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Vesicular Budding and Fusion Cell Biology > Protein Sorting and Vesicular Transport > Protein Sorting and Vesicular Transport

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Vesicular Budding and Fusion Cell Biology > Protein Sorting and Vesicular Transport > Protein Sorting and Vesicular Transport Vesicular Budding and Fusion Cell Biology > Protein Sorting and Vesicular Transport > Protein Sorting and Vesicular Transport STEPS OF VESICULAR BUDDING AND FUSION (+ PROTEINS INVOLVED) • Cargo selection (cargo receptor, adaptor protein) • Vesicular budding (adaptor proteins, coat proteins) • Fission from donor membrane (dynamin) • Vesicular coat dissociates • Vesicular targeting and transport (Rab-GTPase, tethering protein) • Fusion with target membrane (V-snare and T-snare) PROTEINS OF VESICULAR BUDDING AND FUSION • Cargo receptors – select and concentrate molecules to be transported in vesicle • Adaptor proteins - bind cargo receptor and coat proteins • Coat proteins - form protein scaffold around vesicle that facilitate vesicular budding • Dynamin – GTPase involved in vesicular fission • RabGTPase - associates with vesicle after coat has dissociated. Facilitates transport of vesicle to appropriate target membrane. Locks vesicle to target membrane by attaching tethering proteins • Tethering proteins – anchored in target membrane, attach rabGTPase. Move vesicle close to target membrane for vesicular fusion. • V-snares(vesicular) and T-snares(target membrane) - Play role in vesicular fusion COAT PROTEINS DIRECT VESICLE TRANSPORT • Clathrin +adaptin 1: Golgi ? Lysosome • Clathrin + adaptin 2: Plasma membrane ? Endosomes (endocytosis) • COP 1: Cis golgi ? ER AND Later cisternae ? Earlier ones (retrograde transport) • COP II: ER ? Cis golgi 1 / 6 FULL-LENGTH TEXT • Here we will learn how transport vesicles form and fuse with their target membranes. • To begin, start a table to list the key steps that are common to all transport vesicles. • Denote the following steps: - Cargo selection, which is the carefully regulated incorporation of cargo into a vesicle. - Vesicular budding, which involves deformation of the hydrophobic membrane bilayer, allowing the vesicle to break off the membrane into a vesicle. - Vesicular targeting and fusion, which is highly regulated just like cargo selection. We will illustrate the specialized proteins and receptors that facilitate these steps. To begin, let's illustrate step 1, cargo selection. • First, draw a phospholipid bilayer as two lines bending inwards. • Label the bilayer: donor membrane. • Now, donor lumen above it. • And the cytoplasm below it. • Now, draw cargo receptors on the curved portion of the membrane. • Show that they bind soluble cargo; they specifically bind the signal sequences of secretory proteins that are sorted for export from the donor compartment. • Next, draw adaptor proteins that bind to the cytoplasmic side of the cargo receptors. • Write that adaptor proteins maintain the following functions: - They help filter the cargo that binds cargo receptors. 2 / 6 - They function as an interface for coat formation, which we will illustrate shortly. • Cargo selection is highly regulated to ensure that only the correct cargo gets transported. Now let's assemble our vesicular coat. • Show that coat proteins bind to the adaptor proteins, which are in turn bound to cargo receptors. - This creates a "protein scaffold" on the cytoplasmic side of the membrane, a structure that bends the donor membrane and stabilizes the resulting curvature. • Write that coat proteins sculpt the membrane to facilitate vesicular budding, and that they also help select for correct cargo. - Specific coat proteins refer to specific vesicular transport pathways within the cell. Let's introduce these different coat proteins before we move on. • Start a chart to learn the different kinds of coat proteins, their membranes of origin and the destinations of the vesicles that they help shape. • Indicate that clathrin proteins originate in the Golgi and are involved in vesicular transport to lysosomes. • Indicate that these clathrin proteins are specifically paired with adaptin 1 adaptor proteins. Adaptor proteins are often considered inner coat proteins. • Write that clathrin proteins paired with adaptin 2 originate in the plasma membrane and are involved in vesicular transport to endosomes. • Now, indicate that COP 1 proteins originate in the cis Golgi and are involved in transport to the ER. • Write that they also facilitate transport from later cisternal stacks in the Golgi to earlier ones. • Finally, indicate that COP II proteins originate in the ER and facilitate transport to the cis Golgi. Now that we've introduced the diversity of coat proteins within the cell, let's illustrate step 2. of vesicle formation: vesicular budding. 3 / 6 • Extend the donor membrane to illustrate this step, but this time with a more dramatic inward bulge. • Again, draw cargo in the lumen and cargo receptors that bind a membrane scaffold on their cytoplasmic side. • Now, draw an additional protein that spirals around the neck of the budding vesicle. • Label it dynamin. • Write that dynamin family proteins are GTPases, which hydrolyze GTP to GDP. • Indicate via fission, dynamin and associated proteins twist the neck of the vesicle in a GTP-dependent manner, which pinches off the vesicle from the donor membrane. • Now, draw the vesicle suspended in the cytoplasm. • Below it, draw a naked transport vesicle that has shed its coat and adaptor proteins. - These external proteins dissociate from the vesicular membrane after fission occurs. • Label this step uncoating, during which the adaptor and coat proteins that dissociate get recycled via the retrieval pathway and return to the donor compartment. This brings us to our final phase: vesicular targeting and fusion, wherein the vesicle targets a membrane and fuses with it. • First, our vesicle must recognize its target. • Draw a target membrane bending outward and label the target lumen. • Now, in the cytoplasm, draw a simplified naked vesicle with cargo. • This time, include the following proteins in the vesicular membrane: - Rab GTPase, which also hydrolyzes GTP like the dynamin family. - V-snare protein. The "v" stands for "vesicle" SNARE and will bind a complementary t-SNARE on the target membrane. 4 / 6 • Write that the Rab GTPase: - Facilitates the transport of the vesicle to its appropriate target membrane; it does this by associating with cytoskeletal motors. - Write that it also helps the vesicle dock at the correct target membrane. - Lends specificity to the transport vesicle. Different Rab GTPases correspond to different target membranes. - Participates in the fusion of the vesicular and target membranes. We will illustrate the function of V-snare proteins shortly. • Now, on the target membrane, draw a filamentous tethering protein. • Next to it draw a T-snare. Show the tethering step as follows: • Show that the Rab GTPase on our vesicle binds to the tethering protein and moves closer to the target membrane. - Both of these proteins must match in order for this step to proceed. • Finally, indicate that the tethering protein and Rab GTPase match, thus allowing the vesicle to enter the docking phase. - If they do not match, the vesicle dissociates and Rab GTPase continues the search for its correct destination. Show the docking step as follows: • Draw another vesicle even closer to the target membrane, and illustrate the following protein interactions: - Rab GTPase remains bound to the tethering protein. - The v-SNARE binds the membrane-bound t-SNARE tightly. • Show that the tight binding of SNARE proteins facilitates the final step: Fusion. • Show that cargo dissociates from the cargo receptor. • Thus, cargo is finally delivered to the target organelle via fusion. 5 / 6 • As a clinical correlation denote that the tetanus toxin, released by bacteria known as Clostridium tetani, cleaves a SNARE protein in nerve cells. - This prevents the fusion of synaptic vesicles, and as a result, the release of neurotransmitters, which produces muscular spasms. Powered by TCPDF (www.tcpdf.org) 6 / 6.
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