Cell and Molecular Biology Lecture 13 PDF

Cell and Molecular Biology Lecture 13: INTRACELLULAR COMPARTMENTS & PROTEIN SORTING (part 2) CHAPTER 12th Molecular Biology of the Cell, 4th Ed., Alberts et al. Daniele Provenzano, Ph.D.  Recapitulation: 1. Gated transport:...

Cell and Molecular Biology Lecture 13: INTRACELLULAR COMPARTMENTS & PROTEIN SORTING (part 2) CHAPTER 12th Molecular Biology of the Cell, 4th Ed., Alberts et al. Daniele Provenzano, Ph.D.  Recapitulation: 1. Gated transport: - Transport across the nuclear pore Last complex. lecture 2. Transmembrane transport: - Translocation into the mitochondria Today’s - Translocation into peroxisomes lecture - Translocation into the ER 3. Vesicular transport - Proteins traveling from one Next compartment to the other within or on lecture the surface of vesicles. Roadmap to protein sorting Protein translocation into PEROXISOMES: Peroxisomes are “bags” containing high concentrations of oxidative enzymes (catalase and various oxidases). A major function of peroxisomes is to carry out β-oxidation (breakdown) of fatty acids. Peroxisomes are also the cellular compartment where plasmalogen is formed (the major component of neuronal myelin sheets). Peroxisomes are enveloped by a single lipid bilayer (in contrast to plastids and mitochondria). They don’t have their own genomes so all their proteins must be imported. Peroxisomes enzyme crystals Protein translocation into PEROXISOMES: Peroxisomes are believed to grow and multiply by fission depending on the cell’s needs (like mitochondria and plastids). Proteins with peroxisomal signal sequences are taken to the organelle. 23 proteins called peroxins are known to be involved in importing proteins into the peroxisome. The mechanism requires soluble cytosolic receptor proteins as well as docking proteins and energy in form of ATP hydrolysis. The precise mechanism of protein import into the peroxisome is not yet understood. Protein translocation into the ER: We will spend the remainder of this lecture addressing HOW PROTEINS ARE TRANSPORTED INTO THE ENDOPLASMIC RETICULUM The Endoplasmic Reticulum: The ENDOPLASMIC RETICULUM is composed of two portions: SMOOTH and ROUGH ER. The distinction is due to the fact that the ROUGH ER is studded with ribosomes conferring the rough appearance. The nuclear membrane is continuous with the rER that is continuous with the sER. rER sER cisternae  The rER and the sER can be separated: Sucrose-gradient centrifugation separates microsomes from the sER and the rER based on density:  The primary role of the sER is lipid metabolism sER functions: Acquisition, incorporation, processing and breakdown of lipids. Synthesis of LIPOPROTEINS. Sequestration (removal) of calcium from the cytosol and calcium storage. Rapid release of calcium ions from the ER lumen into the cytosol is very important to regulate, among other things, muscle contraction. Smooth ER = notice absence of ribosomes  The Endoplasmic Reticulum’s CISTERNAE occupy over 10% of the space of the cell while the ER membrane takes up ½ of all lipid bilayer of the cell. The ER mediates the selective transfer of molecules between the cytoplasm and the ERs lumen. The ER plays a central role in lipid and protein biosynthesis. The ER membrane is the site of production of all transmembrane proteins and lipids for most of the cell’s organelles (including the ER itself and the plasma membrane). Almost all of the proteins that will be secreted to the outside of the cell and those destined for the lumen of the ER, Golgi and lysosomes are initially delivered to the ER lumen. In other words, the ER functions as a gateway for synthesis of proteins that are going to become transmembrane proteins and those that will be secreted outside the cell as well as those that will be delivered to the inside of vesicles.  Ribosomes synthesize SOLUBLE and INTERGRAL MEMBRANE PROTEINS: While proteins are translocated (transferred) into other organelles after their synthesis is complete, proteins are translocated into the ER while they are being synthesized: Proteins that will become embedded in the cell membrane (and those that will be secreted) are translated directly into the rER by attached ribosomes  Ribosomes can cycle from cytosolic translation to ER membrane-bound translation: The signal sequence of the newly synthesized peptide directs the ribosome(s) to the ER: Simplified view of protein translocation across the ER: When the ER signal sequence emerges the ribosome is directed to a translocator on the ER membrane. The translocator forms a pore across the ER membrane through which the newly synthesized peptide is translocated. The signal peptide is cleaved by signal peptidases.  A Signal-Recognition Particle (SRP) directs the ER signal sequence to a specific receptor on the ER: The SRP consists of six peptides (protein subunits) and an RNA molecule (A). One end of the SRP has a signal-sequence binding pocket (B), the other end binds to the ribosome. When the SRP binds to the signal peptide of the emerging protein translation is paused just long enough to bind the ER membrane before synthesis is complete thereby ensuring that the peptide is not synthesized into the cytosol of the cell. The signal sequence and the signal recognition particle (SRP) bind an SRP receptor on the ER membrane: After the SRP binds the signal peptide translation pauses. The ribosome/SRP complex binds to the SRP receptor on the ER membrane. When the ribosome complex docks on the translocator pore, the SRP and the SRP receptors are released and translation resumes across the translocator. A ribosome bound to the Sec61 ER protein translocator: Translocators are hydrophilic pores formed by proteins. The translocator is PLUGGED on the luminal side to prevent escape of calcium and other ions from the ER. Translocators are dynamic, they open transiently when a ribosome with a growing peptide attach. The signal sequence of the growing peptide is believed to cause opening of the pore. The signal sequence of a peptide is Sec61 therefore recognized twice: by the SRP and the translocator Electron micrograph of a translocator itself. When ribosomes attach to translocators they form a tight seal: The seal that forms between the ribosome and the translocator opens up a space that is continuous from the interior of the ribosome with the ER lumen. In this experiment the light emitted by a fluorescent dye in the interior of the ribosome can only be turned off (quenched) by iodide ions (A). When the iodide ions are in the cytosol during translocation, the fluorescent dye emits light because the iodide ions can’t reach it (B). When the iodide ions are in the lumen of the ER, the fluorescent dye is quenched because the iodide ions travel across the translocator into the ribosome (C).  Three ways to drive protein translocation through similar translocators in different types of cells: (A) Co-translational translocators, as the one we have discussed before are used by all cell types. (B) Proteins can also be translocated into the ER post-translationally, much as they are into mitochondria. This requires additional accessory proteins plus energy in form of ATP hydrolysis. (C) Post-translational translocation in bacteria requires energy and the SecA protein.  ER translocation of soluble proteins: The signal sequence at the N-terminus of the protein is recognized and inserted into the translocator pointing towards the cytosol. The ER signal sequence acts as a “START TRANSFER” signal. The protein is synthesized N’ to C’ through the translocator. A signal peptidase cleaves the signal peptide during/after translation is complete. The translocator opens laterally allowing the signal peptide to diffuse in the ER membrane where it is rapidly degraded while the mature protein is released into the ER lumen Ribosome not shown for clarity  ER translocation of single-pass transmembrane proteins: The signal sequence at the N-terminus of the protein is recognized and inserted into the translocator pointing towards the cytosol. The ER signal sequence acts as a “START TRANSFER” signal and the protein is translocated as before When a “STOP-TRANSFER” sequence reaches the translocator, translocation stops but translation continues until the protein is completely synthesized. Next, the signal peptidase cleaves the N’-terminal signal peptide. The translocator opens laterally and the mature single-pass transmembrane protein is released into the ER membrane. Ribosome not shown for clarity  Orientation of the protein is determined by the distribution of charged amino acids within the signal sequence: In this example the orientation of the charges in an internal “start-transfer” ER signal sequence determine the topology of the protein within the membrane: A) When the signal sequence contains positively charged amino acids pointing towards the N-terminus of the peptide the protein will be oriented with its N-terminus in the cytosol. B) When the signal sequence contains positively charged amino acids pointing towards the C-terminus of the peptide the protein will be oriented with its C-terminus in the cytosol. Ribosomes not shown for clarity  Multipass transmembrane proteins are inserted into the membrane by sequential START and STOP transfer signals: The ribosome docks onto the translocator as soon as the START TRANSFER sequence is translated. The protein is inserted into the translocator in the orientation determined by the charged amino acids composing the START TRANSFER sequence. The protein is translocated across the translocator. When the STOP TRANSFER sequence is translated into translocator translocation stops, but translation continues until the protein is fully synthesized. The translocator opens laterally and releases the mature protein. Ribosome not shown for clarity  Multipass transmembrane proteins are inserted into the membrane by sequential START and STOP transfer signals: Internal (START & STOP) signal sequences are composed of stretches of hydrophobic amino acids that are also the transmembrane domains. Each set of “START TRANSFER” sequence followed by “STOP TRANSFER” sequence forms two transmembrane domains: (A) Hydrophobicity plot of a multipass transmembrane protein. (B) Schematic representation of the START and STOP transfer sequences along the same protein. (C) Final topology of the same protein within the membrane. Most proteins synthesized in the ER are glycosylated by the addition of a common N-linked sugar: One of the major function of the ER is to covalently attach carbohydrates to ER protein proteins. Most proteins translated and translocated into the ER are GLYCOPROTEINS (sugar protein hybrids). A preformed precursor oligosaccharide made up of 14 monosaccharides is attached to N-linkage the amino group of the asparagine side chain of the protein (hence the term oligosaccharide Preformed precursor N-linked). Attachment of the oligosaccharide to a given protein is catalyzed by oligosaccharyl transferase. Most proteins synthesized in the ER are glycosylated by the addition of a common N-linked sugar: N-linked glycosylation in the ER lumen takes place while the protein is translocated into the ER. The precursor oligosaccharide is held in the ER membrane by a lipid called DOLICHOL. The oligosaccharide is transferred from dolichol onto the growing polypetide chain onto (specifically) an asparagine OLIGOSACCHARYL amino acid by the enzyme TRANSFERASE OLIGOSACCHARYL TRANSFERASE. Ribosome not shown for clarity Proteins can be covalently attached to lipids in the ER lumen: Some proteins have an additional signal sequence in the C-terminus. These signal sequences indicate that a protein is destined to be covalently attached to a lipid tail. Glycosylphosphatidyl-inositol (GPI) is a common lipid anchor. As soon as the signal sequence is removed from the protein a GPI anchor is covalently attached.  Proteins that misfold in the ER are translocated back into the cytosol and destroyed: Misfolded proteins in the ER are carried by a chaperone to an ER protein translocator and moved into the cytosol of the cell with the help of additional proteins. An enzyme called N-glycanase de-glycosylates the protein. The protein is then ubiquitinated and targeted for degradation inside the proteasome.  Most membrane lipids are assembled in the ER: Most lipids in the cell are synthesized into the ER membrane, including cholesterol and phospholipids. Phosphatidylcholine is a major phospholipid synthesized in three steps from choline, two fatty acids and glycerol phosphate. (1) Acyl tranferase adds the two fatty acids to glycerol phosphate to produce phosphatidic acid. (2) A phosphatase removes the phosphate group yielding diacylglycerol (3) Choline phosphotranferase adds choline to yield phosphatidyl choline Other major phospholipids including phosphatidylethanolamine and diacylglycerol phosphatidyl serine are also synthesized this way. Note that the “new” phospholipid is inserted into the CYTOSOLIC leaflet of the ER membrane. Phospholipid translocators distribute lipids in the membranes of the cell: Because new lipid molecules are added only to the cytosolic leaflet of the ER membrane and lipid molecules do not readily flip from one leaflet to the other PHOSPHOLIPID TRANSLOCATORS are required to transfer lipids from the cytosolic leaflet to the ER leaflet. Phospholipid translocators of the ER that catalyze flipping of phospholipds are called SCRAMBLASES. Scramblases insure that the lipid bilayer remains symmetrical eventhough new lipids are added only to one of the two leaflets. Phospholipid translocators distribute lipids in the membranes of the cell: Because new lipid molecules are added to the cell membrane by exocytosis and lipid molecules do not readily flip from one leaflet to the other PHOSPHOLIPID TRANSLOCATORS are required to transfer lipids from one leaflet to the other. Phospholipid translocators of the cell membrane that catalyze flipping of phospholipds are called FLIPPASES. Flippases insure that the two leaflets of the lipid bilayer are composed of the appropriate phospholipids as discussed in lecture 10. Phospholipid exchange proteins move lipids from one membrane to the other: QUESTIONS ?