Download MCB Lecture 3 – ER and Golgi

MCB Lecture 3 – ER and Golgi
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What is co-translational translocation?
o The ribosome transfers the growing polypeptide chain through ER
membrane as it is still translating.
What sequence does the polypeptide chain have that targets it for cotranslational translocation?
o An ER Signal Sequence (usually at N-Terminus)
What is the name of the receptor on the ER Membrane that recognizes the ER
Signal Sequence?
o SRP – Signal Recognition Particle
Once the Polypeptide Chain is shuttled into the ER through the translocon,
what protein helps it fold correctly?
o BiP – Binding Protein
What enzyme in the ER Lumen cleaves the N-Terminal Signal from the
Polypeptide chain as it enters the lumen?
o Signal Peptidase
What is needed for a Single-Pass Trans-Membrane Protein?
o Internal signal sequence (Start Transfer Signal)
o An additional hydrophobic region to act as a stop-transfer signal
What is needed for a Multi-Pass Trans-Membrane Protein?
o Internal signal sequence (Start Transfer Signal)
o Stop Transfer Sequence
o Second Start Transfer Sequence
o Second Stop Transfer Sequence
o ETC…
What three chaperones work to correctly fold proteins?
o BiP
o Calnexin
o Calreticulin
Once the protein is inside of the ER Lumen, an oligosaccharide is added.
Answer the questions below regarding this process:
o What enzyme adds this on?
 Dolichol is a membrane-bound lipid that assembles and
transfers the oligonucleotide.
o Via what type of glycosylation is the sugar chain added onto the
protein?
 N-Linked Glycosylation
o What amino acids is involved in the reaction between the sugar chain
and the protein?
 Asparagine (Asn)
o What specific molecule in Asparagine participates in the reaction?
 NH2
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o What two types of sugar molecules are the ones added to the protein?
What are the terminal sugar molecules?
 9 Mannose Sugars
 3 Glucose Sugars
Later on, some of the sugars that were added to the new protein in the ER are
removed. How many/what sugars are removed? Where does this happen?
o 1 Mannose
o 3 Glucose
o Happens in the ER
The processing of this sugar chain continues in the Golgi. How can this chain
be further processed in the Golgi (in general)?
o Further N-Linked trimming or additions
o O-Linked Glycosylation (OH) of selected Serine or Threonine Residues
If a protein enters the ER, and it is folded improperly, chaperones attempt to
re-fold it. If this is also unsuccessful, what happens to the protein?
o Deglycosylated
o Ejected back through the translocon and into the cytosol
o Poly-ubiquinated and degraded in the proteasome
Cystic Fibrosis is an often-fatal disease that first presents in children and
progresses until death at a very early age. Answer the following questions
about Cystic Fibrosis:
o What is the mode of inheritance for Cystic Fibrosis?
 Autosomal Recessive
o What protein has the mutation?
 CFTR
o What is the function of this protein?
 Cl- ion channel
o What is the specific mutation called?
 ∆F508
o How does the mutation affect the protein?
 It causes the protein to fold incorrectly, so it is ejected out of
the ER and degraded in the proteasome. Recent studies have
shown that if the protein could make it to the plasma
membrane, it could still function properly (even though it is
misfolded)
Familial Hypercholesterolemia can be caused by mutations in LDL-R.
Answer the following questions regarding this concept:
o What does an LDL-R Mutation cause?
 Misfolding in the LDL Receptor Protein
o When the protein is misfolded, what is the result?
 The receptor does not make it to the plasma membrane, so the
free LDL outside of the cell cannot come in.
o What are the effects of there being no receptors in the plasma
membrane?
LDL and cholesterol build up outside of the cells leading to
cardiovascular problems.
o Xanthomata are a symptom of high cholesterol levels. What is this?
 Fatty, yellow cholesterol deposits in the body that present as
fatty projections on the skin.
o Xanthelasmata are also a symptom of high cholesterol levels. What is
this?
 Fatty, yellow cholesterol deposits seen on the upper eyelid.
o Corneal Arcus is another symptom of high cholesterol levels. What is
this?
 Presents as a light ring around the iris of the eye. It is
cholesterol deposits.
Targeting Signals direct proteins to a particular cellular location. Answer the
following questions about targeting signals:
o What is the default protein path if the protein has no ER signal
sequence?
 Stays in the cytosol
o If the protein has a NLS Signal Sequence, where does it go?
 Nucleus
o If the protein has a SKL Signal Sequence, where does it go?
 Peroxisome
o If the protein has a KDEL Signal Sequence, where does it go?
 ER
o If the protein has a KKXX Signal Sequence, where does it go?
 ER
o If the protein has a M6P Signal Sequence, where does it go?
 Lysosome
o If the protein has an rER Signal Sequence, but no additional Signal
Sequences, where does it go by default?
 If it has an Internal Hydrophobic Sequence, it goes to the
Plasma Membrane
 If it doesn’t, it gets secreted.
o Is KDEL soluble or membrane bound?
 Soluble
o Is KKXX soluble or membrane bound?
 Membrane Bound
Which side of the Golgi faces the ER?
o Cis-Golgi
Which side of the Golgi faces the Plasma Membrane?
o Trans-Golgi
What type of coat is added to the vesicles leaving the ER and going to the CisGolgi?
o COPII
What type of coat is added to the vesicles that are recycled from the Golgi
back to the ER?
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o COPI
What is the ERGIC?
o ER-Golgi Intermediate Compartment, which is the intermediate stack
between the ER and Golgi that vesicles fuse with as the continue
through the pathway
If KDEL and KKXX are carried forward with the vesicles to the Golgi, what
protein coat is added to bring them back to the ER?
o COP I
Because KDEL is soluble, what is needed to recycle it back to the ER from the
Golgi?
o KDEL Receptors in the membrane that bind and group them together
to be transported back
There is a unique mechanism for creating COP coated vesicles to move them
along the ER and the Golgi. Answer the following questions about COP
coated vesicles:
o What is on the membrane that allows the cargo proteins to congregate
in a particular area to form a vesicle?
 Cargo Receptors
o What is the molecule that binds the receptor and the COP Coat
Proteins to bring them together?
 Adapter
o This process takes energy. What G-Protein is used for this to take
place?
 ARF/SAR-GTP
o After the vesicle buds off, what happens to the G-Protein?
 It is hydrolyzed by GAP and the coat protein falls off, exposing
SNAREs used for fusion.
As explained above, ARF and SAR are G-Proteins that promote coat protein
assembly. What is the difference between ARF and SAR? When is one used
over the other?
o ARF: COP I
o SAR: COP II
What exchanges GDP for GTP on ARF and SAR?
o GEF
What hydrolyzes GTP to GDP on ARF and SAR?
o GAP
Where are GAGs (and other carbohydrates) synthesized?
o Golgi
Explain the difference between the Vesicular Transport Model and the
Cisternal Maturation Model. Which model is correct?
o Vesicular Transport Model: The vesicles exocytose and endocytose
from Golgi Stack to Golgi Stack until it gets from the Cis-Stack to the
Trans-Stack.
o Cisternal Maturation Model: The stacks move from Cis to Trans, with
the vesicles inside of them and then recycle themselves.
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o **It is believed that the correct model is a blend between the two.
Proteins that are destined for lysosomes are tagged with what? In what
location are they tagged with this?
o They are tagged with Mannose-6-Phosphate
o They are tagged in the Cis-Golgi