Molecular Cell Biology Answer Set Chapters 14

Molecular Cell Biology Answer Set Chapters 14 - 14 Review...

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45 14 Vesicular Traffic, Secretion, and Endocytosis Review the Concepts 1. Palade and colleagues chose the pancreatic acinar cell because it is a specialized secretory cell that packages trypsinogen, chymotrypsinogen, and other digestive enzymes into secretory granules that then are released in response to signaling. Most protein synthesis in these cells is devoted to secretion. The vast majority of ribosomes are found in association with the rough endoplasmic reticulum. The cells are also decidedly polarized with a definite gradient in organelle distribution. In his seminal experiments Palade took advantage of radioactive amino acids to label proteins, the majority of which were incorporated into molecules associated with membrane-bound ribosomes and, subsequently, with secretory organelles. After pulse-chase labeling and sectioning the cells for electron microscopy, the sections were covered with photographic emulsion and exposed. In this autoradiographic method, silver grains exposed to the radioactive decay from the newly labeled protein were reduced and, when the emulsion was developed, the appearance of the silver grains was compared under the electron microscope to their subcellular distribution. In a more contemporary method cells can be transfected with a hybrid gene encoding two proteins. In this manner the transcript encodes a membrane glycoprotein from vesicular stomatitis virus (VSV G) that is fused to green fluorescent protein (GFP), which is the tag for detection. Cells expressing this gene rapidly synthesize VSVG-GFP in the ER, which is then transported through the secretory pathway to the cell surface. Using fluorescence microscopy to detect GFP, the investigator can monitor the expression of the chimeric protein and its localization in live cells. Thus both methods require that proteins be labeled in an early compartment so that their processing and transport can be followed over time. The second necessary requirement is to have a way to identify the compartment containing the labeled proteins. In each case a form of microscopy was used that complements the labeling reaction. 2. NSF, through its ATPase activity, catalyzes the dissociation of v-SNARE/t-SNARE complexes. Such complexes are essential in specific membrane fusion at several stages of the secretory and endocytic pathways. Why then does the Sec18 NSF mutation produce a class C phenotype accumulation of ER-to-Golgi transport vesicles? This can be explained readily if one considers the need for NSF to generate free v-SNAREs and t-SNAREs to support multiple rounds of vesicle membrane fusion. In the absence of NSF activity, vesicles bud from the ER but are unable to fuse with downstream membranes because of the lack of v-SNAREs. Vesicles accumulate at what is the first vesicle organelle fusion step within the secretory pathway.
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