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122 Cards in this Set

  • Front
  • Back
Why do proteins need to be targeted?
To establish and maintain the compartmentalized nature of a cell's diverse functions.
Where does all protein synthesis occur?
-All starts on free ribosomes in cytosol
-2 options for finishing:
a)rough ER
b)stay in cytosol
Why are proteins sent to the ER to complete synthesis?
So that they can be secreted or stay in the ER -> but definitely don't want them in the cytosol.
What 2 types of proteins are made in the ER?
1. Transmembrane
2. Water-soluble
What are transmembrane proteins?
Proteins only partially translocated across ER membrane; embedded in it.
What are 2 examples of transmembrane proteins?
LDL receptor, P450
What are water-soluble proteins?
Proteins fully translocated across ER membrane; release into its LUMEN.
What are the 2 types of water-soluble proteins?
1. Secreted
2. Lysosomal
What's one example of a secreted protein?
What are the 3 targets of proteins completed in the cytosol?
1. Mitochondria
2. Peroxisomes
3. Nucleus
What are the 4 targets of proteins completed in RER?
1. Golgi
2. Lysosomes
3. Secretory vesicles
4. Cell surface
Are ribosomes on the ER different from free cytosolic?
Do ribosomes play a role in the targeting of proteins to RER vs. cytosol?
No; all ribosomes come from the same pool.
What is the name of the theory for how proteins are targeted to the rough ER?
Signal recognition particle cycle - "Signal hypothesis".
What are the 6 components of the Signal Hypothesis?
1. SRP (the protein)
2. Signal sequence
3. SRP receptor
4. Ribosome receptor
5. Signal peptidase
6. Translocation process
7. Topography & stop-transfer sequences
What is the main gist of the Signal Hypothesis?
SRP and the SRP receptor function catalytically to target nascent polypeptide chains to the ER membrane.
What exactly is the SRP?
Signal Recognition Particle - a translocation factor.
What is the SRP made of?
-One 7SL RNA molecule (300 nt)
-6 different polypep chains
Where does the SRP function?
It cycles between ER membrane and cytosol
What are the 2 important sites on the SRP?
1. Signal recognition domain
2. Ribosome binding domain
What are the consequence of the 2 sites of specificity on SRP?
-One binds a signal sequence
-One stops protein elongation
What exactly is the Signal Sequence?
-A sequence ranging from 13-48 amino acids, no consensus.
-Usually at N-terminal
-Tripartite domain structure
What happens to proteins with the Signal Sequence at their N-terminal?
The sequence gets cleaved co-translationally.
What is the tripartite domain structure of the signal sequence?
-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.
What exactly is the SRP receptor?
An integral protein of the ER membrane; binds GTP/GDP.
What happens when SRP binds the ribosome making protein?
-It binds the signal sequence
-Arrests elongation
What happens when the SRP receptor binds the SRP-protein-ribosome complex?
-Triggers GTP exchange -> GDP
-Hangs on to SRP tight
-SRP releases Ribosome-protein
What is the consequence of SRP releasing the protein-ribosome complex?
Elongation can once again occur.
What happens after elongation arrest is lifted?
GDP again replaces GTP and the SRP is returned to the pool.
When released from SRP, what happens to the ribosome-protein complex?
It is bound by a Ribosome Receptor to stabilize the complex on the ER membrane.
What happens after the ribosome-protein complex is stable on its receptor?
Signal peptidase cleaves the N-terminal sequence (in the lumen of the ER).
Now that the ribosome-protein is sitting on ER membrane, how does it get inside?
-Remains in unfolded state
-Translocates via aqueous pore using ATP hydrolysis.
When does protein folding occur?
in the ER lumen.
So what is SRP's function?
To stop translation, preventing folding, so that the protein can get into the ER.
What proteins get translocated all the way into the ER lumen?
-Secreted H2O-soluble proteins (e.g., insulin or lysozomal enzymes).
What other class of proteins are targeted to the ER?
Transmembrane (integral)
What allows transmembrane proteins to remain in the ER membrane?
Stop-transfer sequences
What are stop-transfer sequences?
Hydrophobic, alpha-helical sequences that keep the protein associated with the ER membrane.
If proteins are not tagged with a signal sequence, where do they go?
Their ribosomes remain in the cytosol and target the proteins to the Nucleus, Mitochondria, or Peroxisomes.
What are 4 functions of mitochondria?
1. TCA cycle
2. Oxidative phosphorylation
3. B-oxidation of fatty acids
4. Ketone body production
What are the 4 different compartments of mitochondria?
1. Outer mitochond. membrane
2. Inner mitochond. membrane
3. Intermembrane space
4. Mitochondrial matrix
What is the matrix?
The innermost space.
What is the name of the tag that allows proteins to get into the outer mitochondrial membrane?
Mitochondrial entry sequence
Where is the entry sequence usually located?
At N-terminus of protein
How big is the entry sequence typically?
20-80 amino acids
What is the structure of the mitochond. entry sequence?
Amphipathic Alpha Helix - one side has pos charged AA's and one side as hydroxylated AA's.
What recognizes the entry sequence tag?
A receptor on the mitochondr. outer membrane.
What is the preferred conformation of proteins for crossing the mitochondr. membrane?
How are proteins in structure when they arrive at mitochon?
So how do proteins get unfolded?
Via HSP-70!!!
What is HSP-70?
A chaperone
What are the key components involved in mitochondrial import?
-Mitochondrial entry sequence
-Electrochemical gradient across inner membrane
-Processing peptidase
What is the function of the processing peptidase?
To cleave the mitochondrial entry sequence of proteins bound for the inner membrane.
Where is the processing peptidase located?
in the MATRIX - not required for outer membrane proteins.
What defines the nuclear compartment of a cell, and what does it consist of?
The nuclear envelope:
-Inner membrane (contains proteins for interactn w/ chromosomes, nuclear RNA)
-Outer membrane (continuous w/ rough ER)
What types of proteins function in the nucleus?
-DNA and RNA polymerases
-RNA processing proteins
Where are all the nuclear proteins synthesized?
In the cytosol
So how is protein import mediated at the nucleus?
Via nuclear pores
What determines whether a protein can go through the nuclear pore or not?
-Small proteins diffuse (histones)
-Large proteins need a nuclear localization sequence.
What is the name of the tag used for import of large proteins into the nucleus?
Nuclear localization sequence - NLS.
Wait up, what is the outer membrane continuous with?
ROUGH endoplasmic reticulum.
What is the NLS like?
-Rich in basic AA's - proline
-4-8 amino acids long
-Located anywhere on protein
-Not cleaved when done.
Why is the tag not cleaved when done?
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.
How does nuclear import mediated by NLS proceed?
-NLS is recognized by Importin (a/b)
-Receptor in nuclear pore recognizes Importin (bound to protein)
What regulates nuclear pore translocation of protein-receptor complexes?
RAN - a GTP/GDP binding protein.
What transportation system is used for passage between ER, golgi, and lysosomes/cell surface/secretory vesicles?
So what proteins require vesicular transport?
-All trans-membrane proteins
-All secreted proteins
-All proteins that are soluble and go to lysozome.
So what do we KNOW all of these proteins already have? Why?
A signal sequence - because they had to go to the ER first to get INTO the secretory pathway.
What is the order of vesicular transport from ER to the plasma membrane?
Start: ER -> Cis golgi -> medial golgi -> trans Golgi -> trans golgi network -> final destination.
Again, how does movement between the ER, Golgi and TGN occur?
Via vesicular budding.
What is the role of the TGN?
This organelle SORTS the seretory granules and vesicles bombarding it from the ER and golgi apparatus, according to their intended functions.
So what direction do proteins move through the golgi cisternae?
Cis --> trans
To what 3 sites does the TGN direct proteins being sorted?
-Plasma membrane
-Secretory granules
What directs the TGN's sorting?
Signals on the proteins.
What if proteins arrive at TGN without a signal?
They are directed by default to the plasma membrane.
What are the 2 pathways that cells can have for sorting proteins in the TGN?
1. Constitutive pathway
2. Regulated secretory pathway
What is the constitutive pathway? What cells have it?
-The pathway by which all soluble proteins are secreted, not stored.
-ALL animal cells have it.
What is the secretory pathway?
Pathway for storing secretory proteins in granules for release upon regulated stimulation.
What cells utilize the regulated secretory pathway?
Those which use transmittors
What are lysosomes?
Membranous bags of hydrolytic enzymes for digesting macromolcules in the cell.
What hydrolytic enzymes are in lysosomes? What conditions are essential for activity?
Acid hydrolases; require pH 5 within the lysosome.
Where are lysosomal hydrolase and membrane proteins made?
In the ER; transported through Golgi apparatus.
How do lysosomes maintain the acidic pH of 5 within them?
Via the action of PROTON PUMPS on their membrane.
What is the targeting tag for lysosome-bound proteins?
Mannose 6-phosphate
How is the Mannose-6-phosphate tag generated for lysosomal enzymes?
In a 2 step reaction via 2 different enzymes.
What are the enzymes used for making lysosomal protein tags?
-GlcNAc phosphotransferase

-GlcNAc phosphoglycosidase
What happens during the generation of mannose 6-PO4 tags?
1. GlcNAc-phosphate transferred to enzyme's mannose residue.
2. GlcNAc cleaved to leave Mannose-6-phosphate.
What happens to proteins tagged with Mannose-6-phosphate?
Recognized by specific receptors, transferred to a prelysosomal compartment.
WHERE is the mannose-6-phosphate tag generated?
In the golgi.
So starting from precursor protein made in cytosol, what course would a hydrolytic lysosomal enzyme take?
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.
What does the sugar-6-phosphate receptor do?
Takes the lysosomal enzyme from Golgi -> prelysosomal compartment.
What happens to the receptor at the prelysosomal compartment?
It dissociates due to acidic pH. Then recycles back to Golgi.
What particular lysosomal storage disorder are we concerned with?
I cell disease - the most severe one.
What specific defect causes I cell disease?
A lack of GlcNac phosphotransferase - the unusual glycosyltransferase
What happens when patients lack enzyme 1 for lysosomal tagging?
They can't make the mannose-6-phosphate tag, hence no lysosomal enzymes make it to their destination.
What is the result of lack of lysosomes?
Death within the first decade.
What is the molecular basis for vesicular transport specificity?
Vsnares and Tsnares
What are Vsnares?
Receptors on vesicles that are specific for receptors of the same nature on the target membrane.
What allows for the recycling of Vsnares?
What 2 important diseases are associated with inhibition of SNARES?
What is the action of the Clostridium tetani and botulinum?
Peptidase cleavage of SNARES from vesicle membranes
What is the result of Botulinum and Tetani toxins?
Botulism = paralysis because it inhibits ACh transfer.
Tetani = tetani b/c it inhibits ACh inhibitor transfer.
What exactly is receptor-mediated endocytosis (RME)?
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.
What is the basic machinery necessary for RME?
-Macromolecule receptor
-Clathrin-coated pit
-Coated vesicle
What is the process that occurs in RME?
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
What are the 4 possible pathways of RME?
1. Receptor recycles + Ligand degrades
2. Receptor + Ligand recycle
3. Receptor + Ligand degrade
4. Receptor + Ligand transported
What is an example of Pathway #2 where ligand + receptor are recycled?
Class I/II MHC
What is the major protein component of coated pits and vesicles?
What happens to Clathrin during the course of RME?
-Provides site for receptor-ligand accumulation
-Once vesicle forms, clathrin coat leaves and returns to cell membrane.
What side of the cell membrane is Clathrin on?
Internal - it doesn't directly contact the Receptor and/or ligand.
How do receptors know where Clathrin is?
Adaptor proteins allow for this interaction.
What happens after endosome-vesicle fusion?
The acidic pH causes dissociation of ligand from receptor.
What establishes the acidic pH of endosomes?
Special vacuolar ATPases -> proton pumps.
What is a specific example of RME where both ligand adn receptor are recycled?
Once internalized, how does the uncoated vesicle know to fuse with the endosome?
This is where SNARES come into play -> Vsnares and Tsnares and NSF.
What regulates the binding/release of iron by transferrin?
pH - at physiological acidity, Transferrin is actually Apotransferrin..
What are some organisms that take advantage of RME?
-Diphtheria toxins
-Cholera toxin
What are 2 diseases associated with abnormal protein targeting?
-LDL receptor deficiency
-I cell disease
What results from LDL receptor deficiency?
Familial hypercholesterolinemia - seen in 1:500 people
What is deficient in Icell disease?
Lysosomal enzymes.
What is the fate of the accumulated lysosomal enzymes that don't make it to their destination?
They get secreted by the default constitutive pathway.
What drug is associated with cleaving Tsnares and Vsnares?
Botox --> small amts of botulism toxin cleaves T/Vsnares and facilitates paralysis to reduce wrinkles.