Oct 26 Notes to Post

October 26, 2006 1 Vesicular Trafficking, Secretory Pathway, HIV Assembly and Exit from Cell 1. Secretory pathway a. Formation of coated vesicles b. SNAREs and vesicle targeting 2. Membrane fusion a. SNAREs and vesicle fusion 3. Retrograde trafficking a. Steady-state 4. Functions of the Golgi a. Glycosylation b. Protein sorting 5. Viral assembly and exit from the cell Lecture Readings Alberts 498-522 (cont.) 2 Challenges in Vesicular Transport I 1) How is membrane deformed to form a vesicle? 2) How are the correct cargoes enriched in the vesicle and the proteins that should remain in the donor compartment excluded? 3) How do vesicles know what compartment to fuse with? 4) What catalyzes the fusion process? The use of vesicles to move proteins and lipids from one compartment to another raises several challenges that must be overcome. For example, the membrane must be deformed to make a vesicle and this process is energetically unfavorable because the head groups at the neck of the budding vesicle are brought close together, resulting in unfavorable electrostatic interactions. Additionally, the appropriate cargoes must be packaged into vesicles and the proteins that are to remain in the donor compartment must be left behind. Once vesicles are formed, there has to be a mechanism that allows vesicles to know what compartment to fuse with. Finally, as we saw with HIV entry into cells, membrane fusion is energetically unfavorable. In that case, energy stored in the structure of gp41 was used to drive the fusion process. There has to be an analogous mechanism operating for the fusion of vesicles with target membranes in the cell. 3 3 Major Types of Coated Vesicles Proteins coating vesicles that bud from donor compartments play a critical role in addressing the first two questions - vesicle formation and cargo selection. Vesicles bud off as coated vesicles that have a cage of proteins covering their cytosolic surface. There are three major types of coated vesicles: clathrin, COPI, and COPII. The coats have two major functions: (1) deforming membrane/introducing curvature to form a vesicle • Formation of a vesicle requires curvature of the membrane, which brings negatively charged phospholipids into close proximity in places, an energetically unfavorable act. Introduction of curvature is achieved in part by neutralizing the negative charge on the phospholipid head groups, making it more favorable for the membrane to be deformed. Curvature is also introduced by the shape of the coat proteins themselves; some are known to have intrinsic curvature and to polymerize to form a partial sphere. The tight binding of the coat protein to the membrane and coat protein polymerization causes the membrane to deform. (2) concentrating/selecting appropriate cargo • Coats accomplish this by interacting with proteins that are to be enriched in the vesicle. These interactions may be direct or may be mediated through other proteins. Coats are used for different transport steps in the cell: Clathrin - movement of proteins from the Golgi and plasma membrane. COPI and COPII - movement of proteins between the Golgi and the ER. 4 SNAREs and Targeting Vesicles coat protein Vesicles have to recognize the correct target membrane; because there are many membranes, a vesicle is likely to encounter many potential targets. Proteins called SNAREs (which stands for Golgi SNAP receptor complex protein, a name that is not particularly informative) are thought to play a role in determining which vesicles fuse with which compartments. Specifying in targeting is ensured because vesicles display v-SNAREs (vesicle SNAREs) on their surface that identify them according to their origin and type of cargo; target membranes display complementary t-SNAREs (target SNAREs) that recognize the v-SNAREs. For example, in this picture the skin-colored v-SNARE pairs with the light blue t-SNARE on compartment A, targeting cargo a to compartment A. Similarly, the red v-SNARE on vesicles containing cargo b binds to the bright blue t-SNARE on compartment B. This diagram also shows the cargo receptor (brown) and vesicle coat (green); we previously discussed how the coat proteins play a role in deforming the membrane to form a vesicle and in helping to select cargo for inclusion in the vesicle. Cargo selection is achieved indirectly - the coat protein interacts with a cargo receptor, which in turn binds to cargo proteins (yellow) that are to be included in the vesicle. These interactions ensure that the correct cargo is selected for inclusion in the vesicle. 5 SNAREs Promote Membrane Fusion v-SNARE SQUEEZE OUT WATER FORM vesicle STALK target t-SNARE HEMI- FUSION FUSION In addition to their role in targeting vesicles to the correct target compartments, SNAREs are thought to play a role in membrane fusion. The role of SNAREs is analogous to the role played by HIV Gp41 in fusion of the virus with the cell membrane. Recall that in the case of Gp41, the favorable change in Gp41 structure is used to overcome the unfavorable electrostatic repulsion that occurs when the viral and host cell membranes are brought together. v-SNAREs pair with t-SNAREs to form a very stable bundle of four alpha-helices that forces the two membranes into close apposition; like Gp41, the favorable energetics of folding (into a bundle of four alpha-helices) is used to overcome the unfavorable process of expelling water molecules from the interface and bringing charged lipid head groups together (1.5 nm separation of the two membranes). The lipids of the two interacting leaflets then flow between the membranes to form a connecting stalk. Next, the lipids of two other leaflets contact each other, forming a new bilayer which widens the fusion zone (hemifusion state). Finally, rupture of the new bilayer completes fusion of vesicle and target. 6 Challenges in Vesicular Transport II 1) How does the cell maintain organelles and the plasma membrane at a constant size if vesicles are constantly moving lipids from compartments to the plasma membrane? 2) How does the cell ensure that proteins which should remain in a compartment do so, even if the process of cargo selection is not perfect? We talked about how proteins get into the secretory pathway by crossing the ER membrane and how proteins leaving the ER move in vesicles which fuse with the Golgi membrane to deliver their contents. This raises two questions regarding the transport and its consequences. First, how does the cell maintain organelles and the plasma membrane at a constant size if vesicles are constantly moving lipids from compartments to the plasma membrane? This one way transport from the ER to the Golgi and on to the plasma membrane would tend to decrease the size of the ER and increase the size of the plasma membrane. Second, how does the cell ensure that the proteins that are to remain in a compartment do so, given that the process of cargo selection is not perfect? 7 Secretory Pathway: Retrograde Traffic lysosome nuclear envelope plasma late membrane endoplasmic reticulum endosome early endosome CYTOSOL cisternae secretory vesicle Golgi apparatus The answer to both questions lies in the process of retrograde traffic - membrane vesicles move back from plasma membrane into the secretory pathway and from the Golgi to the ER (green arrows) - this provides an opportunity for proteins that were mis-sorted to be sent back to the correct compartment; retrograde traffic also ensures that the balance of lipids and the relative size of the compartments is maintained. Flow of lipids in the secretory pathway is at steady-state. This is true of almost all processes in a living cell - they are steady-state, not at equilibrium. 8 Equilibrium vs. Steady-State equilibrium steady-state What is steady-state and what is the difference between steady-state and equilibrium? Steady-state is similar to equilibrium, but not the same. In steady-state, the system is stable (unchanging), and energy has to be put into the system in order to maintain it. An example of steady-state is the potassium ion concentration within cells - the concentration is stable, but maintenance of this stable state requires the input of ATP to the sodium-potassium pump. In equilibrium, the system is also stable. However, no energy has to be put into the system to maintain this stable state. For example, the ionization of HCl in a beaker of water is a system that quickly reaches equilibrium where HCl is ionized and the system is at its lowest free energy state. This happens without the input of any energy. A simple way to think about this is to consider a bathtub: if the bathtub doesn't leak (left), your bathwater will stay at the same level indefinitely at equilibrium. But if the bathtub does leak, the water level will drop at some rate (middle right). By matching the rate with which you add water (top right) to the rate with which you lose water (middle right), you can achieve a constant, non-equilibrium steady-state (bottom right). Trafficking of vesicles in the secretory pathway is at steady-state - the size of the ER is not changing, even though vesicles are budding and fusing - the rate vesicles bud from the ER is approximately equal to the rate they fuse with it. Since there are net movements of molecules and energy is expended during trafficking, the system is not at equilibrium. Processes in the cell are generally far from equilibrium, but some are at steady-state. Exception: Binding reactions within cells can be at equilibrium. 9 Golgi Apparatus vesicles coming from ER vesicles leaving Golgi Vesicles leaving the ER fuse with the Golgi, a collection of flattened membrane-enclosed sacs called cisternae. Coated vesicles from the ER fuse with the cis-face of the Golgi network, move through the cisternae, and exit from the trans-face of the Golgi network where they are directed to various compartments within the cell.

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