Intracellular Compartments and Protein Sorting Flashcards
Recognize and name the membraneous organelles in eukaryotic cells
1) Nucleus
2) ER (rough and smooth)
3) Golgi
4) Endosome
5) Lysosomes
6) Peroxisomes
7) Mitochondria
8) Secretory Vessicles
Nucleus
controls cell activities
Cell Membrane
- support
- protection
- controls movement of materials in/out of cell
- barrier between cell and its environment
- maintains homeostasis
Nuclear membrane
*Controls movement of materials in/out of nucleus
Cytoplasm
*supports /protects cell organelles
ER
The ER has a central role in lipid and protein biosynthesis. Its membrane is the site of production of all the transmembrane proteins and lipids for most of the cell’s organelles, including the ER itself, the Golgi apparatus, lysosomes, endosomes, secretory vesicles, and the plasma membrane. The ER membrane makes a major contribution to mitochondrial and peroxisomal membranes by producing most of their lipids. In addition, almost all of the proteins that will be secreted to the cell exterior—plus those destined for the lumen of the ER, Golgi apparatus, or lysosomes—are initially delivered to the ER lumen.
- *Functions of the ER:
1) It is the birthplace of membrane proteins.
2) It is the site of lipid biogenesis lipids and cholesterol start in the ER and from there flow to other parts of the cell
3) It is a special environment with respect to its lumen because it is very high in calcium and thus a store house for calcium
4) It is a place where there are enzymes that are involved in lipid and hydrophobic toxin detoxification are present This is very prevalent in the liver
5) It is also this minting environment for making new proteins. Half of the proteins are manufactured into the lumen into the ER
Ribosome
*produces proteins
Mitochondria
Breaks down sugar molecules into energy, they do most of the oxidation but peroxisomes do some too!
There are about 200 mitochondria per average cell. Consider the mitochondrion to be prokaryotic endosymbiont. The inner convoluted membrane can be considered as the membrane of the endosymbiont. The outer membrane could be considered as the vestiges of the membrane that originally engulfed the symbiont. Thus there are four compartments, matrix, inner membrane, inter-membrane and outer membrane.
Mitochondria have their own DNA genome and make some of their own proteins. But two billion years of symbiotic relationships have created interdependencies, such that many mitochondrial proteins are encoded by the nuclear genome. Hence the evolution of systems for getting proteins from cytosol into mitochondria.
Vacuole
*store food, water, waste (plants need to store large amounts of food)
Lysosome
- breaks down larger food molecules into smaller molecules
* digests old cell parts
Peroxisomes
Peroxisomes originally were defined as organelles that carry out oxidation reactions leading to the production of hydrogen peroxide. Because hydrogen peroxide is harmful to the cell, peroxisomes also contain the enzyme catalase, which decomposes hydrogen peroxide either by converting it to water or by using it to oxidize another organic compound. A variety of substrates are broken down by such oxidative reactions in peroxisomes, including uric acid, amino acids, and fatty acids. The oxidation of fatty acids (Figure 10.25) is a particularly important example, since it provides a major source of metabolic energy. In animal cells, fatty acids are oxidized in both peroxisomes and mitochondria, but in yeasts and plants fatty acid oxidation is restricted to peroxisomes.
Functions of Peroxisomes:
1) Oxidation of very long chain fatty acids
2) Oxidation of branched chain fatty acids
3) Oxidation of cholesterol to bile acids (liver)
3) Synthesis of plasmalogens (membrane lipids found in myelin)
4) Oxidation (detoxification) of some metabolic intermediates and foreign substances for elimination (flavoprotein oxidases and catalase)
5) Decomposition of hydrogen peroxide
Compare a signal peptide to a signal patch
Signal peptides are one sequence where the signal patches are distant to one another but come close to each other once the protein has folded.
Recognize features of signal peptides that direct proteins to specific compartments
1) ER signals have a hydrophobic center.
2) Mitochondrial signals are amphipathic alpha helices
3) Nuclear import signals are very basic
4) Perosixomal signals are SKL-COOH (serine, lysine, leucine)
5) Nuclear export signals are leucine-rich
ER signals
ER signals have a hydrophobic center.
Mitochondrial signals
Mitochondrial signals are N-terminal amphipathic alpha helices
Nuclear import signals
Nuclear import signals are very basic
Perosixomal signals
Perosixomal signals are SKL-COOH (serine, lysine, leucine)
Nuclear export signals
Nuclear export signals are leucine-rich
Identify specific organelles that import proteins from the cytosol after synthesis of the protein is completed (post-translationally)
1) Nucleus- Uses Ran-GAP (dephosphorylates to get Ran-GDP in cytosol), Ran-GEF (switches of GDP for GTP to get Ran-GTP in nucleus), Ran-GDP, and Ran-GTP. There is also a nuclear import or export receptor that binds to Ran-GTP or GDP.
2) Mitochondria- Uses the TOM and TIM23 complexes to feed unfolded protein into mitochondria. Also uses the help of cytosolic Hsp70 to keep it unfolded and mitochondrial Hsp70 and Hsp60 to refold protein. The N-terminus amphipathic alpha helix is the
3) Peroxisomes
ER IS CO-TRANSLATIONALLY
Identify nuclear localization signals and their importance in protein import in the nucleus.
Nuclear export signals are leucine-rich. It MUST contain a stretch of Leucines (Leu or L). If one is mutated, it will remain in the cytosol. These are important to bring the necessary proteins into the nucleus. Without these signal, the proteins would have no way of entering.
Compare and contrast protein transport into (and out of) the nucleus with transport into other organelles, with regard to conformation of protein, the dimensions of the pore, the requirement for energy, and the subsequent fate of the import or export signal
1) Nucleus- Uses Ran-GAP (dephosphorylates to get Ran-GDP in cytosol), Ran-GEF (switches of GDP for GTP to get Ran-GTP in nucleus), Ran-GDP, and Ran-GTP. There is also a nuclear import or export receptor that binds to Ran-GTP or GDP. Here the fully folded protein is transported into the nucleus and thus the nuclear pore is much larger. The nuclear localization signals STAY ON because the nucleus breaks down and reforms after cell division or mitosis. They need to know where to go again. This way the cell is not having to remake protein! This requires GTP to be moved.
2) Mitochondria- Uses the TOM and TIM23 complexes to feed unfolded protein into mitochondria. Also uses the help of cytosolic Hsp70 to keep it unfolded and mitochondrial Hsp70 and Hsp60 to refold protein. The N-terminus amphipathic alpha helix is the signal. It is fed into the mitochondria BEFORE folding using Hsp70 to assist this. There is a requirement for ATP TWICE, once at TOM and once at TIM23 because the Hsp70 are ATPases that require ATP to ratchet it in. The signal peptide is cleaved because the protein will remain there! Smaller pore.
3) Peroxisomes- They acquire some proteins via budding so they are fully folded proteins. Other proteins are also introduced post-translationally when folded. Larger pore.
4) ER- Proteins are inserted CO-TRANSATIONALLY using a SRP (signal recognition particle) that binds to a receptor on the ER. The pore is thus relatively smaller to fit a chain into it. This process using the Sec61 translocon. It is not using ATP, rather the ribosome is pushing it in.
Describe the roles of Ran, the accessory proteins Ran-GEF and Ran-GAP, and import and export receptors in regulated transport through the nuclear pore
Ran-GDP is in excess in the cytosol and Ran-GTP is in excess in the nucleus. When a protein binds to a nuclear import receptor, it will be taken into the nucleus. In order to dissociate from the nuclear import receptor a Ran-GTP needs to bind. This causes the protein to be dissociated and then the Ran-GTP bound nuclear import protein will be exported. Once it gets into the cytosol, Ran-GTP will dissociate from the nuclear import protein. The Ran-GTP will then quickly encounter at Ran-GAP in the cytosol will then convert the Ran-GTP to Ran-GDP.
When a protein needs to to be exported from the nucleus, it will encounter a nuclear export receptor that binds to its signal. It will also bind Ran-GTP. It will then be exported from the nucleus and then when the Ran-GTP is dephosphoylated via a Ran-GAP, the protein dissociates.\
Ran-GEF is essential in reestablishing Ran-GTP from Ran-GDP that is produced in these two steps but exchanging a GDP for GTP
Explain how the regulation of the transport of transcription factors through the nuclear pore can lead to changes in the expression of specific genes
If the transcription factors are being shuttled in with a nuclear import signal, then there will be an increased in transcription. However, if the transcription factors have a nuclear export signal that develops, they will be signaled for export and gene expression will thus be decreased.
Identify the components of the mitochondrial import machinery and their functions. Compare the functions of chaperone proteins (heat shock protein families, Hsp60 and Hsp70), ATP and electrochemical energy in mitochondrial protein import.
Uses the TOM and TIM23 complexes to feed unfolded protein into mitochondria. Also uses the help of cytosolic Hsp70 to keep it unfolded and mitochondrial Hsp70 and Hsp60 to refold protein. The N-terminus amphipathic alpha helix is the signal. It is fed into the mitochondria BEFORE folding using Hsp70 to assist this. There is a requirement for ATP TWICE, once at TOM and once at TIM23 because the Hsp70 are ATPases that require ATP to ratchet it in. The signal peptide is cleaved because the protein will remain there! Smaller pore.
So, an UNFOLDED peptide with a signal sequence associates with TOM and Hsp70 chaperones. ATP is required to ratchet it into the intermembrane space. Then it goes to TIM23 and mitochondrial Hsp70 on the INSIDE of the mitochondria help pull it through once it gets going via electrophoretic processes (inside is negative and the N-terminus of peptide is + so it wants to go in). It requires ATP because the Hsp70s are ATPases. There are also Hsp60s that are in the mitochondria and take in the protein in a barrel like structure to help it fold in the mitochondria like Hsp70.
Identify 5 important functions of the peroxisomes in mammalian cells
Functions of Peroxisomes:
1) Oxidation of very long chain fatty acids
2) Oxidation of branched chain fatty acids
3) Oxidation of cholesterol to bile acids (liver)
3) Synthesis of plasmalogens (membrane lipids found in myelin)
4) Oxidation (detoxification) of some metabolic intermediates and foreign substances for elimination (flavoprotein oxidases and catalase)
5) Decomposition of hydrogen peroxide
Compare Peroxisomes and mitochondria with respect to their structure, biogenesis, and the way in which they carry out the oxidation of substances
Peroxisomes are created via the budding of them off the ER. Mitochondria replicate themselves on their own but are thought to have originated via the endosymbiosis theory. Mitochondria also have two membranes, an inner and an outer where the peroxisomes only have one membrane. Peroxisomes DO NOT look like mitochondria, they lack the inner cristae and have a dense core. Finally, mitochondria are crucial for utilizing oxidation of sugars to create ATP where the peroxisomes undergo oxidation for detoxification reasons.
Identify the specific functions of the rough ER, smooth ER and transitional ER
Rough ER: This is the site of protein synthesis for proteins that will be exported or membrane proteins. It also glycosylates proteins or marks them.
Smooth ER: It is the site of lipid biogenesis. This is the site of Calcium release and reuptake. It is also where lipid-solube drugs and poisons are detoxified.
Transitional ER: It is very similar to the smooth ER. It is the site where the vesicular transport occurs via budding off.
Identify the major types and ultimate destinations of proteins that are inserted into the ER
The two major times of proteins that are in the ER are free or soluble proteins and membrane-bound proteins. The soluble proteins will ultimately be destined to be escorted to the outside of the cell or extracellular space via vesicular transport. The membrane-bound proteins will become a part of the cell membrane.
Identify and describe the feature of proteins that directs them to the rough ER
The feature that directs proteins to the rough ER is their 20-30 amino acid signal peptide on the N-terminus. The protein starts to be synthesized on a free ribosome. Then, when this signal becomes present, A SRP (signal recognition particle) will bind to the signal and bring it to the rough ER by associated with an SRP receptor and binding. This brings the whole ribosome and mRNA complex to the rough ER. The ribosome is places above Sec61 (translocon) and “pushes” the protein into the ER via translational force.
Explain how single pass membrane proteins can have either their amino terminal (type I) or carboxyl terminal (type II) directed toward the lumen of the ER.
This is because the start-transfer sequence can be in any orientation. If the start-transfer sequence is on the N-terminus, it will place the N-terminus in the ER lumen. But if the start transfer sequence is in the middle of the protein, then it could go either way! It depends on the charge of the amino acid on the ends of the hydrophobic span. The positive amino acid will be on the cytosol side and the negative amino acid on the Lumen side. So if the + is on the N-terminus side, then the N-terminus will be in the cytosol and the C-terminus in the lumen!
Describe how multi-pass membrane proteins are inserted into the ER membrane
These are inserted because the protein will have two consecutive start translocation sequences and the followed by a stop transfer sequence. This will cause it to be embedded twice. But multiple pairs of start/stop transfer sequences can be added to get more and more “stitching”
Describe in general terms the attachment of of the mannose-rich oligosaccharide units to asparagine residues in N-linked glycoproteins.
A very important cotranslational modification is N-glycosylation. Here, dolichol lipids are substrates for the buildup of a large 14-sugar carbohydrate. An enzyme, oliogosaccharyltransferase, transfers the sugar to asparagine (Asn) residues that are in the N-X-T/S context. This “marks” the protein with a big carbohydrate. This happens in the ER.
Identify the specific lipid that participates in the assembly and transfer of the high mannose-oligosaccharide chain in N-linked glycoproteins
dolichol lipids
List 3 possible functions for protein glycosylation
1) They will protect the protein once it is fully folded because it forms a “sugar cloud” around the protein
2) A lot of these proteins will end up on the surface of epithelial cells in the lungs for example. These sugars help to trap water and keep it moist.
3) The sugars themselves are signals for the proper folding of the protein into its native state. The ER has lots of proteins fluxing in its lumen and this is important to have it fold correctly.
Describe the systems that participate in the unfolded protein response, which acts to limit the accumulation of unfolded proteins in the ER, block their transport to the Golgi, and promote their removal and degradation
Protein folding is challenging. Membrane proteins have complex multidomain structures with many disulfide bonds, glycans, various other posttranslational modifications. The process is slow and the pathway to the native folded protein state is an arduous one. There are many “off the track” pathways. Proteins that go off track have to be eliminated. This takes place via communication with the glycans, and ends with the improperly folded proteins being retrotranslocated back into the cytosol and degraded by proteasomes.
But this is only part of the story. The ER is a dynamic organelle and is responsive to the buildup of misfolded proteins in its lumen. These buildups stimulate the Unfolded protein response, or UPR. The final three slides illustrate the UPR.
1) Even though the N-glycans help nascent ER proteins to fold up properly, and even though there are many chaperones in the ER such as calenexin, Hsp70 / BiP, and protein disulfide isomerase, the ER sometimes becomes overwhelmed with unfolded proteins. What happens when the ER becomes overloaded? The cell will respond. When proteins become very abundant in the ER, they might fail to fold properly and therefore will aggregate (1). The unfolded proteins can be recognized by sensors (purple), which will dimerize and form a ribonuclease (2). The nuclease splices some RNA out of a pre-mRNA (3), which makes a new mRNA (4) that encodes a gene regulatory protein (5). The regulatory protein enters the nucleus (6) and drives transcription of ER chaperone genes (7) which are translated into the ER and resupply the ER with enough potential to get the proteins properly folded. This is a beautiful feedback loop and when it works, the cell can return to homeostasis and can keep doing its job, producing transmembrane and secreted proteins. When feedback loops of this sort are not enough, the cell suffers and eventually will die by apoptosis. A classic example of this process in human disease is type II diabetes, where the call for insulin production in islet cells can be so constant that the unfolded protein responses cannot hold up. The islet cells die by apoptosis and the resultant insulin deficiency causes diabetes.
2) There is an analogous sort of feedback from ER / Golgi to nucleus and then back to ER in controlling cholesterol levels. Cholesterol is made in the ER, and ER enzymes involved in cholesterol biosynthesis are under the control of sensors that reside in the ER and Golgi, sensing cholesterol levels and feeding back, via transcription factors, to the nucleus to regulate the expression of the sterol biosynthetic genes.
The detail points are that abundant cholesterol binds SCAP and holds SREBP in the ER. When cholesterol levels drop, SCAP and SREBP are allowed to transport out of ER to Golgi. In Golgi, a protease cuts SREBP and the released fragment goes to nucleus to activate cholesterol biosynthesis genes.
There are many patients with hyper cholesterolemia. New drugs on the horizon may block SREBP action at one or more of these stages, thus lowering cholesterol.
Describe the possible consequences to cells caused by a prolonged exposure to unfolded proteins in the ER
If the unfolded proteins continue to accumulate, they will eventually aggregate. This aggregation is detrimental to the cell and eventually leads to apoptosis. When the cells die, they no longer perform their function and thus leads to disease. Such an example is diabetes.
