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Techniques for Studying the Secretory Pathway - I
Synthesis of proteins bearing an ER signal sequence is completed on the rough ER and the newly made polypeptide chains are inserted into the ER membrane or cross it into the lumen. Pulse-chase experiments are particularly useful for tracing changes in the intracellular location of proteins or the transformation of a metabolite into others over time. In this experimental protocol, a cell sample is exposed to a radiolabelled compound, the “pulse” for a brief period of time, then washed with buffer to remove the labelled pulse, and finally incubated with a nonlabelled form of the compound, the “chase”. A gene encoding an abundant membrane glycoprotein (G protein) from vesicular stomatitis virus (VSV) is introduced into cultured mammalian cells. Use of a mutant encoding a temperature-sensitive VSV G protein allows researchers to turn subsequent protein transport on and off. The clever use of a temperature-sensitive mutation in effect defines a protein cohort whose subsequent transport can be followed. Many secretory proteins leaving the ER contain one or more copies of the N-linked oligosaccharide Man8(GlcNAc)2, which are synthesized and attached to secretory proteins in the ER. Scientists can use a specialized carbohydrate-cleaving enzyme known as endoglycosidase D to distinguish glycosylated proteins that remain in the ER from those that have entered the cis-Golgi.
Techniques for Studying the Secretory Pathway - II
Yeast mutants define major stages and many components in vesicular transport. Genetic studies with yeast have been useful in confirming the sequence of steps in the secretory pathway and in identifying many of the proteins that participate in vesicular traffic. The best-studied of these, invertase, hydrolyzes the disaccharide sucrose to glucose and fructose. A large number of yeast mutants initially were identified based on their ability to secrete proteins at one temperature and inability to do so at a higher, non-permissive temperature. Analysis of double mutants permitted the sequential order of the steps to be determined. In Cell-free Transport Assays, cultured mutant cells lacking one of the enzymes that modify N-linked oligosaccharide chains in the Golgi are infected with vesicular stomatitis virus (VSV). When golgi membranes isolated from such mutant cells are mixed with golgi membranes from wild-type, uninfected cells, the addition of N-acetylglucosamine to VSV G protein is restored. Under appropriate conditions a uniform population of the retrograde transport vesicles that move N-acetylglucosamine transferase-I from the medial- to cis-Golgi can be purified away from the donor wild-type Golgi membranes by centrifugation. By examining the proteins that are enriched in these vesicles, scientists have been able to identify many of the integral membrane proteins and peripheral vesicle coat proteins that are the structural components of this type of vesicle.
Molecular Mechanisms of Vesicular Traffic - I
Budding Vesicles bud from the membrane of a particular “parent” organelle and fuse with the membrane of a particular “target” organelle. The polymerization of soluble protein complexes onto the membrane to form a proteinaceous vesicle coat. The integral membrane proteins in a budding vesicle include v-SNAREs. Three types of coated vesicles have been characterized, each with a different type of protein coat and each formed by reversible polymerization of a distinct set of protein subunits. COPII vesicles transport proteins from the rough ER to the Golgi. COPI vesicles mainly transport proteins in the retrograde direction. Clathrin vesicles transport proteins from the plasma membrane and the trans-Golgi network to late endosomes. For both COPI and clathrin vesicles, the GTP-binding protein is known as ARF. A different but related GTP-binding protein known as Sar1 is present in the coat of COPII vesicles. ARF and Sar1 proteins, like Ras, belong to the GTPase superfamily of switch proteins that cycle between inactive GDP-bound and active GTP-bound forms. Sar1 attached to the membrane serves as a binding site for the Sec23/Sec24 coat protein complex. The coat is completed by assembly of a second type of coat complex composed of Sec13/and Sec31. Release of Sar1·GDP from the vesicle membrane causes disassembly of the coat in cells expressing mutant versions of Sar1 or ARF that cannot hydrolyze GTP, vesicle coats form and vesicle buds pinch off. In addition to sculpting the curvature of a donor membrane, the vesicle coat also functions in selecting specific proteins as cargo. The primary mechanism by which the vesicle coat selects cargo molecules is by directly binding to specific sequences, or sorting signals, in the cytosolic portion of membrane cargo proteins.
Molecular Mechanisms of Vesicular Traffic - II
Binding of Rab GTP to a Rab effector on target membrane docks the vesicle on an appropriate target membrane. After vesicle fusion occurs, the GTP bound to the Rab protein is hydrolyzed to GDP. The fusion of early endosomes with each other in cell-free systems requires the presence of Rab5 and GTP. A different type of Rab effector appears to function for each vesicle type and at each step of the secretory pathway. One of the best-understood examples of SNARE-mediated fusion occurs during exocytosis of secreted proteins. The cytosolic region in each of the three SNARE proteins contains a repeating heptad sequence that allows four helices one from VAMP, one from syntaxin, and two from SNAP-25 to coil around one another to form a four-helix bundle. Fusion of a vesicle and target membrane occurs much more rapidly and efficiently in the cell than it does in liposome experiments in which fusion is catalyzed only by SNARE proteins. Because of the stability of SNARE complexes, which are held together by numerous non-covalent intermolecular interactions, their dissociation depends on additional proteins and the input of energy. The predominant glycoprotein of the influenza virus is hemagglutinin (HA), which forms the larger spikes on the surface of the virus. Following endocytosis of an influenza virion, the low pH within the enclosing late endosome triggers fusion of its membrane with the viral envelope. Each HA molecule participates in only one fusion event, whereas the cellular fusion proteins, such as SNAREs, are recycled and catalyze multiple cycles of membrane fusion.
Early Stages of the Secretory Pathway
Anterograde transport from the ER to Golgi, the first step in the secretory pathway, is mediated by COPII vesicles, whereas the reverse retrograde transport from the cis-Golgi to the ER is mediated by COPI vesicles. Formation of COPII vesicles is triggered when Sec12, a guanine nucleotide–exchange factor, catalyzes the exchange of bound GDP for GTP on Sar1. After the complex forms on the ER membrane, a second complex comprising Sec13 and Sec31 proteins then binds to complete the coat structure. Certain integral ER membrane proteins are specifically recruited into COPII vesicles for transport to the Golgi. In some cells, small vesicles form from the ER, move less than 1 m, and then fuse directly with the cis-Golgi. In other cells, in which the ER was located several micrometres from the Golgi complex, several ER-derived vesicles fuse with each other shortly after their formation, forming “ER-to-Golgi intermediate compartment.” COPI coated vesicle coat is formed from large cytosolic complexes, called coatomers. The ER contains several soluble proteins dedicated to the folding and modification of newly synthesized secretory proteins. Most soluble ER-resident proteins carry a Lys-Asp-Glu- Leu (KDEL in the one-letter code) sequence at their C terminus. The KDEL sorting signal is recognized and bound by the KDEL receptor. The selective entry of proteins into membrane-bounded transport vesicles, the recycling of membrane phospholipids and proteins, and the recycling of soluble luminal proteins between the two compartments are fundamental features of vesicular protein trafficking that also occur in later stages of the secretory pathway. Forward transport of cargo proteins from the cis- to the trans-Golgi occurs by a non-vesicular mechanism, called cisternal progression.