1
Q
what is the endomembrane system composed of
A
The endomembrane system is comprised of:
- The ER
The Golgi - Lysosomes
- And some intermediates and transporters
2
Q
what are the main functions of the ER
A
- Proteins destined for plasma membrane, organelles, and export
- biosynthesis of lipids
3
Q
describe the rough ER
A
- ribosomes embedded in the ER give the rough appearance
- site of protein production and modification and lipid synthesis
- ribosomes attached non covalently to the ER, but very strong attachment
4
Q
what can happen to proteins made by free ribosomes
A
- Proteins made by free ribosomes are released as completed polypeptides into the cytosol
- afterwards they can:
- remain in cytosol
- get imported into an organelle such as the ER, nucleus, mitochondria, chloroplast, peroxisomes (post-translational import)
5
Q
what can happen to proteins made by bound ribosomes
A
- Proteins made by bound ribosomes are released through a pore into the ER lumen (Co-translational import)
- afterwards they can:
- remain in the ER
- head to the golgi
6
Q
What are the six steps of the SRP signal mechanisms
A
- The SRP recognizes and binds both the signal sequence and large ribosomal subunit: translation stops momentarilySRP = signal recognition protein
- Docking of the ribosome to the ER membrane occurs through two key mechanisms… one between the SRP and the SRP receptors and the other between the ribosome and the translocon
- SRP and SRP recepto each bind a molecule of GTP, triggering the restart of translation, opening of the pore and the insertion of the signal sequence into the pore
- Both GTP and hydrolysed and SRP is released
- Signal sequence is cleaved by signal peptidases as polypeptide elongates and translocates into the ER lumen
- Completed polypeptide is released into the ER lumen, ribosome is release and translocon pore closes.
7
Q
What does SRP do
A
ER signal sequence is the first part of the amino acid.
- First SRP binds to the ER signal and blocks translation
- After that SRP binds to SRP receptor; ribosome docks on the membrane
- Then GTP binds to SRP and SRP receptor; pore opens as the polypeptide is inserted
SRP receptor + pore + signal peptidase = translocon
- GTP hydrolysis results in the release of the SRP
- During translocation the signal sequence is typically removed by a signal peptidase and degraded
- Results in a protein that is now a soluble protein with the ER lumen; futher processing typically occurs, including glycosylation and various modifications that may occur in the ER or golgi
8
Q
what happens to polypeptides with an internal stop-transfer sequence and a terminal ER signal sequence
A
- Polypeptide with an internal stop-transfer sequence and a terminal ER signal sequence. The stop-transfer sequence halts the process of translocation moves out through a side opening in the translocon to anchor the polypeptide in the membrane, creating a transmembrane protein with its N-terminus in the ER lumen and its C-terminus in the cytosol
9
Q
what happens to polypeptides with only a single, internal start-transfer sequence
A
Polypeptide with only a single, internal start-transfer sequence. This single start-transfer sequence starts polypeptide tranfer and then moves through a side opening in the translocon to anchor itself in the membrane. (if this polypeptide also had a stop-tranfer sequence that prevented complete tranfer of the polypeptide through the translocon, the result would be a transmembrane protein with both its N-terminus and its C-terminus in the cytosol.
10
Q
How does N-Glycosylation work
A
- Most proteins made at the RER are N-glycosylated in a cotranslational manner by oligosaccharyltransferase, a transmembrane protein complex of the RER
- all proteins receiving an N-linked oligosaccharide are given the same “core oligosaccharide”
- oligosaccharide added by an oligosaccharyltransferase to an Asn (Asparagine) that is part of the signal sequence Asn-X-Ser/Thr
- core oligosaccharide is modified in the Er and or Golgi
11
Q
what does the smooth ER do
A
- involved in lipid metabolism production of lipoprotein particles (carry lipids via bloodstram to other parts of the body) and detoxification of lipid soluble drugs and other harmful products (cytochrome p450)
- transition ER are areas of the smooth ER which form transport vesicles for delivery to the golgi
- important for intracellular Ca2+ storage
12
Q
how does drug detoxification work
A
RH + NAD(P)H + H+ + O2 → ROH + NAD(P)+ + H2O
- often takes place in liver cells
- hydroxylation often the first step increases solubility and introduces a site for further modification (often conjugation)
13
Q
What are the two processes in metabolism and excretion
A
Oxidation and conjugation
14
Q
explain metabolic oxidation
A
Oxidation:
P450 in the liver, plays a major role
Ex An -H group in Ibuprofen has an oxygen added making it an -OH group
15
Q
explain metabolic conjugation
A
Conjugation:
- Replacement of a hydrogen, functional group, or section of a molecule with an R group. Usually an exchange between RX or ROH and the larger molecule
16
Q
What is a positive outcome for detoxification
A
The molecule is modified to have an -OH group through oxidation
Then conjugation adds a large structure such as glucuronide or SO3 2- that deactivates the drug
17
Q
what is a negative outcome for detoxification
A
When acetaminophen is hydroxilated it forms NAPQI
Glutathionine is suppose to attach to NAPQI
When you take excess acetaminophen, there is not enough Glutathionine in the liver to take care of the NAPQI. Then NAPQI can attach to, and kill liver cells, which can end up killing the liver protein
18
Q
What are three other functions of the SER
A
Carbohydrate Metabolism:
- Glucose-6-phosphatase localizes to SER
- involved in releasing glucose from glycogen storage molecule
- allows glucose to leave liver
Calcium storage:
- pumped into SER by ATP-dependent calcium ATPases
- important for signalling
Steroid biosynthesis:
- Biosynthesis of cholesterol and steroid hormones
19
Q
where do most important biosynthesis enzymes occur
What is the result of this
A
- Most important biosynthesis enzymes exclusive to the ER
- this means most phospholipids and cholesterol are manufactured on cytosolic face of ER
- require flippases, even distribution and asymmetry
- moved by endomembrane system and cytosolic exchange proteins (phospholipid transfer proteins)
20
Q
what is Anterograde and Retrograde transport
A
- Anterograde movement is movement from the ER through the Golgi towards the plasma membrane
- how does this affect the overall membrane balance
- retrograde transport is the flow of vesicles from the Golgi back toward the ER
20
Q
Describe the golgi
A
- Cis face orientated towards ER, trans face away from the ER
- compartments are biochemically and functionally distinct
- two models of formation:
1. stationary cisternae model
2. cisternal maturation model (above)
21
Q
What role does the Golgi play in glycosylation
A
- N-linked glycosylation initiated in the ER
- role in protein folding
- as proteins move through the Golgi, N-glycosyl group is modified
- contain many glucan synthetases and glycosul transferases
22
Q
what are some challengers in protein trafficking
A
- proteins localize to ER, Golgi, endosomes, lysosomes, plasma membrane and extracellular space
- protein utilize a variety of tags for sorting
- membrane lipids may also be tagged to direct trafficking of vesicles
23
Q
what happens to resident ER proteins
A
- Resident ER proteins are either retained or retrieved
- kin recognition may allow proteins to be retained
- retention tags may also keep proteins in the ER
- In contrast, retrieval tags such as KDEL and KKXX may be used to return ER proteins
24
What are resident Golgi proteins
- resident Golgi proteins are integral membrane proteins with one or more transmembrane regions
- does membrane thickness determine which subcellular compartment of the golgi they occupy?
- membrane increases in thickness from about 5nm to 8nm
25
How are lysosome proteins sorted
- mannose-6-phosphate tag on lysosomal proteins
- binding to mannose-6-phosphate receptor (MPR) at pH 6.4 (Golgi)
- release at pH 5.5 (endosomes)
26
How does secretion normally work
Constitutive: default pathway unless signalled to do otherwise
Secretory: signals upon receiving a signal
27
What is Constitutive Secretion
- unregulated and continuous secretion
- thought to be a default pathway (no tag, no retention in endomembrane system)
- may not be quite this simple, as short amino acids tags may be implicated in constitutive secretion
- glycosylation may play a role in secretion
28
What is Regulated Secretion
- Secretory vesicles accumulate in the cell, but only fuse with the plasma membrane in response to a specific signal
- vesicles bud from TGN and undergo maturation
- maturation involves concentrating the proteins, and sometimes also modifying the proteins
29
what is exo and endocytosis
Exocytosis → transporting materials across the PM, OUT of the cell
Endocytosis → transporting materials across the PM, INTO the cell
30
describe exocytosis
1. Approach of secretory vesicles to plasma membrane
2. Fusion of membranes
3. Rupture of plasma membrane
4. Discharge of vesicle contents to the outside of the cell. Vesicle membrane becomes integrated into plasma membrane
31
how do vesicles move
not fully understood
evidence that vesicles move along microtubule tracks
vesicular trafficking stopped in presence of colchicine (which is a microtubule assembly inhibitor)
32
what is the cytoskeleton
- cytoskeleton is a network of proteins within a eukaryotic cell
- polymeric proteins
33
what are motor proteins
- utilize ATP hydrolysis to move along microtubules
- dyneins move towards minus end
- kinesins move towards plus end
34
How does endocytosis work
- may be important for bring material into a cell (membrane transport) or play a role in defence (phagocytosis)
1. Membrane invaginates, formign a pocket containing macromolecules or other materials from teh exterior of the cell
2. pocket begins to pinch off, enclosing the extracellular material
3. membrane closes around the invaginated material, forming a vesicle
4. Vesicle separates from the plasma membrane, carrying material from the exterior within a membrane derived from the plasma membrane.
35
How much exocytosis is there relative to endocytosis
- exocytosis and endocytosis must balance out to keep plasma membrane roughly the same size
36
what is Phagocytosis
Ingestion of large particles (can be mroe than 0.5 um in diameter)
- in animals, mainly carried out by neutrophils, macrophages (components of the immune system)
37
what are endolysosomes
- also called clathrin-dependent endocytosis
- requires receptors on the outer surface of the PM
38
what are coat proteins
Clathrin was the first coat protein identified
triskelion - 3 large and 3 small polypeptides
Others such as COPI and COPII
Clathrin polymerizes into a “soccer ball” shape
39
describe the clattering coat assembly
Coat assembly is initiated by ARFs (GTPases)
Adaptin binds to target receptor and begins coat assembly
Clathrin assembly is tightly associated
Dynamin (GTPase) mediates membrane piching off
40
What is Dynamin
Dynamin - PI(4,5)P2 binding domain and GTPase domain
- GTP hydrolysis is require for pinching off
41
What are G proteins, GAPs, RGSs, GDIs, GEFs
G proteins are a form of molecular switch
All G proteins are GTPases
GAPs = GTPase-accelerating protein
RGSs = regulators of G protein signaling
GDIs = guanine nucleotide dissociation inhibitors
GEFs = guanine nucleotide-exchange factors
42
how do COPI and COPII coats form
- For COPI and clathrin at Golgi: initiated by ARF protein
- For COPII: utilizes Sar1 for initiation
- release may be caused by self-hydrolysis of GTP to GDP
43
How does Receptor Mediated Endocytosis work
- can take up specific molecules
- more than 25 different receptors have been identified
- a single clathrin-coated pit can probably hold about 1000 receptors
- despite the fact that the ultimate cell fate of all the endocytosed molecule may be different, they all enter the same endosomal compartment
44
What is LDL receptor mediated endocytosis
- LDL = Low density lipoprotein
- Cholesterol largely manufactured in liver, transported in lipoproteins which hold about 1500 cholesteryl molecules (immature) also 800 phospholipids and one protein
- released when pH drops, hydrolyzed to cholesterol in lysosomes
45
how does receptor mediated endocytosis work
- not all ligands will dissociate in the early endosomes
- receptor-ligand complexes may also be sorted into different transport vesicles
- alternative fates include:
1. carried to lysosomes for degradation
2. carried to TGN, sorted by Golgi with a variety of destinations
3. transcytosis
46
How does receptor-independent endocytosis work
- pinocytosis or “cell sipping” → non-specific
- vesicles generally about 100 nm in diameter
- generally start with clathrin-coated pits or caveolae
- caveosomes form from lipid rafts
- proteins that enter the cell by caveosomes avoid endosomes and lysosomes
- examples of pinocytosis rates: macrophages ingest 3% of their plasma membrane / min; fibroblasts 1% / min
- cell volume and surface area remain constant, therefore endocytic and exocytic pathways are equivalent
47
How do SNAREs work
1. the proper vesicle is recognized and bound by particular membrane anchored tethering proteins
2. a Rab GTPase bound to the incoming vesicle stimulates association of v-SNARe with t-SNARE
3. membrane fusion is promoited by the interaction of v-SNARE with t-SNARE
4. Binding of NSF and SNAPs promotes dissociation of the SNARE complex
48
what is one role of SNARE complexes
one role of SNARE complexes is in neurons where they result in neurotransmitter vesicles fusion and release of neurotransmitter into the synaptic cleft
Bio 225
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