Module 3 Flashcards
(104 cards)
1
Q
What do targeting sequences do?
A
direct a protein to a different organelle
2
Q
Which proteins follow the secretory pathway?
A
- membrane proteins
- secreted proteins
- any protein transported to the ER or any protein made in the ER that is processed in and trafficked out of the Golgi
3
Q
What is the function of the ER?
A
the ER is the site of synthesis and folding of all proteins that enter the secretory pathway
4
Q
What is the function of the Golgi?
A
- remodeling of carbohydrate modifications of secretory proteins
- distributing proteins to their final destination at the plasma membrane or lysosome
5
Q
What is a lysosome?
A
an organelle with an acidic interior that is used for degradation of proteins and storage of small molecules such as amino acids
6
Q
What is the difference between the rough ER and the smooth ER?
A
- rough ER has ribosomes bound to it
- smooth ER has no ribosomes bound and is usually where vesicles bud before they have to move to the Golgi
- smooth ER is responsible for making lipids and hormones (typically steroid based)
- if cells secrete a lot of substances (such as hormones) they typically have more smooth ER
7
Q
How is the ER purified from cells in order to be studied?
A
- when a cell is homogenized, the membranes of the ER are disrupted but reseal to form small vesicles known as microsomes
- microsomes are authentic miniature versions of the ER and function the same (interior is biochemically equivalent)
- rough microsomes have ribosomes on the outside
8
Q
How can you separate rough microsomes from smooth microsomes?
A
- rough and smooth microsomes differ in density
- you can separate them using equilibrium density centrifugation (ex: sucrose gradient)
- rough microsomes are higher in density than smooth microsomes so they will be closer to the bottom of the tube
9
Q
How are secretory proteins directed to the ER?
A
- synthesis begins on cytosolic free ribosomes
- while synthesis is going on, the polyribosome (messenger RNA bound by several ribosomes) is directed to the ER through a signal sequence
- the sequence can be anywhere in the protein
- as synthesis continues, the signal sequence is cleaved off by signal peptidase in the ER
- called the signal hypothesis
10
Q
What experiment was done to support the signal hypothesis?
A
- took mRNA encoding secretory proteins and allowed in vitro translation to occur in the presence and absence of microsomes
- the products were placed on an SDS page gel
- in the absence of microsomes, the proteins were larger than predicted
- in the presence of microsomes, the proteins were the correct size
- this supported that the signal sequence was cleaved in the ER
11
Q
What experiment shows that translation and translocation occur simultaneously?
A
- take mRNA bound to ribosomes
- allow the synthesis of this protein in the absence of microsomes
- add microsomes after protein synthesis and look to see if the protein is in the microsome
- the result is that the protein will be outside of the microsome
- once synthesis is completed, the protein can not be inserted into the lumen of the microsome
- if you allow the synthesis of the protein to go on in the presence of the microsome you will find that the proteins are inside of the microsomes at the end
12
Q
How can you tell if a protein has been extruded into a microsome?
A
- if you are allowing the synthesis of a secretory protein in the presence of microsomes, you are assuming that it gets translocated in the lumen of the microsome
- you can use SDS page to look at the size of the proteins
- if the proteins have been inserted into the microsome, the signal sequence will have been cleaved by signal peptidase
- therefore, if the protein was inside the microsome, it will be smaller
- proteins in the microsome will also be resistant to proteases because they are protected by the membrane of the microsome
- in the presence of a detergent, the membrane is disrupted and the proteins inside can be degraded by proteases
13
Q
Which features of the signal peptide are conserved?
A
- 30-35 amino acids in length
- stretch of hydrophobic amino acids
- hydrophobic region is followed by a few negatively charged amino acids and one or more positively charged amino acids
14
Q
What is the process of a secretory protein being trafficked to the ER?
A
- Protein is being synthesized on a cytosolic free ribosome
- Signal sequence becomes exposed
- Signal sequence binds to SRP (a riboprotein)
- Binding to SRP temporarily halts translation until the polyribosome can be directed to the ER
- SRP interacts with the SRP receptor in the ER membrane
15
Q
What is the structure of SRP?
A
- very large riboprotein complex
- RNA associated with 5-6 other proteins
- P54 subunit of the protein is the signal recognition protein
16
Q
What happens after SRP binds to the SRP receptor?
A
- Ribosome translating the secretory protein becomes disassociated with SRP and its receptor becomes associated with the translocon (disassociation requires the hydrolysis of GTP)
- The translocon opens and synthesis is reinitiated
- Protein becomes extruded into the lumen of the ER
- The signal sequence is cleaved by signal peptidase found in the lumen of the ER
- End result is a mature soluble secreting protein
17
Q
What does Sec61a in yeast do?
A
- it is a translocon component
- contacts nascent secretory proteins as they pass through the translocon into the ER lumen
18
Q
What is translocation driven by?
A
- the translational elongation of the protein
- energy comes from the ATP used in translation to push the polypeptide through the translocon
- the translocon by itself does not need to use ATP/energy
19
Q
What is the structure of Sec61?
A
- has two gating mechanisms using an hourglass shaped channel through the center of the pore
- the first gating mechanism is a short helical plug that moves out of the way when for an incoming peptide (translocation)
- the second gating mechanism is a ring of isoleucine residues at the constricted waist of the pore that keeps the channel sealed to small molecules during translocation
20
Q
How is translocation of secretory proteins different in yeast?
A
- many secretory proteins are translocated into the ER after they are completely made in the cytosol (post-translational translocation)
- after the protein is synthesized, it is fed through a translocon in the ER
- may be some soluble secretory proteins in higher eukaryotes made this way (but rare)
- SRP and SRP receptor are not used
- the signal sequence of the protein becomes associated with the translocon and moves into the lumen of the ER
- to prevent protein backsliding, BiP (binding protein) in the lumen of the ER binds to the protein (requires the hydrolysis of ATP)
- Sec63 facilitates the binding of BiP to the protein
- once the protein is completely translocated into the ER lumen, BiP faciliates proper folding of the protein
- end result is a mature soluble secretory protein in the lumen of the ER
21
Q
Why is there no risk of back sliding in traditional secretory protein translocation?
A
the position of the ribosome prevents the protein from moving backwards out of the ER lumen
22
Q
What is the difference between a single pass membrane protein and a multiple pass membrane protein?
A
- single pass transmembrane proteins pass through the membrane once
- the majority of the protein is on one side of the membrane or the other or it can be equally distributed
- the part that passes through the membrane is hydrophobic and has an alpha helical structure
- multiple pass transmembrane proteins pass through the membrane pass through the membrane multiple times
- they can have their C terminus on the cytosolic side or their N terminus on the cytosolic side
- when they pass through the membrane an even number of times, the N and C terminus will be on the same side of the membrane
- when they pass through the membrane an odd number of times, the N and C terminus will be on opposite sides of the membrane
23
Q
What is a Type 1 transmembrane protein?
A
- has a small portion of their C terminus on the cytosolic side
- majority of the protein is found on the other side of the membrane (extracellular space, lumen of ER, lumen of Golgi)
- signal sequence is cleaved
24
Q
What is a Type 2 transmembrane protein?
A
- a small portion of the N terminus is on the cytosolic face
- majority of the protein is found on the other side of the membrane (extracellular space, lumen of ER, lumen of Golgi)
25
What is a Type 3 transmembrane protein?
- C terminus is on the cytosolic side
| - majority of the protein is found on the cytosolic side
26
What is a Type 4 transmembrane protein?
- aka tail anchored protein
- N terminus on the cytosolic side
- majority of the protein on the cytosolic side
- C terminus is inserted into the membrane
27
What is a Type 5 transmembrane protein?
- multiple pass membrane proteins
- form alpha helices in the membrane
- can be further divided into Type 5A and Type 5B
- Type 5A: N and C terminus on the same side (even number of passes)
- Type 5B: N and C terminus on opposite sides (odd number of passes)
28
What is a GPI anchored protein?
- N terminus on the exoplasmic side
| - anchored into the membrane via lipids
29
How are Type 1 transmembrane proteins synthesized and what is an example?
1. Synthesis begins on cytosolic free ribosome
2. When the signal sequence becomes exposed it is bound by the signal recognition particle (SRP) which guides the polyribosome to the ER membrane by its interaction with the SRP receptor
3. The protein enters the translocon
4. Synthesis proceeds and the N terminal signal sequence is cleaved by signal peptidase in the ER
5. As synthesis proceeds, a string of amino acids known as the stop transfer anchor sequence or anchoring segment/sequence of the protein is synthesized. This sequence represents the transmembrane region of the protein.
6. This sequence will form the alpha helical transmembrane portion of the protein
7. Transmembrane protein diffuses out of the translocon and synthesis is completed forming a Type 1 transmembrane protein
Example: insulin receptor
30
How are Type 2 transmembrane proteins synthesized and what is an example?
1. Synthesis begins on a cytosolic free ribosome but there is no N terminal signal sequence
2. When the signal anchor sequence is synthesized it is bound to an SRP that guides the polyribosome to the ER through its interaction with the SRP receptor
3. Protein is inserted into the translocon
4. A string of positively charged amino acids are oriented just to the N terminus of the signal anchor sequence. These amino acids regulate the mechanism by which the protein is inserted into the translocon. The positively charged amino acids are always on the cytsolic side.
5. The transmembrane region of the protein diffuses out of the translocon and synthesis is completed forming a Type 2 transmembrane protein
Example: transferrin receptor
31
How are Type 3 transmembrane proteins synthesized and what is an example?
1. Synthesis begins on a cytosolic free ribosome
2. It is unknown if SRP is involved, so by some unknown mechanism, the polyribosome becomes associated with the transolcon in the ER membrane
3. A string of positively charged amino acids line up on the cytosolic side to ensure that the protein is in the correct orientation (N terminus in the exoplasmic space and C terminus in the cytosolic space)
4. Transmembrane region diffuses out of the translocon and synthesis is completed leaving you with a Type 3 transmembrane protein
Example: Cytochrome P450
32
How are tail anchored transmembrane proteins synthesized and what is an example?
1. Synthesis of protein is completed in the cytosol
2. C-terminal sequence appears once translation is completed and the protein is released from the ribosome
3. A complex of Sgt2, Get4, and Get5 sequesters the protein's hydrophobic C-terminal anchor sequence and transfers it to the Get3-ATP complex
4. The Get3-ATP complex docks onto the ER membrane on the Get1-Get2 dimeric receptor
5. When bound to the receptor, ATP is hydrolyzed and Pi is released
6. The ATP hydrolysis releases the protein's C-terminal tail into the Get1-Get2 receptor which releases the anchor sequence into the ER membrane
7. The release of ADP and binding of ATP to Get3 releases Get3 from the Get1-Get2 complex
Example: SNARE proteins
33
How are Type 4 transmembrane proteins synthesized and what is an example?
1. Synthesis begins on a cytosolic free ribosome
2. There is no N terminal signal sequence, but there is an internal anchor signal sequence, which SRP binds to
3. SRP guides the polyribosome to the ER membrane and interacts with the SRP receptor
4. The first and second membrane regions are synthesized and found in the lumen of the translocon
5. The remainder of the transmembrane regions are synthesized in the cytosol, inserted into the translocon, and diffused out 2 at a time until the protein is completely synthesized. How the insertion happens is not well known, but it is hypothesized that there are cytosolic proteins involved because the transmembrane regions are very hydrophobic and would have unfavorable interactions with the aqueous cytosolic environment.
Example: GLUT1
34
How are GPI-anchored proteins synthesized and what is an example?
1. Synthesis begins just like a Type 1 transmembrane protein
2. There is usually an N-terminal signal sequence that is cleaved in the ER lumen
3. There is a string of amino acids close to the transmembrane region that is recognized by an enzyme known as GPI trans-amidase
4. GPI trans-amidase cleaves the protein at that position and makes a covalent attachment with that protein to a GPI anchor that is preformed in the membrane
5. End result is a GPI-anchored protein
Example: plasminogen activator receptor
35
What is the purpose of GPI-anchored proteins?
- more mobile
| - have an affinity for lipid rafts (small domains in the membrane that are cholesterol rich and sphingolipid rich)
36
What is the topogenic sequence of a Type 1 transmembrane protein?
NH3+ - signal sequence - lumen - STA - cytosol - COO-
37
What is the topogenic sequence for a Type 2 transmembrane protein?
NH3+ - cytosol (+++) - SA - lumen - COO-
38
What is the topogenic sequence for a Type 3 transmembrane protein?
NH3+ - lumen - SA - (+++) cytosol - COO-
39
What is the topogenic sequence for a Type 4A transmembrane protein?
NH3+ - cytosol (+++) - SA - lumen - STA - cytosol (+++) - SA - lumen - STA - cytosol - COO-
40
What is the topogenic sequence for a Type 4B transmembrane protein?
NH3+ - lumen - SA - (+++) cytosol (+++) - SA - lumen - STA - cytosol (+++) - SA - lumen - STA - cytosol - COO-
41
What is the difference between STA and SA?
- STA is a single internal stop-transfer anchor and is present in Type 1 proteins that have N-terminal signal sequences
- SA is a single internal signal-anchor whose orientation depends on the positively charged amino acids on the cytosolic side of the proteins
- SA is found in Type 2 and 3 transmembrane proteins
- Type 4A proteins have no N-terminal signal sequence, so they have alternating Type 2 SA and STA sequences and the positively charged amino acids are on the N-terminal side of the SA
- Type 4B proteins also have no N-terminal signal sequence, so they have an SA sequence followed by alternating Type 2 SA and STA sequences and the positively charged amino acids are on the N-terminal side of the SA
42
What do positive and negative values represent on a hydrophobicity profile?
- positive: relatively hydrophobic
| - negative: relatively hydrophilic
43
Which sequences in a protein are hydrophobic?
- signal sequence
- stop transfer sequence
- signal anchor sequence
- transmembrane sequences
44
What are some examples of ER resident proteins?
- Binding protein (BiP)
- Peptidyl-prolyl isomerase (PPI)
- Protein Disulfide Isomerase (PDI)
- Glycosyltransferases (GTs)
45
What does BiP do?
- a chaperone protein
- binds to proteins to help them fold properly
- can assist in the formation of protein complexes (multimeric assembly)
- also used in the post-translational translocation of secretory proteins in yeast to prevent back sliding
- involved in the unfolded protein response
46
What does PPI do?
- key enzyme that can interconvert cis-trans isomers with proline
- due to the conformationally restrained peptide bond in proline, both the cis and trans isomers exist in nature
- important for protein folding
47
What does PDI do?
- catalyzes the formation of disulfide bridges in proteins (free SH groups to S-S-S bridges)
- aids in appropriate folding
48
How does PDI form/rearrange protein disulfide bonds?
Formation:
1. Start with reduced substrate protein that has SH groups
2. PDI starts in the oxidized state and has 2 closely spaced cysteines
3. PDI forms a disulfide bond with the one of the SH groups on the protein to form a PDI-substrate-protein intermediate
4. The substrate protein's second ionized thiol reacts with the intermediate to form a disulfide bond within the substrate protein, releasing PDI
5. The PDI disulfide bond is reformed by electron transfer to reduce Ero1 (ER lumen protein) that regenerates oxidized PDI
Rearrangement:
1. Start with protein with incorrect disulfide bonds
2. Reduced PDI forms a complex with the protein, corrects the bonds, and is released
3. Left with reduced PDI and protein with correct disulfide bonds
49
What is the unfolded protein response?
- occurs when proteins are synthesized incorrectly, folded incorrectly, or the cell made too much of a certain protein
- this response prevents the protein from continuing on the secretory pathway to the Golgi and its final destination
Process:
1. BiP starts off bound to an Ire1 monomer in the ER membrane
2. BiP binds to unfolded proteins in the ER lumen, causing Ire1 to form a dimer (activated form)
3. The Ire1 endonuclease binds to the Hac1 messenger RNA and splices it so that it is a mature, functional RNA
4. The transcription factor is translated, moves to the nucleus, and regulates the expression of genes involved in the unfolded protein response
5. Unfolded proteins are moved out of the ER and degraded by cytoplasmic proteases (by an unknown mechanism)
50
What do GTs do?
glycosylate proteins in the ER
51
How are secretory proteins glycolated?
- there is a large, preformed oligosaccharide complex and it is transferred en masse to the secretory proteins
- oligosaccharide modification is found specifically on asparagine residues (not every single one)
- typical sequence: Asn-X-Ser/Thr
- type of sugar modification is N-linked oligosaccharides (N for asparagine)
- complex is made up of glucose, mannose, and N-acetyl glucosamine
52
How is the oligosaccharide precursor synthesized?
1. Start with dolichol phosphate, which is a strongly hydrophobic lipid containing 75-95 carbon atoms that is embedded into the ER membrane.
2. First N-acetylglucosamine is brought up by the nucleotide sugar phosphate UDP and is attached to dolichol phosphate by the high energy pyrophosphate linkage. This step is blocked by tunicamycin.
3. Then, the second N-acetylglucosamine is added by another UDP followed by the addition of 5 mannose residues, each brought by a GDP
4. Once these 7 residues are added to the dolichol phosphate it flips in the membrane and additional mannose residues are delivered using GDP. Glucose is added in the same way, but UDP is used instead of GDP.
53
Which sugars are trimmed during protein glycosylation in the ER?
three glucose and one mannose
54
How do vesicles move from the ER to the Golgi?
- through vesicle trafficking
- a vesicle buds from a donor compartment and fuses with a target compartment
- this is a highly regulated process
55
What is the structure of the Golgi apparatus? x
- the cis side of the Golgi is where the vesicles fuse (entrance)
- flattened stacks called cisterna: cis cisterna, media cisterna, trans cisterna
- the trans side of the Golgi is the exit
56
What are the two classes of N-linked oligosaccharides?
- complex
| - high mannose
57
How are complex N-linked oligosaccharides processed to be sent to the Golgi?
1. Start with an asparagine residue with sugar residues added
2. Glucosidase 1 removes one glucose residue
3. Glucosidase 2 removes two additional glucose residues
4. Mannosidase removes a mannose residue
5. A glucose is re-added if the protein needs to stay in the ER for longer before moving to the Golgi (usually because of improper folding)
6. Calnexin (CNX) and calreticulin (CRT) bind to N-linked oligosaccharides where a glucose is added and act as a chaperone until it is properly folded
7. If the protein fails to fold properly, glucosidase 2 will remove the added glucose and ER alpha mannosidases will remove 4 mannose
8. This trimming pattern promotes the binding of OS-9 which dislocates the misfolded protein out of the ER where it is degraded by cytosolic proteasomes
58
How are complex N-linked oligosaccharides processed in the Golgi?
1. Start with 2 N acetyl glucosamines and 9 mannose
2. Golgi mannosidase 1 removes 3 mannoses
3. GlcNlc transferase adds 1 N acetyl glucosamine
3. Golgi mannosidase 2 removes 2 additional mannoses
4. Then 2 N acetyl glucosamine, 3 galactose, and 3 sialic acid residues are added
59
How is endoglycosidase D used in trafficking studies?
- we hypothesize that misfolded proteins are retained in the ER while properly folded proteins move out to the Golgi
- we also hypothesize that the glycans on the proteins will be trimmed as it moves through the Golgi stacks and it will become sensitive to endo D
- if the protein is misfolded, it is retained in the ER and Golgi specific trimming of glycans does not happen
- if the protein is folded correctly, it will move through the Golgi stacks and there will be Golgi specific trimming of the 3 mannose residues
- properly folded proteins are cleaved by endo D and will have a lower molecular weight
60
How did the experiment to test endoglycosidase D sensitivity work?
- the VSV-G membrane glycoprotein was being studied, but it was a temperature sensitive mutant (ts045)
- the ts045 VSV mutant grown at 40 degrees will not transport proteins out of the ER but at 32 degrees the protein will be transported to the Golgi
- cell used in the experiment was from a Chinese hamster ovary labeled with 35S methionine
- cells were split into 2 groups
- group 1 was the control group and the cells were maintained at 40 degrees C and will be misfolded (retained in the ER)
- group 2 will have the experimental cells shifted to 32 degrees C and the protein will move through the secretory pathway
- cells were harvested at different time points and ice cold buffer was immediately added to stop vesicle trafficking
- the cells were homogenized and the ER and Golgi were purified and incubated with clarified cytosol and ATP to reconstitute microsomes
- proteins were extracted from the ER and Golgi microsomes and purified by immunoprecipitation
- in immunoprecipitation, VSV-G specific antibodies are added to the protein mixture which specifically bind the VSV-G protein
- staphylococcus protein A attached to sepharose beads was added to the mixture
- staphylococcus protein A will bind to the antibody
- a short centrifuge precipitates the beads and all unbound proteins will be in the supernatant
- this step was repeated a few times
- the result is your purified protein from different time points, which is digested with endo D
- if you see 2 bands on the SDS page gel, it means that the protein is sensitive to endo D
- the time where 2 bands shows up tells you when the protein crossed the medial Golgi
- at 32 degrees C, most of the protein passes through the medial Golgi
- at 40 degrees C, almost none of the protein passes through the medial Golgi
61
How is endoglycosidase H used in trafficking studies?
- the protein is sensitive to endo H before crossing the medial Golgi and becomes resistant afterwards
- you should get 2 bands on your SDS page gel that are different sizes before crossing the medial Golgi
- you should get 2 bands on your SDS page gel that are the same size after crossing the medial Golgi
- another way to tell when the protein crossed the medial Golgi
- can also use endo H to tell whether high mannose or complex N-linked sugars were used
- complex N-linked sugars are resistant to endo D after crossing the medial Golgi while high mannose sugars are always sensitive to endo D
62
How is microscopy used to track secretory protein trafficking?
- VSV G is a protein that traffics to the plasma membrane
- use the same temperature mutant of VSV G
- at 40 degrees C, trafficking is halted in the ER
- this synchronizes the synthesis of the protein
- then relieve the block and move the cells to 32 degrees C and continue synthesis
- this allows VSV G to move from the ER to the Golgi and then to the plasma membrane
- you can also take fluorescence measurements
- resolution is not as fine as if you used endoglycosidases because you can't say when for sure the protein crossed the medial Golgi
- advantage: looking at the trafficking in real time
63
How does the cell keep ER resident proteins in the ER?
- most of the ER resident proteins have a specific sequence on their C-terminus that is made up of lysine, aspartic acid, glutamic acid, and leucine (KDEL sequence)
- the KDEL sequence tells the cell that the proteins belong in the ER
- when a vesicle buds, you have some soluble secretory proteins being made that need to move on to the Golgi
- you also have the ER resident proteins that may get caught up in these vesicles
- if in the vesicle, ER resident proteins will end up in the Golgi
- the vesicles, Golgi, and ER have KDEL receptors that bind to ER resident proteins and signal for the return to the ER
- acts as a "safety net"
64
What are O-linked oligosaccharides?
- bound to the OH groups on serine and threonine on secretory proteins
- ex: collagen, glycophorin
- sequence: X-Ser/Thr-X
65
What is equivalent to the extracellular space?
- ER lumen
- Golgi lumen
- lumen of transport vesicles
66
What is the role of glycosylation?
- hypothesized that it is important in folding
- aid in transport (very rarely, ex: targeting to lysosomes)
- resistance to proteases (stability)
- protein-protein interactions
67
What are lysosomes?
- lysosomes are small membrane bound organelles in the cell that can degrade macromolecules
- filled with nucleases, proteases, glucosidases, lipases, etc
- the optimum pH for the activity of the hydrolases in lysosomes is acidic, meaning that these hydrolases are acid hydrolases
- lysosome lumen (pH 5) is acidic relative to the cytosol (pH 7.2)
- the fact that the hydrolases work best in acidic environments is a protective mechanism because if they were to get out into the cytosol, they wouldn't work as well
- pH of lysosome is maintained by a V class pump that brings in hydrogen ions and hydrolyzes ATP
- the membrane of lysosomes is protected from degradation through the dense population of glycoproteins in the membrane
- the hydrolases don't randomly degrade macromolecules
68
How do vacuoles serve as versatile lysosomes in plant and fungal cells?
- vacuoles function like lysosomes in the degradation of macromolecules
- also serve as storage organelles for waste and nutrients
- in plant cells, the vacuole regulates the turgor pressure necessary to maintain plant structure
69
How do lysosomes act as a functional "hub"?
- many cellular trafficking pathways go through lysosomes
- ex: pinocytosis, phagocytosis, autophagy, secretory pathway
- pinocytosis and phagocytosis are types of endocytosis
- autophagy is the mechanism by which cells degrade spent organellular components
70
How does the Golgi mark proteins to go to lysosomes?
- all lysosomal hydrolases (proteins destined for the lysosome) have a mannose 6 phosphate modification on them
- the phosphorylation of mannose residues on lysosomal hydrolases provides the signal for the hydrolase to move from the Golgi to the lysosome
- the signal is added by an enzyme in the Golgi called N-acetyl glucosamine phosphotransferase
- this enzyme catalyzes the transfer of a phosphate from an N-acetyl glucosamine to the 6th carbon of a mannose residue
- in order to be recognized by the enzyme, the protein needs to have a signal patch (two leucine or tyrosine)
- after the phosphate group and N-acetyl glucosamine are added, a phosphodiesterase cleaves off the sugar leaving you with mannose-6-phosphate
71
How are proteins trafficked from the Golgi to the lysosome?
- there are receptors in the Golgi that bind specifically to mannose-6-phosphate called mannose-6-phosphate receptors
- lysosomal hydrolase binds to the M6P receptor in the Golgi
- a bud forms in the Golgi to make a transport vesicle
- the receptor is caught up in this bud and is going to be trafficked to the lysosome
- the bud becomes coated with clathrin and is called a clathrin-coated bud that becomes a clathrin-coated vesicle
- the vesicle then becomes uncoated
- then the vesicle fuses with a late endosome (the coat needs to be taken off in order to get close interaction between the membrane of the vesicle and the membrane of the late endosome
- the late endosome has phosphatases that will remove the phosphate group from M6P
- this causes the protein to detach from the M6P receptor
- the M6P receptor gets put into its own transport vesicle using the recycles clathrin coat and returns back to the Golgi
- the protein is then trafficked to the lysosomes
72
What safety net exists for when lysosomal hydrolases accidentally get secreted out of the cell?
- there are M6P receptors on the plasma membrane that capture lysosomal hydrolases if they escape and bring them back to the late endosome in a clathrin coated vesicle
- still have to remove the coat to fuse with the late endosome
- from the late endosome, theses proteins are trafficked to the lysosomes
- it would be detrimental to let the hydrolases be secreted, because they can still function at a neutral pH
73
What are lysosomal storage diseases?
- a group of genetic diseases due to the absence of 1 or more lysosomal hydrolases or to the mistargeting of lysosomal hydrolases
- there are about 40 of these diseases
- if the lysosome is missing certain hydrolases, the macromolecules that are normally degraded by these hydrolases will pile up in the lysosome
74
What is I Cell Disease?
- enzyme deficiency: GlcNac phosphotransferase
- causes severe tissue destruction
- causes issues with trafficking hydrolases to the lysosomes
- GlcNac phosphotransferase is what catalyzes the M6P modification
- if lysosomal hydrolases do not have M6P, they will not bind to M6P receptors to be trafficked to the lysosome
- no accumulation of macromolecules in the lysosome
- hydrolases will end up in the secretion pathway or outside the cell
- liver cells in these patients demonstrate normal distribution/targeting of hydrolases without M6P
- different cells use different targeting methods
75
What is Tay Sachs Disease?
- enzyme deficiency: hexosaminidase A
- causes accumulation of ganglioside GM2
- causes intellectual impairment, enlarged liver, skeletal involvement, and is fatal (age 2)
76
What is Gaucher's Disease?
- enzyme deficiency: glucocerebrosidase
- accumulation of glucocerebroside
- causes liver and spleen enlargement, long bone erosion, and intellectual impairment
77
What is the constitutive secretion pathway?
- vesicles bud from the Golgi and fuse with the plasma membrane
- the pathway by which plasma membrane proteins are delivered and the process by which components of the extracellular space are released
- unregulated
78
What is the regulated secretion pathway?
- still travelling from the Golgi to the plasma membrane
- the cell receives a signal such as a hormone or neurotransmitter that is needed for the vesicle to fuse to the plasma membrane
- cells that make hormones, neurotransmitters, or digestive enzymes use this pathway
79
What are the endocytic pathways to lysosomes?
- phagocytosis: uptake of large particles
- pinocytosis: fluid phase endocytosis
- autophagy: brining in old organelles for degradation
80
What is phagocytosis?
- the process by which large particles (as large as bacteria) can be ingested into the cells
- very common in lower, single cell eukaryotes such as amoeba or protozoa because it is used for feeding and obtaining nutrients
- in multicellular organisms, it is used in immune system cells to defend against invading microbes
- macrophages (monocytes, agranulocytes): recycle other cells and phagocytose pathogens such as bacteria
- neutrophils (leukocytes, granulocytes): migrate into tissue spaces looking gor invading pathogens)
81
How does phagocytosis occur?
- requires a surface receptor
- does not happen randomly
- the phagocytic target must bind to a specific receptor on the cell surface in order to be taken up
- an induced/triggered event
- once a target binds to its receptor, this triggers the cell to extend its membrane, forming pseudopods around the particle being ingested
- in our immune system, the macrophages or neutrophils have Fc receptors for the Fc portion of certain antibodies on the pathogen
- when you have a bacterial or viral infection, your body produces antibodies that bind and coat the bacterium/virus and allow for phagocytosis
82
How many old red blood cells can be scavenged by macrophages per day?
10^11
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What is fluid phase endocytosis (pinocytosis)?
- the way that small particles and fluids are taken up into the cell
- sometimes cell surface receptors are also taken up during this process and that is called receptor mediated endocytosis
- a continuous process, unlike phagocytosis
- non-specific
- cell is constantly carrying this out
- no trigger
- rate of uptake depends on cell type
- lower eukaryotes carry out pinocytosis at a faster rate and use it to obtain nutrients in addition to phagocytosis
84
How does pinocytosis occur?
- begins at clathrin coated pits, which are small invaginations of the cell that are coated with a group of proteins including clathrin
- clathrin coated pits are short lived
- shortly after the invagination forms, it extends and pinches off to form clathrin-coated vesicles filled with fluid from the extracellular space
- this is continuously happening along the plasma membrane
- clathrin-coated pits occupy 2% of the total plasma membrane
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What is receptor mediated endocytosis?
-occurs when cell surface receptors are taken up by the clathrin-coated pits in pinocytosis
-the proteins in the pit are able to attract cell surface receptors or bind to them so that they become trapped in the pit
-remember that cell surface receptors are not the same thing as antibodies
-they are typically transmembrane proteins that bind to specific macromolecules in the extracellular space
-if the receptors bind a ligand in the pit, then it invaginates and pinches off
so that the receptors and the ligand will be in the clathrin-coated vesicle
-not a specific event
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How is receptor mediated endocytosis seen in the uptake of cholesterol in mammalian cells?
- receptor mediated endocytosis is used to acquire cholesterol which is important for building membranes
- if the process does not occur cholesterol build up in the blood stream and damages blood vessels, high risk of heart disease
- most cholesterol is carried in low density lipoprotein (LDL) particles
- LDL particles have a phospholipid monolayer that carry cholesterol molecules, core can carry about 1500 cholesterol molecules
Process:
1. LDL binds to Apo-B protein, which is the receptor in this case
2. LDL bound to Apo-B ends up in the clathrin-coated vesicle which eventually becomes uncoated and known as the early endosome
3. The early endosome fuses with the late endosome which has an pH of 5
4. The acidic pH causes LDL to detach from Apo-B
5. LDL is then trafficked to the lysosome where it is degraded and the cholesterol and fatty acids are used for membrane building
6. The amino acids from Apo-B are recycled
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What are some genetic disorders of cholesterol uptake?
- missing the LDL receptor (Apo-B)
- genetic mutation that causes the LDL receptor to be unfolded and removed from the secretory pathway by the unfolded protein response
- mutation in LDL receptor that prevents their association with clathrin-coated pits
- can lead to arterial disease, heart attack, stroke, and sudden cardiac arrest
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What happens to the surface receptors that are taken up by endocytosis?
- 3 possible fates
- receptor can be recycled back to the plasma membrane to the same domain from which they came
- can be trafficked to a lysosome and be degraded along with their cargo
- transcytosis: receptors are taken up in one domain and trafficked into a different domain in the cell (ex: moved from apical side to basolateral side)
89
What is the Transferrin cycle?
- transferrin is a major blood glycoprotein that is responsible for delivering iron from the liver to practically all cells in the body
- involved in receptor mediated endocytosis
- apotransferrin is not bound to iron and ferrotransferin is bound to iron
- apotransferrin binds at acidic pH and ferrotransferrin binds at neutral pH
Process:
1. Ferrotransferrin has a high affinity for its surface receptor at a neutral pH
2. The receptor along with ferrotransferrin are taken up by the clathrin-coated pit to form a clathrin-coated vesicle
3. The vesicle becomes uncoated and known as an early endosome
4. The early endosome binds with the late endosome which has an acidic interior
5. The acidic interior causes the iron to come off which is transferred by a transferrin transporter into the cytosol
6. Once the iron is off, the previous ferrotransferrin is now an apotransferrin bound to its receptor
7. The apotransferrin and receptor are trafficked back to the cell surface
8. Once at the surface, we are in a neutral environment, so the apotransferrin detaches from its receptor
9. The apotransferrin can pick up more iron to become ferrotransferrin and repeat the cycle
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What is an example of a receptor that gets degraded with its cargo in the lysosome?
- the epidermal growth factor (EGF) receptor
- EGF is an important growth promoting signaling molecule
- EGF and receptor complex go through receptor mediated endocytosis and are both trafficked to the lysosome for degradation
91
What is an example of transcytosis?
- antibodies moving across epithelial cells of the intestine
- a way of bypassing tight junctions
Process:
1. Newborn mammals obtain antibodies from their mother's milk
2. Antibody binds to the Fc receptor facing the lumen of the intestine which has a pH of 6
3. The antibody and receptor are taken up by a clathrin-coated pit, which becomes a clathrin-coated vesicle
4. The clathrin-coated vesicle loses its coat, becoming the early endosome
5. The endosome is trafficked to the opposite side of the cell where the antibody is released into the blood and interstitial fluid (pH 7)
6. The receptor travels back to the lumen side to be reused
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What are the 3 types of coated vesicles?
- clathrin-coated vesicles
- COP I- Coatomer-coated vesicles
- COP II- Coatomer-coated vesicles
93
When are clathrin-coated vesicles used?
- pathway from the Golgi to the lysosome
| - receptor mediated endocytosis
94
When are COP 1 vesicles used?
- inter-Golgi trafficking
| - backward flow of vesicles from the Golgi to the ER (brining back ER resident proteins)
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When are COP 2 vesicles used?
regulates vesicle trafficking from the ER to the Golgi network
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Why do clathrin-coated vesicles have a more regular geometric pattern that COP vesicles?
- the major protein of clathrin-coated vesicles is clathrin
- clathrin is evoluntioarily conserved
- 6 polypeptide chains (3 heavy and 3 light)
- assembles into a 3 armed structure (triskelion shape)
- the triskelions form a regular geometric basket around the vesicles
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What are the functions of the clathrin coat?
- mechanical force to form vesicles
| - captures membrane receptors
98
How is the clathrin coat assembled and disassembled?
1. Start with an organellar membrane
2. Clathrin and other proteins begin to bind to the membrane
3. Proteins assemble into a basket-like structure spontaneously
4. Basket structure causes the bending of the membrane
5. Membrane pinches off to form a clathrin-coated vesicle
6. Coat is taken off of the vesicle so that it can bind/fuse to another membrane
7. Components of the clathrin coat are recycled and reused
99
How does the clathrin coat bind receptors?
- the protein adaptin, which is a part of the clathrin coat, mediates the binding of receptors
- adaptin is bound directly to the surface receptors and to clathrin
- a different adaptin is used depending on whether you are in the Golgi or the plasma membrane
- in the plasma membrane, the adaptin has an affinity for receptors with the sequence: Phe, Arg, X, Tyr
- in the Golgi, the adaptin has an affinity for a specific sequence of phosphorylated amino acids (these are found on the MP6 receptor)
100
What pinches off the clathrin-coated vesicle once it forms?
- protein called dynamin
- GTP binding protein
- intrinsic GTP activity
- dynamin forms a collar on the clathrin-coated vesicles as they form
- dynamic hydrolyzes GTP to GDP which leads to a conformation change that is similar to a spring tightening up
- this causes the vesicle to be pinched off
101
How do coats become attached to the membrane?
- involves small molecular weight GTPases that can hydrolyze GTP
- when a GTPase is bound to GDP, the protein is inactive
- when a GTPase is bound to GTP, the protein is active
- this allows proteins to toggle between the inactive and active forms (molecular switches)
- the exchange of GTP for GDP is facilitated by the guanine exchange factor protein (GEF) aka guanine nucleotide releasing protein (GNRP)
- the exchange of GTP for GDP is facilitated by the GTPase activating protein (GAP)
- SAR 1 is a GTPase involved in the formation of COP II coats
- SAR 1 interacts with the protein Sec12 which is its GEF to become activated
- Becoming activated exposes the hydrophobic N terminus on SAR 1 which binds to the membrane
- this recruits the components of COP II
- COPII coat assembles using Sec23 and Sec24 as its component proteins
- the coat forming creates the mechanical forces needed to form the vesicle
- before fusing with another membrane the coat needs to be removed, which uses a GTPase to go from GTP to GDP (inactivating it)
- the inactivation causes the coat to come off of the membrane
-ARF is used in the same way as SAR 1 but for COP I and clathrin coats
102
How do SNARE proteins regulate the specificity of vessicle trafficking?
- two classes of snares: vesicle snares (V-snares) and target snares (T-snares)
- V-snares become associated with the vesicles that are forming on donor organelles
- T-snares interact with the V-snares at the target organelle to help with vesicle fusion
- V-snares and T-snares are specific, meaning that a V-snare can only bind to its specific T-snare
- fits like a lock and key
103
How do Rab GTPases regulate the specificity of vesicle trafficking?
- assist in the recognition of T-snare
- help to dock the vesicle with the target organelle
- function in the same way as ARF and SAR 1 (activated when bound to GTP)
- Rab on the transport vesicle binds to its receptor/effector on the target organelle to become activated
- the activation causes the V-snare and T-snare to become intertwined
- this allows for spontaneous fusion of the two membranes
- there are many different Rabs in eukaryotic cells
104
What happens to the SNARE proteins after vesicle fusion with the target organelle?
- NSF and alpha-SNAP use the energy stored in ATP by harnessing the energy of ATP hydrolysis to disassemble the SNARE complexes
- VAMP (the V-snare) is recycled back to the donor membrane
