translocated across the inner membrane do so only after their synthesis in the cytosol is completed but before they are folded into their final conformation.

The post-translational translocation of bacterial proteins across the inner membrane cannot involve a ratchet mechanism similar to that mediated by BiP in the ER lumen (see Figure 16-9) because the ATP required for such a mechanism would be lost by diffusion through the outer membrane. Rather, the driving force for translocation of bacterial proteins is generated by SecA, which binds to the cytosolic side of the translocon and hydrolyzes cytosolic ATP. In the model depicted in Figure 16-23, SecA binds to the unfolded translocating polypeptide, and then a confor-mational change in SecA, driven by the energy released

Cytosol Inner membrane

Periplasmic space

Cytosol Inner membrane

Periplasmic space

Translocon (SecY, SecE, SecG)

▲ FIGURE 16-23 Post-translational translocation across inner membrane in gram-negative bacteria. The bacterial inner membrane contains a translocon channel composed of three subunits that are homologous to the components of the eukaryotic Sec61 complex. Translocation of polypeptides from the cytosol to the periplasmic space is powered by SecA, a cytosolic ATPase that binds to the translocon and to the translocating polypeptide. In the model shown here, binding and hydrolysis of ATP cause conformational changes in SecA that push the bound polypeptide segment through the channel (steps hi, 12). Repetition of this cycle results in movement of the polypeptide through the channel in one direction. Current evidence indicates that the N-terminal signal sequence moves from the channel into the bilayer but at some point is cleaved by a signal peptidase, so that the mature polypeptide enters the periplasmic space. [See A. Economou and W. Wickner, 1994, Cell 78:835, and J. Eichler and W. Wickner, 1998, J. Bacteriol. 180:5776.]

from ATP hydrolysis, acts to push the bound polypeptide segment through the translocon pore toward the periplas-mic side of the membrane. Repetition of this cycle eventually pushes the entire polypeptide chain through the translocon into the periplasmic space, where disulfide bonds are formed and the polypeptide folds into its proper conformation.

Several Mechanisms Translocate Bacterial Proteins into the Extracellular Space

Quite different mechanisms from the one shown in Figure 16-23 are used to translocate bacterial proteins from the cy-tosol across both the inner and outer bacterial membranes to the extracellular space. These secretion mechanisms are particularly important for pathogenic bacteria, which commonly use secreted extracellular proteins to colonize specific tissues within the host and to evade host defense mechanisms. Well-known examples of extracellular proteins that promote the growth and dissemination of pathogenic bacteria include protein toxins (e.g., cholera toxin and tetanus toxin) and pili, which are proteinaceous fibers that project from the outer membrane and assist enteric bacteria in adhering to the epithelium of the gut.

The numerous specialized bacterial secretion systems that have been identified can be classified into four general types based on their mechanism of operation. Both the type I and the type II secretion systems involve two steps. First, substrate proteins are translocated across the inner membrane into the periplasmic space, where they fold and often acquire disulfide bonds. Second, the folded proteins are translocated from the periplasmic space across the outer membrane by complexes of periplasmic proteins that span the inner and outer membranes. The energy for this translocation comes from hydrolysis of ATP in the cytosol, but the mechanisms that couple ATP hydrolysis and translocation across the outer membrane are not well understood.

Translocation by the type III and type IV secretion systems, on the other hand, entails a single step. These systems consist of large protein complexes that span both membranes, allowing proteins to be translocated directly from the cytosol to the extracellular environment. The type III system is adapted not only for secreting proteins but also for injecting them into target cells, a very useful property for pathogenic bacteria.

Pathogenic Bacteria Can Inject Proteins into Animal Cells via Type III Secretion Apparatus

Yersinia pestis is the bacterial species responsible for the bubonic plague, one of the deadliest diseases in human history. One reason Yersinia is such a virulent pathogen lies in its ability to disable host macrophage cells that might otherwise engulf and destroy the invading bacterial cells. The incapacitating effect of Yersinia is mediated primarily by a small set of proteins that the bacterial cells inject into macrophage cells.

Various pathogenic bacteria inject proteins into host cells via a complicated syringe-like machine composed of more than 20 different proteins. This type III secretion apparatus, shown in Figure 16-24, has ringlike components embedded in both the inner and outer membranes of the bacterial cell wall and a hollow needlelike structure (pilus) that projects

FIGURE 16-24 Type III secretion apparatus for injecting bacterial proteins into eukaryotic cells. (a) Schematic diagram of the type III secretion apparatus, which is similar in size and morphology to the bacterial flagellum. Bacterial proteins with targeting sequences (red) that allow interaction with specialized chaperones (orange) enter the cytosol-facing portion of the type III secretion apparatus, travel down the hollow core of the pilus in an ATP-dependent process, and ultimately are delivered to the cytoplasm of the target eukaryotic cell. (b) Electron micrograph of isolated type III secretion apparatuses. Long needlelike pili can be seen extending from the widened basal portions, which are embedded in the outer and inner membranes. See the text for discussion. [Part (a) adapted from D. G. Thanassi and S. J. Hultgren, 2000, Curr. Opin. Cell Biol. 12:420. Part (b) from T Kubori et al., 1998, Science 280:602.]

out from the outer membrane. Proteins at the outer (distal) end of the pilus can penetrate the plasma membrane of certain mammalian cells, thus creating a conduit that spans three membranes, providing a connection between the bacterial cytoplasm and that of the target host cell.

A key insight into how the type III secretion apparatus may operate came from the observation that many of the components of the secretion apparatus are homologous to proteins in the base of the bacterial flagellum. The flagellar base is located in the inner membrane and functions as a motor to drive rotation of the attached flagellum, which extends outward from the cell surface. The flagellum, which is a hollow helical tube made up entirely of a repeating polymer of the protein flagellin, grows by addition of new fla-gellin subunits at the distal tip. Several proteins in the flagellar base are thought to use the energy from ATP hydrolysis to push new flagellin subunits through the central channel of the flagellum toward the distal end. Quite possibly, the type III secretion apparatus uses a similar ATP-driven mechanism to push proteins through the central channel of the pilus into target cells.

Recent experiments have identified the signal sequences that target bacterial proteins for transport through the type III secretion apparatus. For instance, recombinant DNA methods were used to express in Yersinia cells chimeric proteins containing adenylate cyclase attached to different portions of YopE, which normally is secreted via the type III apparatus. An amphipathic sequence at the N-terminus of YopE was capable of directing adenylate cyclase, which normally resides in the cytosol, to the type III secretion apparatus for injection into mammalian cells. Other experiments showed that YopE proteins in which this amphipathic tar geting sequence is mutated still are secreted normally. This surprising finding eventually lead to discovery of a second, independent targeting signal that allows YopE to bind to a small chaperone molecule. The chaperone-YopE complex can be successfully secreted even in the absence of an N-terminal amphipathic sequence. Each of the proteins secreted by the type III apparatus is thought to interact with one of a set of small chaperone proteins (see Figure 16-24). These chaperones may act to keep secreted proteins in a partially unfolded state as they pass through the central channel of the type III apparatus. Once the transported proteins have been released into the target cell, folding can be completed by cy-tosolic target-cell chaperones. I

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