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122 Cards in this Set
- Front
- Back
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Why do proteins need to be targeted?
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To establish and maintain the compartmentalized nature of a cell's diverse functions.
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Where does all protein synthesis occur?
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-All starts on free ribosomes in cytosol
-2 options for finishing: a)rough ER b)stay in cytosol |
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Why are proteins sent to the ER to complete synthesis?
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So that they can be secreted or stay in the ER -> but definitely don't want them in the cytosol.
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What 2 types of proteins are made in the ER?
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1. Transmembrane
2. Water-soluble |
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What are transmembrane proteins?
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Proteins only partially translocated across ER membrane; embedded in it.
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What are 2 examples of transmembrane proteins?
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LDL receptor, P450
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What are water-soluble proteins?
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Proteins fully translocated across ER membrane; release into its LUMEN.
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What are the 2 types of water-soluble proteins?
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1. Secreted
2. Lysosomal |
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What's one example of a secreted protein?
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Insulin
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What are the 3 targets of proteins completed in the cytosol?
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1. Mitochondria
2. Peroxisomes 3. Nucleus |
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What are the 4 targets of proteins completed in RER?
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1. Golgi
2. Lysosomes 3. Secretory vesicles 4. Cell surface |
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Are ribosomes on the ER different from free cytosolic?
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No
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Do ribosomes play a role in the targeting of proteins to RER vs. cytosol?
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No; all ribosomes come from the same pool.
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What is the name of the theory for how proteins are targeted to the rough ER?
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Signal recognition particle cycle - "Signal hypothesis".
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What are the 6 components of the Signal Hypothesis?
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1. SRP (the protein)
2. Signal sequence 3. SRP receptor 4. Ribosome receptor 5. Signal peptidase 6. Translocation process 7. Topography & stop-transfer sequences |
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What is the main gist of the Signal Hypothesis?
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SRP and the SRP receptor function catalytically to target nascent polypeptide chains to the ER membrane.
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What exactly is the SRP?
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Signal Recognition Particle - a translocation factor.
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What is the SRP made of?
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-One 7SL RNA molecule (300 nt)
-6 different polypep chains |
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Where does the SRP function?
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It cycles between ER membrane and cytosol
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What are the 2 important sites on the SRP?
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1. Signal recognition domain
2. Ribosome binding domain |
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What are the consequence of the 2 sites of specificity on SRP?
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-One binds a signal sequence
-One stops protein elongation |
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What exactly is the Signal Sequence?
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-A sequence ranging from 13-48 amino acids, no consensus.
-Usually at N-terminal -Tripartite domain structure |
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What happens to proteins with the Signal Sequence at their N-terminal?
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The sequence gets cleaved co-translationally.
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What is the tripartite domain structure of the signal sequence?
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-Nterminal is hydrophilic w/ a net positive charge.
-Core domain is hydrophobic with at least 7 residues and alpha helix -C-terminal is polar w/ 4-6 residues. |
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What exactly is the SRP receptor?
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An integral protein of the ER membrane; binds GTP/GDP.
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What happens when SRP binds the ribosome making protein?
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-It binds the signal sequence
-Arrests elongation |
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What happens when the SRP receptor binds the SRP-protein-ribosome complex?
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-Triggers GTP exchange -> GDP
-Hangs on to SRP tight -SRP releases Ribosome-protein |
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What is the consequence of SRP releasing the protein-ribosome complex?
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Elongation can once again occur.
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What happens after elongation arrest is lifted?
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GDP again replaces GTP and the SRP is returned to the pool.
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When released from SRP, what happens to the ribosome-protein complex?
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It is bound by a Ribosome Receptor to stabilize the complex on the ER membrane.
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What happens after the ribosome-protein complex is stable on its receptor?
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Signal peptidase cleaves the N-terminal sequence (in the lumen of the ER).
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Now that the ribosome-protein is sitting on ER membrane, how does it get inside?
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-Remains in unfolded state
-Translocates via aqueous pore using ATP hydrolysis. |
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When does protein folding occur?
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in the ER lumen.
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So what is SRP's function?
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To stop translation, preventing folding, so that the protein can get into the ER.
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What proteins get translocated all the way into the ER lumen?
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-Secreted H2O-soluble proteins (e.g., insulin or lysozomal enzymes).
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What other class of proteins are targeted to the ER?
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Transmembrane (integral)
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What allows transmembrane proteins to remain in the ER membrane?
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Stop-transfer sequences
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What are stop-transfer sequences?
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Hydrophobic, alpha-helical sequences that keep the protein associated with the ER membrane.
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If proteins are not tagged with a signal sequence, where do they go?
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Their ribosomes remain in the cytosol and target the proteins to the Nucleus, Mitochondria, or Peroxisomes.
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What are 4 functions of mitochondria?
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1. TCA cycle
2. Oxidative phosphorylation 3. B-oxidation of fatty acids 4. Ketone body production |
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What are the 4 different compartments of mitochondria?
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1. Outer mitochond. membrane
2. Inner mitochond. membrane 3. Intermembrane space 4. Mitochondrial matrix |
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What is the matrix?
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The innermost space.
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What is the name of the tag that allows proteins to get into the outer mitochondrial membrane?
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Mitochondrial entry sequence
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Where is the entry sequence usually located?
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At N-terminus of protein
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How big is the entry sequence typically?
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20-80 amino acids
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What is the structure of the mitochond. entry sequence?
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Amphipathic Alpha Helix - one side has pos charged AA's and one side as hydroxylated AA's.
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What recognizes the entry sequence tag?
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A receptor on the mitochondr. outer membrane.
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What is the preferred conformation of proteins for crossing the mitochondr. membrane?
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UNFOLDED
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How are proteins in structure when they arrive at mitochon?
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Folded
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So how do proteins get unfolded?
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Via HSP-70!!!
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What is HSP-70?
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A chaperone
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What are the key components involved in mitochondrial import?
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-Mitochondrial entry sequence
-Receptor -Electrochemical gradient across inner membrane -Processing peptidase |
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What is the function of the processing peptidase?
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To cleave the mitochondrial entry sequence of proteins bound for the inner membrane.
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Where is the processing peptidase located?
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in the MATRIX - not required for outer membrane proteins.
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What defines the nuclear compartment of a cell, and what does it consist of?
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The nuclear envelope:
-Inner membrane (contains proteins for interactn w/ chromosomes, nuclear RNA) -Outer membrane (continuous w/ rough ER) |
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What types of proteins function in the nucleus?
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-Histones
-DNA and RNA polymerases -RNA processing proteins |
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Where are all the nuclear proteins synthesized?
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In the cytosol
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So how is protein import mediated at the nucleus?
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Via nuclear pores
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What determines whether a protein can go through the nuclear pore or not?
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Size!
-Small proteins diffuse (histones) -Large proteins need a nuclear localization sequence. |
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What is the name of the tag used for import of large proteins into the nucleus?
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Nuclear localization sequence - NLS.
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Wait up, what is the outer membrane continuous with?
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ROUGH endoplasmic reticulum.
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What is the NLS like?
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-Rich in basic AA's - proline
-4-8 amino acids long -Located anywhere on protein -Not cleaved when done. |
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Why is the tag not cleaved when done?
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Because during mitosis the nuclear membrane breaks down; if the sequence were cleaved, the proteins would have to be re-synthesized because they wouldn't get taken up again.
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How does nuclear import mediated by NLS proceed?
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-NLS is recognized by Importin (a/b)
-Receptor in nuclear pore recognizes Importin (bound to protein) |
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What regulates nuclear pore translocation of protein-receptor complexes?
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RAN - a GTP/GDP binding protein.
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What transportation system is used for passage between ER, golgi, and lysosomes/cell surface/secretory vesicles?
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VESICULAR TRANSPORT.
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So what proteins require vesicular transport?
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-All trans-membrane proteins
-All secreted proteins -All proteins that are soluble and go to lysozome. |
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So what do we KNOW all of these proteins already have? Why?
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A signal sequence - because they had to go to the ER first to get INTO the secretory pathway.
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What is the order of vesicular transport from ER to the plasma membrane?
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Start: ER -> Cis golgi -> medial golgi -> trans Golgi -> trans golgi network -> final destination.
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Again, how does movement between the ER, Golgi and TGN occur?
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Via vesicular budding.
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What is the role of the TGN?
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This organelle SORTS the seretory granules and vesicles bombarding it from the ER and golgi apparatus, according to their intended functions.
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So what direction do proteins move through the golgi cisternae?
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Cis --> trans
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To what 3 sites does the TGN direct proteins being sorted?
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-Lysosomes
-Plasma membrane -Secretory granules |
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What directs the TGN's sorting?
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Signals on the proteins.
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What if proteins arrive at TGN without a signal?
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They are directed by default to the plasma membrane.
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What are the 2 pathways that cells can have for sorting proteins in the TGN?
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1. Constitutive pathway
2. Regulated secretory pathway |
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What is the constitutive pathway? What cells have it?
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-The pathway by which all soluble proteins are secreted, not stored.
-ALL animal cells have it. |
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What is the secretory pathway?
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Pathway for storing secretory proteins in granules for release upon regulated stimulation.
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What cells utilize the regulated secretory pathway?
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-Exocrine
-Endocrine -Neurons Those which use transmittors |
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What are lysosomes?
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Membranous bags of hydrolytic enzymes for digesting macromolcules in the cell.
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What hydrolytic enzymes are in lysosomes? What conditions are essential for activity?
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Acid hydrolases; require pH 5 within the lysosome.
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Where are lysosomal hydrolase and membrane proteins made?
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In the ER; transported through Golgi apparatus.
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How do lysosomes maintain the acidic pH of 5 within them?
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Via the action of PROTON PUMPS on their membrane.
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What is the targeting tag for lysosome-bound proteins?
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Mannose 6-phosphate
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How is the Mannose-6-phosphate tag generated for lysosomal enzymes?
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In a 2 step reaction via 2 different enzymes.
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What are the enzymes used for making lysosomal protein tags?
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-GlcNAc phosphotransferase
-GlcNAc phosphoglycosidase |
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What happens during the generation of mannose 6-PO4 tags?
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1. GlcNAc-phosphate transferred to enzyme's mannose residue.
2. GlcNAc cleaved to leave Mannose-6-phosphate. |
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What happens to proteins tagged with Mannose-6-phosphate?
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Recognized by specific receptors, transferred to a prelysosomal compartment.
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WHERE is the mannose-6-phosphate tag generated?
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In the golgi.
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So starting from precursor protein made in cytosol, what course would a hydrolytic lysosomal enzyme take?
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1. Start in cytosol
2. Signal sequence allows signal recognition to transfer protein to ER. 3. In the ER N-linked glycosylation occurs 4. Glycosylated protein is transferred to Golgi 5. Protein is tagged for lysosome in the Golgi, then sorted by TGN. |
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What does the sugar-6-phosphate receptor do?
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Takes the lysosomal enzyme from Golgi -> prelysosomal compartment.
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What happens to the receptor at the prelysosomal compartment?
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It dissociates due to acidic pH. Then recycles back to Golgi.
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What particular lysosomal storage disorder are we concerned with?
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I cell disease - the most severe one.
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What specific defect causes I cell disease?
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A lack of GlcNac phosphotransferase - the unusual glycosyltransferase
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What happens when patients lack enzyme 1 for lysosomal tagging?
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They can't make the mannose-6-phosphate tag, hence no lysosomal enzymes make it to their destination.
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What is the result of lack of lysosomes?
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Death within the first decade.
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What is the molecular basis for vesicular transport specificity?
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Vsnares and Tsnares
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What are Vsnares?
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Receptors on vesicles that are specific for receptors of the same nature on the target membrane.
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What allows for the recycling of Vsnares?
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NSF
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What 2 important diseases are associated with inhibition of SNARES?
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-Tetanus
-Botulism |
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What is the action of the Clostridium tetani and botulinum?
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Peptidase cleavage of SNARES from vesicle membranes
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What is the result of Botulinum and Tetani toxins?
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Botulism = paralysis because it inhibits ACh transfer.
Tetani = tetani b/c it inhibits ACh inhibitor transfer. |
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What exactly is receptor-mediated endocytosis (RME)?
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An efficient way for cells to take up molecules in low concentration, w/out taking up a large vol of ECF at the same time.
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What is the basic machinery necessary for RME?
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-Macromolecule receptor
-Clathrin-coated pit -Coated vesicle -Endosome |
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What is the process that occurs in RME?
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1. Macromolecule binds specific cell surf. receptor
2. Several of these complexes accumulate in Clathrin-coated pit. 3. Coated vesicle forms 4. Coat is taken off 5. Endosome fuses with vesicle 6. One of 4 possible pathways |
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What are the 4 possible pathways of RME?
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1. Receptor recycles + Ligand degrades
2. Receptor + Ligand recycle 3. Receptor + Ligand degrade 4. Receptor + Ligand transported |
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What is an example of Pathway #2 where ligand + receptor are recycled?
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Transferrin
Class I/II MHC |
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What is the major protein component of coated pits and vesicles?
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Clathrin
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What happens to Clathrin during the course of RME?
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-Provides site for receptor-ligand accumulation
-Once vesicle forms, clathrin coat leaves and returns to cell membrane. |
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What side of the cell membrane is Clathrin on?
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Internal - it doesn't directly contact the Receptor and/or ligand.
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How do receptors know where Clathrin is?
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Adaptor proteins allow for this interaction.
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What happens after endosome-vesicle fusion?
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The acidic pH causes dissociation of ligand from receptor.
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What establishes the acidic pH of endosomes?
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Special vacuolar ATPases -> proton pumps.
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What is a specific example of RME where both ligand adn receptor are recycled?
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Transferrin
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Once internalized, how does the uncoated vesicle know to fuse with the endosome?
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This is where SNARES come into play -> Vsnares and Tsnares and NSF.
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What regulates the binding/release of iron by transferrin?
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pH - at physiological acidity, Transferrin is actually Apotransferrin..
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What are some organisms that take advantage of RME?
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VIRUSES
-Influenza -Semlike -Diphtheria toxins -Cholera toxin |
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What are 2 diseases associated with abnormal protein targeting?
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-LDL receptor deficiency
-I cell disease |
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What results from LDL receptor deficiency?
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Familial hypercholesterolinemia - seen in 1:500 people
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What is deficient in Icell disease?
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Lysosomal enzymes.
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What is the fate of the accumulated lysosomal enzymes that don't make it to their destination?
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They get secreted by the default constitutive pathway.
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What drug is associated with cleaving Tsnares and Vsnares?
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Botox --> small amts of botulism toxin cleaves T/Vsnares and facilitates paralysis to reduce wrinkles.
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