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5. Membrane Trafficking.pdf

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McGill University
Anatomy & Cell Biology
ANAT 262
John Presley

Membrane Trafficking: The secretory pathway is made of the ER, Golgi, endosomes and lysosomes, while the endocytic pathway is made from the PM, endosomes, Golgi and lysosomes From the inside out, the secretory pathway contains: o The ER o Pre-Golgi intermediates, also called intermediate compartments or VTCs o The Golgi o Post-Golgi carriers (several kinds) and secretory vesicles o The cell surface and cell exterior The endocytic pathway is made of, from the outside in (oversimplified, but all we need to know for now) o The PM o Early endosomes (though alternate pathways exist) o Late endosomes o Lysosomes, which are degrading organelles Also, direct pathways between Golgi and endosomes create a linkage between the secretory and endocytic pathways Keith Portman first discovered the ER in 1945 through obtaining some crude EM pictures stained with carbon (not a quality image in todays standards) Proper staining techniques really began to shape in the 1950s As we can see, there is a fine network in the cytoplasm which is actually the ER, though barely visible here Also, the wormlike structure in the cell are the mitochondria Discovery of the Golgi was by Camillo Golgi in 1898, who initially called it the reticular network The staining process actually involved drying water from Italian rivers, which was high in silver (which is coincidentally an excellent EM stain) Recent EM pictures are much clearer, showing each stack of the Golgi membrane, which looks like a stack of pancakes You can also see the ER spread all over the cell as a fine network Cells with large amounts of secretion, such as goblet cells with tons of mucous secretion, have a very prominent Golgi because there a lot of secretory proteins being sorted Additionally, many secretory vesicles and granules can be seen near the cell surface Studies in 1923, before the discovery of the central dogma, were very confused by secretory vesicle formation and the Golgi because they had no idea how transcription and translation work o But, they were able to make a close association between the Golgi and the appearance of secretory vesicles Work by George Palada in the 1950s and 60s done using EM and some other techniques allowed him to define the secretory pathway The pathway he determines was the ER Golgi secretory granule cell surface o He first noticed that some ribosomes were free in the cytoplasm, while some others were bounds and secreted their contents right into the ER lumen Using autoradiography and EM, he determined that proteins were first secreted in the ER and then they were brought to the Golgi Autoradiography involved labelling proteins with 3H leucine in a rat pancreas o The rat pancreas works well because almost all the proteins are secretory o By taking readings of the protein location at various time intervals, the proteins could be followed along their path in the secretory apparatus Thus, after 3 minutes, the 3-H leucine proteins were seen all almost all in the ER through silver staining o Some were also seen in the mitochondria because mtDNA have their own proteins After 10 minutes, many of these proteins could be seen in the Golgi o The process is quite fast After 40 minutes, the proteins were almost all in vacuoles, which are secretory vesicles o The light vacuoles are condensing vesicles, which during their development condense the proteins and acidify the contents, and turn into dark zymogenic granules After 120 minutes, the proteins were seen in the very dark zymogen granules The ER has several functions including the folding of translocated proteins and their N-linked glycosylation, but what does the Golgi do? Evidence accumulated that proteins from the ER were not only funnelled through the Golgi apparatus prior to secretion but were also chemically modified o Most notably, the final sugar residues were added to N-linked oligosaccharides Tritiated galactose is also able to stain with silver, and was shown to appear over the Golgi after 2 minutes of staining o After 10 minutes, these 2-T galactose were found in the microvilli, which means they must have been attached to N-linked glycoproteins in the Golgi Uncharged, high mannose N-linked proteins come in from the ER, which are modified on asparginine (N) o When they get to the Golgi, they go through a series of modification in turns the uncharged sugars into negative ones Palade proposed that vesicle transport was involved in the transport of proteins from one organelle to another, but the technology he had prevented him from proving it In the 1980s, the use of yeast genetics (Schekman group) and in vitro systems (Rothman group) identified many of the molecules involved in transport through the secretory pathway and provided a first picture of how carrier vesicles transported proteins from one compartment to another Surprisingly, the two sets of results from completely different organism shows very similar results, which means that the secretory pathway must be almost identical in most eukaryotes o Mammalian have a more elaborate system than yeast because they are more complex Thus, the summary of the secretory organelle functions are: The ER synthesizes secretory proteins, fold them, and mediates N-linked glycosylation o It also has a quality control mechanism to make sure misfolded proteins dont get sent on The Golgi Takes N-linked glycoproteins from the ER and modifies them with various enzymes o It sorts the various proteins according to their destination The destinations are numerous; they can e
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