Dl

Clathrin-coated vesicle

▲ FIGURE 17-22 Formation of mannose 6-phosphate (M6P) residues that target soluble enzymes to lysosomes. The M6P

residues that direct proteins to lysosomes are generated in the c/s-Golgi by two Golgi-resident enzymes. Step 1: An N-acetylglucosamine (GlcNAc) phosphotransferase transfers a phosphorylated GlcNAc group to carbon atom 6 of one or more mannose residues. Because only lysosomal enzymes contain sequences (red) that are recognized and bound by this enzyme, phosphorylated GlcNAc groups are added specifically to lysosomal enzymes. Step 2|: After release of a modified protein from the phosphotransferase, a phosphodiesterase removes the GlcNAc group, leaving a phosphorylated mannose residue on the lysosomal enzyme. [See A. B. Cantor et al., 1992, J. Biol. Chem. 267:23349, and S. Kornfeld, 1987, FASEB J. 1:462.]

lysosomal enzymes from the trans-Golgi network to the late endosome is a carbohydrate residue, mannose 6-phosphate (M6P), which is formed in the cis-Golgi. The addition and initial processing of one or more preformed A-linked oligosaccharide precursors in the rough ER is the same for lysosomal enzymes as for membrane and secreted proteins, yielding core Man8(GlcNAc)2 chains (see Figure 16-18). In the cis-Golgi, the A-linked oligosaccharides present on most lysosomal enzymes undergo a two-step reaction sequence that generates M6P residues (Figure 17-22). The addition of M6P residues to the oligosaccharide chains of soluble lyso-somal enzymes prevents these proteins from undergoing the further processing reactions characteristic of secreted and membrane proteins (see Figure 17-3).

As shown in Figure 17-23, the segregation of M6P-bearing lysosomal enzymes from secreted and membrane proteins occurs in the trans-Golgi network. Here transmembrane mannose 6-phosphate receptors bind the M6P residues on lysosome-destined proteins very tightly and specifically. Clathrin/AP1 vesicles containing the M6P receptor and bound lysosomal enzymes then bud from the trans-Golgi network, lose their coats, and subsequently fuse with the late endosome by mechanisms described previously. Because M6P receptors can bind M6P at the slightly acidic pH («6.5) of the trans-Golgi network but not at a pH less than 6, the bound lysosomal enzymes are released within late endo-somes, which have an internal pH of 5.0-5.5. Furthermore, a phosphatase within late endosomes usually removes the phosphate from M6P residues on lysosomal enzymes, preventing any rebinding to the M6P receptor that might occur in spite of the low pH in endosomes. Vesicles budding from late endosomes recycle the M6P receptor back to the trans-

M FIGURE 17-23 Trafficking of soluble lysosomal enzymes from the trans-Golgi network and cell surface to lysosomes.

Newly synthesized lysosomal enzymes, produced in the ER, acquire mannose 6-phosphate (M6P) residues in the cis-Golgi (see Figure 17-22). For simplicity, only one phosphorylated oligosaccharide chain is depicted, although lysosomal enzymes typically have many such chains. In the trans-Golgi network, proteins that bear the M6P sorting signal interact with M6P receptors in the membrane and thereby are directed into clathrin/AP1 vesicles (step 1). The coat surrounding released vesicles is rapidly depolymerized (step 2|), and the uncoated transport vesicles fuse with late endosomes (step 3). After the phosphorylated enzymes dissociate from the M6P receptors and are dephosphorylated, late endosomes subsequently fuse with a lysosome (step 4). Note that coat proteins and M6P receptors are recycled (steps 2a| and 4a|), and some receptors are delivered to the cell surface (step 5). Phosphorylated lysosomal enzymes occasionally are sorted from the trans-Golgi to the cell surface and secreted. These secreted enzymes can be retrieved by receptor-mediated endocytosis (steps 6- 8), a process that closely parallels trafficking of lysosomal enzymes from the trans-Golgi network to lysosomes. [See G. Griffiths et al., 1988, Cell 52:329; S. Kornfeld, 1992, Ann. Rev. Biochem. 61:307; and G. Griffiths and J. Gruenberg, 1991, Trends Cell Biol. 1:5.]

Golgi network or, on occasion, to the cell surface. Eventually, mature late endosomes fuse with lysosomes, delivering the lysosomal enzymes to their final destination.

The sorting of soluble lysosomal enzymes in the trans-Golgi network (see Figure 17-23, steps 1 - 4) shares many of the features of trafficking between the ER and cis-Golgi compartments mediated by COPII and COPI vesicles. First, man-nose 6-phosphate acts as a sorting signal by interacting with the luminal domain of a receptor protein in the donor membrane. Second, the membrane-embedded receptors with their bound ligands are incorporated into the appropriate vesi-cles—in this case, AP1-containing clathrin vesicles—by interacting with the vesicle coat. Third, these transport vesicles fuse only with one specific organelle, here the late endosome, as the result of interactions between specific v-SNAREs and t-SNAREs. And finally, intracellular transport receptors are recycled after dissociating from their bound ligand.

Study of Lysosomal Storage Diseases Revealed Key Components of the Lysosomal Sorting Pathway

0A group of genetic disorders, termed lysosomal storage diseases, are caused by the absence of one or more lysosomal enzymes. As a result, undigested glycolipids and extracellular components that would normally be degraded by lysosomal enzymes accumulate in lysosomes as large inclusions. I-cell disease is a particularly severe type of lysosomal storage disease in which multiple enzymes are missing from the lysosomes. Cells from affected individuals lack the A-acetylglucosamine phosphotrans-ferase that is required for formation of M6P residues on lysosomal enzymes in the cis-Golgi (see Figure 17-22). Biochemical comparison of lysosomal enzymes from normal individuals with those from patients with I-cell disease led to the initial discovery of mannose 6-phosphate as the lyso-somal sorting signal. Lacking the M6P sorting signal, the lysosomal enzymes in I-cell patients are secreted rather than being sorted to and sequestered in lysosomes.

When fibroblasts from patients with I-cell disease are grown in a medium containing lysosomal enzymes bearing M6P residues, the diseased cells acquire a nearly normal in-tracellular content of lysosomal enzymes. This finding indicates that the plasma membrane of these cells contain M6P receptors, which can internalize extracellular phosphorylated lysosomal enzymes by receptor-mediated endocytosis. This process, used by many cell-surface receptors to bring bound proteins or particles into the cell, is discussed in detail in the next section. It is now known that even in normal cells, some M6P receptors are transported to the plasma membrane and some phosphorylated lysosomal enzymes are secreted (see Figure 17-23). The secreted enzymes can be retrieved by receptor-mediated endocytosis and directed to lysosomes. This pathway thus scavenges any lysosomal enzymes that escape the usual M6P sorting pathway.

Hepatocytes from patients with I-cell disease contain a normal complement of lysosomal enzymes and no inclusions, even though these cells are defective in mannose phosphory-lation. This finding implies that hepatocytes (the most abundant type of liver cell) employ a M6P-independent pathway for sorting lysosomal enzymes. The nature of this pathway, which also may operate in other cells types, is unknown. I

Protein Aggregation in the Trans-Golgi May Function in Sorting Proteins to Regulated Secretory Vesicles

As noted in the chapter introduction, all eukaryotic cells continuously secrete certain proteins, a process commonly called constitutive secretion. Specialized secretory cells also store other proteins in vesicles and secrete them only when triggered by a specific stimulus. One example of such regulated secretion occurs in pancreatic p cells, which store newly made insulin in special secretory vesicles and secrete insulin in response to an elevation in blood glucose (see Figure 15-7). These and other secretory cells simultaneously utilize two different types of vesicles to move proteins from the trans-Golgi network to the cell surface: regulated transport vesicles, often simply called secretory vesicles, and unregulated transport vesicles, also called constitutive secretory vesicles.

A common mechanism appears to sort regulated proteins as diverse as ACTH (adrenocorticotropic hormone), insulin, and trypsinogen into regulated secretory vesicles. Evidence for a common mechanism comes from experiments in which recombinant DNA techniques are used to induce the synthesis of insulin and trypsinogen in pituitary tumor cells already synthesizing ACTH. In these cells all three proteins segregate into the same regulated secretory vesicles and are secreted together when a hormone binds to a receptor on the pituitary cells and causes a rise in cytosolic Ca2 + . Although these three proteins share no identical amino acid sequences that might serve as a sorting sequence, they obviously have some common feature that signals their incorporation into regulated secretory vesicles.

Morphologic evidence suggests that sorting into the regulated pathway is controlled by selective protein aggregation. For instance, immature vesicles in this pathway—those that have just budded from the trans-Golgi network—contain diffuse aggregates of secreted protein that are visible in the electron microscope. These aggregates also are found in vesicles that are in the process of budding, indicating that proteins destined for regulated secretory vesicles selectively aggregate together before their incorporation into the vesicles.

Other studies have shown that regulated secretory vesicles from mammalian secretory cells contain three proteins, chro-mogranin A, chromogranin B, and secretogranin II, that together form aggregates when incubated at the ionic conditions (pH «6.5 and 1 mM Ca2+) thought to occur in the trans-Golgi network; such aggregates do not form at the neutral pH of the ER. The selective aggregation of regulated secreted proteins together with chromogranin A, chromogranin B, or secre-togranin II could be the basis for sorting of these proteins into regulated secretory vesicles. Secreted proteins that do not associate with these proteins, and thus do not form aggregates, would be sorted into unregulated transport vesicles by default.

Some Proteins Undergo Proteolytic Processing After Leaving the Trans-Golgi

For some secretory proteins (e.g., growth hormone) and certain viral membrane proteins (e.g., the VSV glycoprotein), removal of the N-terminal ER signal sequence from the nascent chain is the only known proteolytic cleavage required to convert the polypeptide to the mature, active species (see Figure 16-6). However, some membrane and many soluble secretory proteins initially are synthesized as relatively long-lived, inactive precursors, termed proproteins, that require further proteolytic processing to generate the mature, active proteins. Examples of proteins that undergo such processing are soluble lysosomal enzymes, many membrane proteins such as influenza hemagglutinin (HA), and secreted proteins such as serum albumin, insulin, glucagon, and the yeast a mating factor. In general, the proteolytic conversion of a proprotein to the corresponding mature protein occurs after the proprotein has been sorted in the trans-Golgi network to appropriate vesicles.

In the case of soluble lysosomal enzymes, the proproteins are called proenzymes, which are sorted by the M6P receptor as catalytically inactive enzymes. In the late endosome or lysosome a proenzyme undergoes a proteolytic cleavage that generates a smaller but enzymatically active polypeptide. Delaying the activation of lysosomal proenzymes until they reach the lysosome prevents them from digesting macromol-ecules in earlier compartments of the secretory pathway.

Normally, mature vesicles carrying secreted proteins to the cell surface are formed by fusion of several immature

► EXPERIMENTAL FIGURE 17-24 Proteolytic cleavage of proinsulin occurs in secretory vesicles after they have budded from the trans-Golgi network. Serial sections of the Golgi region of an insulin-secreting cell were stained with (a) a monoclonal antibody that recognizes proinsulin but not insulin or (b) a different antibody that recognizes insulin but not proinsulin. The antibodies, which were bound to electron-opaque gold particles, appear as dark dots in these electron micrographs (see Figure 5-51). Immature secretory vesicles (closed arrowheads) and vesicles budding from the trans-Golgi (arrows) stain with the proinsulin antibody but not with insulin antibody. These vesicles contain diffuse protein aggregates that include proinsulin and other regulated secreted proteins. Mature vesicles (open arrowheads) stain with insulin antibody but not with proinsulin antibody and have a dense core of almost crystalline insulin. Since budding and immature secretory vesicles contain proinsulin (not insulin), the proteolytic conversion of proinsulin to insulin must take place in these vesicles after they bud from the trans-Golgi network. The inset in (a) shows a proinsulin-rich secretory vesicle surrounded by a protein coat (dashed line). [From L. Orci et al., 1987, Cell 49:865; courtesy of L. Orci.]

ones containing proprotein. Proteolytic cleavage of proproteins, such as proinsulin, occurs in vesicles after they move away from the trans-Golgi network (Figure 17-24). The proproteins of most constitutively secreted proteins (e.g., albumin) are cleaved only once at a site C-terminal to a dibasic

(a) Proinsulin antibody

(a) Constitutive secreted proteins

Proalbumin

NH3+

Arg Arg

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