MCB 380 Lecture Outline - Spring 2007
Lecture 10 (Chapter 17: Vesicular traffic, secretion and endocytosis)
Section 17.3: Early stages of the secretory pathway
1. Figure 17-14, Lodish 5e. Vesicle-mediated protein trafficking between the ER and cis-Golgi. This is a good overview of the forward pathway employing COPII vesicles and the backward pathway employing COPI vesicles. Note the coat formation that drives budding and the coat protein-cargo protein interactions that specifically load the vesicles. This diagram also clearly shows the disassembly of the coats prior to docking and fusion with the target membrane. The same holds for the backward pathway. Even though the SNARE pairs are given the same color, each v-SNARE and t-SNARE in the pair is a different protein. Although not shown here, the COPII vesicles travel along microtubule pathways to the cis-Golgi.
2. Figure 17-15, Lodish 5e. 3D structure of ternary complex (3 components) comprising the COPII coat proteins (Sec23 and Sec 24) and Sar1(GTP). For initiation of budding, Sar1(GTP) is the first protein to bind to the membrane. To form a stable complex for structural studies, the nonhydrolyzable GTP analog GppNHp was used. Complexes of coat proteins Sec23 and Sec 24 were recruited to the bound Sar1(GppNHp). Sec24 is the binding site for transmembrane cargo proteins that have di-acidic signals on their cytosolic domains (Asp-X-Glu). Yeast mutants with alterations in their coat protein genes accumulate proteins in the rough ER and are thus class B sec mutants.
3. Figure 17-16, Lodish 5e. Role of KDEL receptor in retrieval of ER-resident luminal proteins from the Golgi. Yeast cells with mutated COPI proteins are also class B sec mutants, because they accumulate proteins in the rough ER. However, this phenotype is produced in a very different way from the mutants described above. Because COPI vesicles are needed to carry proteins that function in the ER back to the ER, disruption of this pathway leads to depletion of essential ER proteins needed for COPII vesicle formation. Secretory proteins then accumulate in the ER, the hallmark of class B mutants. Why do ER proteins escape from the rough ER if they do not have the sorting signals to be incorporated specifically into COPII vesicles? Many of these proteins are very abundant and simply are incorporated into transport vesicles because there are some many of them in the ER. How do these ER resident protein get into COPI vesicles in the cis-Golgi then? The soluble ones have a sorting sequence, the KDEL sequence, at their C-termini and this sorting signal binds to the KDEL receptor in the COPI vesicles. How do these receptors get into COPI vesicles? They have KKXX sorting signals at their cytosolic C-termini which bind to sites on the COPI coat proteins.
4. Figure 17-17, Lodish 5e. Electron micrograph of the Golgi complex in an exocrine pancreatic cell reveals both anterograde (forward) and retrograde (backward) transport vesicles. In this particularly clear micrograph, a large secretory vesicle is shown as it forms at the trans-Golgi network. Also clear are transitional elements of the ER from which smooth protrusions seem to be budding. These buds likely form the small COPII vesicles that move secretory proteins from the rough ER to the Golgi complex. The small vesicles located near the Golgi stacks are mostly COPI vesicles involved in the backward or retrograde flow of enzymes and proteins that function in younger Golgi cisternae.
5. Figure 6-20, Lodish 5e. Major events in biosynthesis of fibrillar collagens. The currently favored model for movement of material forward through Golgi stacks is the cisternal progression model. Early evidence, which was largely ignored, came from micrographs of algal scales in the Golgi. These are relatively large structures in the land of the Golgi, being 20 times larger than typical transport vesicles that bud from the Golgi cisternae. This was thought to be a special case. However, more recently it was found that large assemblies of procollagen form in the lumen of the cis-Golgi, as shown in this figure. These assemblies are too large to form in transport vesicles and in fact they are not observed by microscopy in these vesicles. This finding suggested that movement by another method, maturation of Golgi cisternae (cisternal progression), may be more general than previously thought. It is of course difficult to rule out the possibility that some smaller protein complexes move forward in vesicles.
Section 17.4: Later stages of the secretory pathway
6. Figure 17-18, Lodish 5e. Involvement of the three major types of coat proteins in vesicular traffic in the secretory and endocytic pathways. The late pathways are dominated by clathrin-coated vesicles, shown in red here. These vesicles may bud from the trans-Golgi network or inward from the plasma membrane during endocytosis. The destination is the same, release of clathrin coat followed by fusion with the late endosomes (a heterogeneous collections of vesicles with varying degrees of acidic lumens).
7. Figure 17-19, Lodish 5e. Structure of clathrin coats. Coated vesicles are about 50 nm in diameter. a) triskelion structure- each leg contains a heavy and a light polypeptide chain. The larger chain ends in a globular domain (binds a subunit of AP complex) involved in assembly of the coat. b) Assembly intermediate. These structures can form into a cage in vitro even without vesicles. When the clathrin triskelions polymerize, they form a polygonal lattice with an intrinsic curvature. c) Reconstructed images from about 1000 electron micrographs of clathrin particles assembled in vitro. These are the average structures created by computerized image analysis. AP2 complexes are shown packed into the interior of the left image. They have been subtracted by the computer in the image on the right.
Media Connection: Birth of a Clathrin Coat
8. Figure 17-20, Lodish 5e. Model for dynamin-mediated pinching off of clathrin/AP-coated vesicles. Assembly particles (AP complex) are located in the space between the cytosolic face of the membrane and the clathrin layer. They bind the globular domain of clathrin heavy chain and stimulate the polymerization of triskelions into cages. There are three types, AP1, AP2, and AP3. The signal sequence on the plasma membrane proteins incorporated into coated pits, ie the Tyr-X-X-Phe sequence and Leu-Leu sequence, bind to a subunit of the assembly particle AP2, the plasma membrane-associated adaptor. Interestingly, the M6P receptor, which is incorporated into vesicles that bud from the trans-Golgi network as well as the plasma membrane, recognizes both AP1 and AP2. Dynamin subunits polymerize around the neck of a pit, and GTP hydrolysis is involved in the contraction of this 'collar', causing pinching off or budding of the vesicle. How are clathrin coated vesicles uncoated? This appears to be a regulated process, since the coated vesicles are stable at the pH and ionic concentration of cytosol. The molecular chaperone Hsc70 is thought to be involved in the depolymerization of clathrin coats into triskelions, which can then be recycled by cells.
9. Figure 17-21, Lodish 5e. Evidence that GTP hydrolysis by dynamin is required for pinching off of clathrin-coated vesicles. If a nonhydrolyzable analog of GTP, GTP-gamma-S (sulfur substituted for a phosphorus atom, eg sulfate group for phosphate group), is added to a preparation of nerve terminals, and then treated with gold-labeled anti-dynamin antibody, the following image is seen in the electron microscope. Clathrin coated pits with long necks surrounded by polymerized dynamin are observed. The GTP analog binds to dynamin, allowing it to polymerize and form the neck of the vesicle. In the absence of GTP hydrolysis, this structure is stable.
10. Figure 17-22, Lodish 5e. Formation of mannose 6-phosphate (M6P) residues that target soluble enzymes to lysosomes. Phosphorylation of mannose residues on lysosomal proteins. Another function of some N-linked oligosaccharides is to target lysosomal enzymes to lysosomes and to prevent their secretion. The mannose-6-phosphate sorting signal is added to lysosomal proteins in the cis-Golgi in a two-step process. The first step involves N-acetylglucosamine phosphotransferase, an enzyme that specifically binds to signal sequences (a conformational patch) on lysosomal proteins. The enzyme also binds UDP-GlcNAc and transfers the N-acetylglucosamine phosphate group to a mannose residue on the end of an oligosaccharide side chain on the lysosomal protein. The second step is a nonspecific N-acetyl glucosamine phosphoglycosidase that works on a variety of proteins carrying oligos. It cleaves the GlcNAc(red box), leaving the M6P tag.
11. Figure 17-23, Lodish 5e. Trafficking of soluble lysosomal enzymes from the trans-Golgi network and cell surface to lysosomes. How are newly made lysosomal hydrolases targeted to lysosomes? They use the unique marker consisting of mannose 6-phosphate groups which are added to N-linked oligos in the lumen of the cis Golgi network as just described. This tag is recognized by M6P receptor proteins which are transmembrane proteins located in the trans Golgi network. These receptors direct the lysosomal enzymes into vesicles coated with clathrin. Clathrin baskets surround these vesicles and may help to drive the vesicle budding process. In order to fuse with the late endosomal compartment, the clathrin coat must be lost, which occurs via a regulated depolymerization reaction. How does the M6P receptor work? It binds to its target oligosaccharide at pH 7 in the trans Golgi network and releases it at pH 6, which is the pH of the interior of late endosomes. Note the function of the endosomal ATPase in acidification. Phosphate is also removed from the tag in endosomes to add to the irreversibility of the pathway. Note recycling of the receptor. The lysosomal proteins that appear in endosomes are mostly inactive precursors or proenzymes. In the acidic late endosome or in the lysosomes, these proteins are proteolytically cleaved and activated. This delay in activation assures that any lysosomal proteins that escape will not destroy the cell.
Some lysosomal hydrolases escape the targeted packaging mechanism and follow the default pathway to the cell surface. Fortunately, some of the M6P receptors also escape and can retrieve some the hydrolases by receptor-mediated endocytosis, which also involves the formation of clathrin cages, and return them to lysosomes through endosomes. This is sometimes called a scavenger pathway. Its presence has allowed study of mutant fibroblasts in culture from humans with genetic defects in individual lysosomal hydrolase genes, e.g. Hurler's disease. Normal hydrolase can be added to mutant cells and can be taken up into lysosomes by the surface M6P receptors to replace the defective enzyme.
Defects in GlcNAc phosphotransferase are responsible for a rare but deadly human genetic disease known as I-cell disease, inclusion cell disease. Almost all of the hydrolytic enzymes are missing from the lysosomes from these individuals and instead they are secreted out of the cell. The hydrolases lack the M6P tag and are secreted out of the cell instead of following the targeted pathway to lysosomes. Interestingly, hepatocytes and some other cell types in these individual have a normal complement of hydrolases, indicating the existence of an M6P-independent pathway. Also, the membrane proteins of lysosomes are sorted properly, indicating an M6P-independent pathway for them as well.
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