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| 3442258885 | What mechanisms control exit of proteins from the ER? | Quality control and Active cargo selection. | 0 | |
| 3442255649 | Quality control | Is the protein folded? Is the protein complex assembled? If not it is actively retained by ER-localized chaperones. | 1 | |
| 3442315728 | Active cargo selection | Specific cargo are collected in regions of the ER that pinch off to form a transport vesicle. Soluble cargo are recognized by membrane proteins that span the ER bilayer. Membrane cargo can be recognized by cytosolic proteins that will aid in vesicle formation. |  | 2 |
| 3336518190 | uncoating of clathrin | Disassembly of the clathrin coat is dependent upon lipid composition (removal of phosphate groups from inositol phospholipids leads to uncoating). | 3 | |
| 3442358538 | How to keep ER proteins in the ER | Some proteins that are resident ER proteins may mistakenly exit the ER. The soluble proteins contain the targeting signal *KDEL* at the C-terminus that interacts with the *KDEL receptor*. The receptor cycles between the Golgi and the ER, binding KDEL-containing proteins at the Golgi and releasing them in the ER. For resident ER membrane proteins the retrieval signal is *KKXX* at the C- terminus in the cytosol. It is recognized by the COP I coat (a set of proteins that are needed to form transport vesicles from the Golgi to the ER). | 4 | |
| 3442405942 | coat protein complexes | Set of proteins that coat the outside of the newly-formed vesicle drives formation of transport vesicles | 5 | |
| 3442422902 | Clathrin | mediates transport vesicle formation at the trans-Golgi (for transport to lysosomes via endosomes) and at the plasma membrane (for transport to endosomes). | 6 | |
| 3442426465 | COP I | mediates transport from the cis-Golgi to the ER and between various Golgi cisternae | 7 | |
| 3336496629 | Vesicle-mediated protein transport | conserved among all eukaryotes, including yeast. Yeast temperature-sensitive mutants were used to identify many of the proteins needed for this process and to understand the mechanism |  | 8 |
| 3336501342 | COP II-coated vesicle | Mediates transport from the ER to the cis-Golgi The elctron-dense region surrounding the membrane is composed of proteins making up the COP II coat complex. |  | 9 |
| 3442445087 | Two functions of protein coat on the cytosolic surface of budding vesicles | 1. Shapes the donor membrane into a bud 2. Helps to capture cargo proteins (membrane-bound and soluble) into budding vesicles | 10 | |
| 3336505183 | Rab in protein coat formation | Coat formation and other steps of vesicle transport require small GTP binding proteins called *Rab protein* that cycle between active (GTP-bound) and inactive (GDP-bound) forms. Binding of GTP (activation) requires a protein called a *GEF* (guanine nucleotide exchange factor) and hydrolysis of GTP to GDP (inactivation) requires a *GAP* (GTPase activating protein). |  | 11 |
| 3336510498 | COP II coat formation | The Rab protein Sar1 is activated by its GEF. It then inserts into the membrane and begins to curve the membrane. Activated Sar1 recruits the inner portion of the COP II coat made up of the proteins Sec23 and Sec24. These proteins further bend the membrane. Sec24 acts as a cargo receptor for membrane proteins. Sec23 and Sec24 recruit the outer layer of the COP II coat made up of the proteins Sec13 and Sec31. Continuous recruitment of Sar1 and the COP II coat proteins eventually deforms the membrane to the point of vesicle release. |  | 12 |
| 3442457944 | Fusion of all three types of transport vesicles with their target membranes exhibits several common features: | 1. The vesicle coats must be completely or mostly removed from the vesicle. 2. The vesicle must be specifically recognized by the correct destination membrane. 3. The vesicle and target membrane must fuse and mix to deliver the contents of the vesicle to the target organelle. | 13 | |
| 3336514751 | COP I coat formation | Arf1 (a Rab protein) activation COP I complex composed of 7 subunits that are recruited en bloc (as one unit) | 14 | |
| 3336514305 | uncoating of COP II and COP I | requires inactivation of the Rab protein (hydrolysis of GTP to GDP and Pi). |  | 15 |
| 3442506110 | Rab proteins | Proteins that distinguish each membrane within a cell. Fusion of vesicles displays great specificity. In their activated (GTP-bound) form, they can bind to effector proteins. Rab proteins in the vesicle and target membrane can bind effectors that contribute to vesicle tethering | 16 | |
| 3442520649 | The main steps in vesicle-mediated transport, after vesicle formation (budding): | 1. Tethering - mediated by Rabs and their effectors, tethering factors and SNAREs 2. Docking - mediated by SNAREs pairing 3. Fusion - driven by SNARE "zippering" | 17 | |
| 3336521512 | tethering | the recognition/ initial contact between the vesicle and the target membrane Occurs over a long distance (>diameter of the transport vesicle) 2 classes of tethers/ tethering factors- can be Rab effectors |  | 18 |
| 3336522736 | Multiprotein tethering complexes | composed of up to 10 proteins, localize to distinct organelles | 19 | |
| 3336523053 | Coiled-coil proteins | long a-helical proteins that project great distances from the target membrane | 20 | |
| 3336525170 | Docking | Stronger interaction between the vesicle and the target
membrane. Occurs over a short distance (< 21 | | |
| 3445180057 | SNARE pairing | Drives membrane fusion. Energy released after SNARE pairing is sufficient to bring the vesicle and target membrane into close proximity and to displace the water molecules surrounding the polar head groups at the outer leaflet. | 22 | |
| 3336527874 | Membrane fusion | Fusion happens in three stages: 1. Outer leaflet mixing between the vesicle and target membranes produce a hemifusion intermediate. 2. Expansion of the hemifusion intermediate provides a surface for the inner leaflets to fuse 3. Fusion of the inner leaflets allows access of the soluble material in the vesicle and target membrane to mix. |  | 23 |
| 3442537991 | vesicle-mediated transport reactions | require SNAREs (v-SNARE and t-SNARE) as well as Rabs and their effectors. These will be specific for each vesicle-mediated transport reaction. Also require factors that are common to each transport step: 1. NSF 2. SNAP proteins | 24 | |
| 3336533046 | NSF and SNAP proteins | NSF is a hexameric (6 copies of the same polypeptide) ATPase that attaches to trans SNARE complexes using accessory proteins called SNAP proteins. Hydrolysis of ATP breaks apart the stable cis SNARE complexes and allows the SNAREs to be reused in another round of fusion. |  | 25 |
| 3336535417 | Vesicle transport model | Golgi cisternae are static, stable compartments that receive and transport cargo in anterograde- directed (ER-Golgi-PM) vesicles (i.e. the secretory cargo moves, Golgi enzymes do not move). Cargo is packaged into vesicles that first bud from the cis Golgi and fuse with the medial Golgi. Vesicles from the medial Golgi, containing the same cargo, then bud and fuse with the trans Golgi. In such a manner, the cargo is transported through the various stacks of the Golgi. Note that the cargo is physically transported in vesicles while the Golgi compartments never move. |  | 26 |
| 3336537178 | Cisternal maturation model | secretory cargo is static and passively matures as Golgi enzymes from later compartments travel in retrograde-directed (trans→cis) vesicles (i.e. Golgi enzymes move, the secretory cargo does not move). Vesicles bud from each Golgi cisterna and contain Golgi enzymes specific to that cisterna. The vesicles move backwards, to an earlier Golgi cisterna and deliver their contents there. Thus, over time, the cis Golgi acquires medial Golgi enzymes, converting it into a medial Golgi. At the same time, the medial Golgi "sheds" its enzymes in vesicles and acquires trans Golgi emzymes, eventually becoming the trans Golgi. The trans Golgi morphs into the trans Golgi network (TGN) from which vesicles will bud and fuse with the plasma membrane, endosomes or lysosomes. Note that the cargo never moved. Rather, its surroundings changed. This can explain how very large cargo (e.g. collagen), too big to fit into a vesicle, can move through the Golgi. |  | 27 |
| 3336539706 | Where do different modifications of proteins take place in the golgi? | Addition of galactose and other carbohydrates takes place in the trans Golgi. Addition of GlcNAc, fucose and additional mannose trimming takes place in the medial Golgi. Mannose trimming takes place in the cis Golgi. |  | 28 |
| 3336543948 | glycosylation factory pt 2 | A unique modification takes place on soluble lysosomal enzymes resulting in the production of mannose-6-phosphate. Addition of phospho-GlcNAc to one or more mannose residues Removal of GlcNAc, leaving mannose-6-phosphate |  | 29 |
| 3336548817 | Delivery from the TGN to lysosomes | Soluble lysosomal enzymes containing mannose-6-phosphate (M6P) are recognized by the membrane-bound M6P receptor which: • binds to the M6P residue at pH 6.5-6.7 (pH of the TGN) • releases the residue at pH 6 (pH of the endosome). From the endosome, the M6P receptor recycles back to the TGN. The phosphate is removed from the soluble enzyme which is then transported from the endosome to lysosomes. |  | 30 |
| 3336551643 | Bulk-phase endocytosis | (pinocytosis) - non-selective, can be clathrin-dependent or clathrin-independent | 31 | |
| 3445185297 | Receptor-mediated endocytosis | selective, clathrin- dependent, initiated by the binding of a ligand to its receptor (e.g. transferrin, LDL, EGF are ligands that bind to specific receptors) | 32 | |
| 3336555598 | vesicle coat formation | Clathrin forms the outer layer of the coated vesicles and has a distinctive triskelion appearance. Adaptor proteins form the inner layer of the coated vesicles and engage the cytoplasmic tails of receptors. Their recruitment to the "coated pit" is facilitated by a lipid called phosphatidylinositol (4,5) bisphosphate |  | 33 |
| 3336557652 | dynamin | Small GTP binding protein. Binds as a ring around the emerging stalk as the coated pit invaginates. Using the energy of GTP hydrolysis, it breaks the vesicle free from the plasma membrane. If a non-hydrolyzable form of GTP is used, the stalk continues to grow with a dynamin ring. |  | 34 |
| 3445192081 | Uncoating of a clathrin coated vesicle requires: | 1. Modification of the lipids that bind the adaptor proteins 2. Energy provided by the hydrolysis of ATP by Hsc70 | 35 | |
| 3336559528 | early endosome | Result of fusion of uncoated clathrin vesicles. |  | 36 |
| 3445195199 | late endosomes | Early endosomes go through maturation and become late endosomes | 37 | |
| 3445198062 | Early vs late endosomes | • Late endosomes have a lower pH than early endosomes (dissociation of the ligand from its receptor happens in the endosome) • Late endosomes associate with a Rab protein called Rab7 while early endosomes associate with Rab5 • Late endosomes are found near the Golgi in the cell interior while early endosomes are found near the plasma membrane • Late endosomes are round or oval while early endosomes have a more complex structure (tubulo-vacuolar) |  | 38 |
| 3445202703 | Three fates for the receptor/ligand complex: | 1. The low pH of the early endosome causes dissociation of the ligand from the receptor (e.g. LDL/LDL receptor). The receptor is then returned to the cell surface and the ligand is routed to the lysosome. 2. The ligand and receptor do not dissociate and the receptor shuttles the ligand back to the cell surface (e.g. transferring/transferrin receptor). 3. In some cases, the ligand and receptor are both sent to the lysosome for degradation (e.g. EGF/EGF receptor). | 39 | |
| 3336563275 | Dissociation of the receptor and ligand: receptor recycling to cell surface and ligand delivery to the lysosome |  | 40 | |
| 3336565005 | Receptor and ligand do not dissociate: both are recylced to cell surface |  | 41 | |
| 3336566653 | Receptor and ligand are delivered to the lysosome | The receptor is tagged with a small protein called ubiquitin. In the maturing endosome, invagination takes place and the ubiquitin-tagged receptor-ligand complex enters the invagination area and ends up in an intralumenal vesicle. The multivesicular body eventually fuses with a lysosome where the proteases and hydrolases will degrade the receptor and the ligand. |  | 42 |
