Protein Sorting: Co-Translational:1. Suppose that slices from a tissue that actively secretes proteins were incubated with radioactive leucine in a suspension culture for five minutes and then prepared for autoradiography. What might you expect to see on the developed autoradiograms (based on the studies by George Palade)? How would the slices have been processed for autoradiography, by the ways?Expect to see: You would see the path of the radioactive leucine, they could be in the rER, golgi, secretory vesicles depending on how long you waited to view them. The developed autoradiograms would show secretory proteins in rough ER after 3 minutes, then in cytoplasm between ER and golgi after 10 minutes, then in golgi between 30-60min, then apical region of acinar cell after 2 hours.Process:1. The tissue would be incubated with the radioactive leucine 2. Cells would be fixed to slide with various buffers (alcohol/formaldehyde) +++++++++++++++++++++++3. In a dark room, the slide is dipped in a photographic emulsion of AgBr. During incubation, silver granules interact with the photons emitted by the decay of the radioactive leucine.4. The slide is developed. The radioactive decay would show the silver granules that interacted with the photons. This would look like black specs on the prepared slide under a microscope. (After a period of labeling with leucine, the tissue is fixed, sectioned for electron microscopy, and subjected toautoradiography. The radioactive decay of leucine in newly synthesized proteins produces autoradiographic grains in an emulsion placed over the cell section (which appear in the micrograph as dense, wormlike granules) that mark the positionof newly synthesized proteins. At the end of a 3-minute labeling period autoradiographic grains are over the rough ER. Following a 7-minute chase period with unlabeled leucine, most of the labeled proteins have moved to the Golgi vesicles. After a 37-minute chase, most of the proteins are over immature secretory vesicles. After a 117-minute chase, the majorityof the proteins are over mature zymogen granules.)2. Suppose that mRNA for a secreted pancreatic enzyme were translated in vitro using a cell-free protein synthesis preparation, and later the amino acid sequence of the protein produced in vitro were compared with that of the purified secreted protein: Comment of what the results might be and provide interpretations for each of the possibilities you include.It would be larger because a cell-free protein would not involve passage through the ER which cleaves the signal peptide. The main point of this question is to know that there is a signal peptide that remains in the membrane of the RER when the protein is produced. The 1st protein in the question would be longer, because it is NOT fed through the rER when it was produced. The 2nd protein would be shorter, because the signal peptide remains embedded in the rER when it is fed through into the RER lumen. +Not all proteins translated on the RER are integral RER membrane proteins, some are released into the RER lumen for secretion. Secreted proteins weigh less than their precursors because the signaling sequence that binds to SRP (signal recognition particle) is cleaved inside the translocation channel by a signal peptidase. In the first case, the protein would weigh more than expected because it still has the signaling sequence, rather than the authentic protein whose sequence is cleaved during co-translational translocation. 3. Discuss potential outcomes for the newly made protein if the translation reaction described above had included:(a) Plasma membrane derived vesicles: (b) ER derived vesicles: This would be in the presence of microsomes (ER vesicles). A protein of the correct size is created because N-terminal leader peptide present in vitro is cleaved by signal peptidase (c) Golgi derived vesicles: 4. What would you expect the traits of a signal peptide for a protein secreted from liver to be (size, sequence, location)?The signal peptide is a small section of 5-30 peptides on the N-terminal end of a protein. It has hydrophobic residues down one side and tends to form a single alpha helix with a grouping of positive charges to one side of it.5. Which amino acid(s) on an integral plasma membrane protein would be linked to oligosaccharide chains attached to the protein during synthesis on the rER?Two types of linkages:N-linked: sugars attch to asparagine (rER and Golgi)O-linked: sugars attch to serine and threonine (Golgi)* not every asparagine is glycosylated. +Must have specific sequence -- proceeded by either Serine or Threonine6. Explain the genetic causes of the I-cell disease and the biochemical consequences of the deficits.An individual has genes for defective phosphotransferases, enzymes used to phosphorylate mannose in glycoproteins during sorting in the Golgi. This enzyme transfers phosphate to+mannose+residues on specific proteins, and serves as a marker for them to be targeted to+lysosomes+within the cell. Without this marker, the proteins are instead excreted outside the cell -- the default pathway for proteins moving through the Golgi apparatus. Lysosomes cannot function without theseproteins, which function as catabolic enzymes for the normal breakdown of substances+throughout the body. As a result, a buildup of these substances occurs within lysosomes because they cannot be degraded, resulting in the characteristic "I cells," or "inclusion cells." This leads to a broad variety of problems including enlarged organs, restricted joint movement,abnormal skeletal development, and coarse facial features. It’s very similar to Hurler’s syndrome, so similar problems canbe anticipated.7. Outline the roles of SRP and of its receptor on progression of translation of albumin mRNA, starting with eventsin the cytoplasm and ending with the insertion of the growing protein through the rER membrane.Translocon/sec61→ channel protein through which the nascent polypeptide inserted 1. Free Ribosome in cytosol starts to translate mRNA with signal sequence2. Signal sequence is recognized by signal recognition particle (SRP) and binds to it and stops translation 3. SRP (with the mRNA and ribosome attached) binds to SRP receptor on ER membrane; then binds to translocon, where the signal sequence causes it to open and anchors protein to channel; the SRP and SRP receptor leave due to GTP hydrolysis and are recycled; translation continues and translated sequence begins to be
View Full Document
Unlocking...