BMB 462 Lecture 36 Outline of Last Lecture I. Continuing the 3 steps of elongationII. Termination of translationIII. Translation factors used by the cellIV. Coupling transcription and translationV. Chaperones and posttranslational modificationsVI. Inhibitors of translationVII. Translocating proteins across a membraneOutline of Current Lecture I. Role of glycosylation in protein targetingII. Targeting for secretion, membrane integration, or degradationIII. Targeting to mitochondria and chloroplastsIV. Targeting to nucleusV. Basic principles of nuclear transportVI. Amino-terminal signal sequences for targetingVII. Protein degradationVIII. Ubiquitin and Proteasomes in degradationCurrent LectureConcepts to remembers from previous courses/lectures:- The order of protein translocation across the membrane.o The signal sequence on the emerging peptide is recognized by the SRP.o The SRP brings the translating ribosome to the SRP receptor on the ER membrane.o The peptide translocates across the ER membrane.o The signal sequence is cleaved by peptidase in the ER lumen.- Translocation happens while the protein is still stuck to the ribosome, so it is not fully folded yet. This makes it easier for it to be threaded through the peptide translocation complex. Then the peptide is cleaved and folded inside the ER lumen.- For membrane proteins, a latter opening in the translocation complex allows the membrane sections to incorporate into the ER membrane.These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.o As will be discussed, the ER then buds of a vesicle, which incorporates into the plasma membrane so that the transmembrane protein can be part of the plasma membrane.I. Role of glycosylation in protein targetinga. Glycosylation occurs in the ER and the Golgi. All membrane proteins and secretedproteins are glycosylated.i. N-linked glycosylation occurs on an asparagine residue. This type of glycosylation is only done in the ER.1. The sugar is attached to the protein while it is still attached to the ribosome. They are built as precursors on the cytosolic side of the ER membrane, and then transferred into the ER to be attached to the protein.a. The sugars are attached to a Dolichol-phosphate moiety, which is inserted in the membrane to provide an anchor.b. Once the polysaccharide structure is complete, it is translocated to the luminal side of the membrane.c. Next, more sugars are attached to the polysachharide, and then the core of the polysaccharide is transfered to an asparagine in the polypeptide. 2. The glycosylated polypeptide is then released from the ribosome into the lumen, and the dolichol-phosphate is recycled and translocated back to the luminal side.a. The polypeptide can then be moved to the Golgi for further N-linked glycosylation, though primarily the golgi performs O-linked glycosylation. II. Targeting for secretion, membrane integration, or degradationa. After processing, the polypeptides can be incorporated into the membrane, secreted, or transferred to a lysosome for degradation.i. The clathrin coat of the vesicles determines which direction they will travel with the proteins.b. N-acetylglucosamine phosphotransferase uses UDP N-acetylglucosamine to transfer a phosphate to a mannose residue on an enzyme in order to target it to the lysosome.c. Defects caused by an inability to target proteins.i. I-cell disease: results in inclusion of carbohydrates and glycolipids in lysosomes due to lack of hydrolytic enzymes.III. Targeting to mitochondria and chloroplastsa. The key difference in transport to mitochondria/chloroplasts is that targeting of the protein occurs post-translation (after the protein has been released from the ribosome).i. Those proteins targeted for mitochondria and chloroplasts are bound by cytosolic chaperones post-translation, which prevent them from folding into their final 3D state.1. The protein is then brought to a membrane receptor and translocated by fully unfolding it and threading it through a channel in the membrane.2. The signal sequence is cleaved off, then organelle chaperones helpfold the protein into its final form.IV. Targeting to nucleusa. For a protein to be targeted to the nucleus, it requires a signal sequence called a Nuclear Localization Signal (NLS).i. Unlike other targeting sequences, this does not need to be on the N-terminus; it can be located almost anywhere on the protein.1. It also doesn't get cleaved off of the protein but remains in the mature structure.b. The protein also does not have to be unfolded for translocation into the nucleus; the nuclear pores are large enough for the protein to be transported through as is.i. The nuclear pores are constructed of many different proteins, including nuclear porins, which form a basket structure.1. Proteins of approximately 40kD can passively diffuse in and out. Anything larger than that has to be targeted.c. The nuclear pore complex has to import and export a variety of things.i. It imports ribosomal proteins, polymerases, histones, etc.ii. It exports mRNA, tRNA, and immature 40S and 60S ribosomal subunits.V. Basic principles of nuclear transporta. Classic NLSs are typically from pieces of regular amino acids, and there are usually 2 that are spaced by about 10 amino acids.i. Thus, these signals are called bipartite NLS.1. i.e. KK---10a.a.---KKRKb. It was discovered there is a family of karyopherin-BETA proteins that are either importins or exportins.i. As importins, they only bind their cargo in the absence of RanGTP.1. Ran is a small GTPase that regulates transport.ii. The exportins require RanGTP to bind their cargo.iii. There is high RanGTP in the nucleus and low RanGTP concentrations in the cytoplasm.c. Importin-β was the founding member of the family of karyopherin-β.i. It binds its cargo in the cytosol, and then transports it through the nuclearpore channel to release the cargo in the nucleus.1. In this case, the importin doesn't bind the NLS directly but uses the adaptor protein, importin-α.a. The importin-ALPHA is responsible for recognizing the NLS.2. RanGTP mediates release.ii. All other importins found later do not require an adaptor protein but instead bind the NLS directly; they bind to other NLS though, not the classic one.d. An exportin binds its cargo only in the nucleus of the cell, when it is in the presence of RanGTP. Then RanGTP is hydrolyzed in the cytosol,
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