Week 7 Flashcards
Two models for the mechanism of protein transport from the cis-Golgi to the trans-Golgi network (TGN)
- Vesicular transport between stationary cisterna
- Cisternal maturation
Which model for the mechanism of protein transport from the cis-Golgi to the trans-Golgi network (TGN) is more favored?
The cisternal maturation model.
In the vesicular transport model, what is required for bringing proteins from one cisternae to the next?
Intermediate COPI vesicles.
What does the signaling pathway of secretory (exocytic) vesicles typically lead to?
The signaling pathway typically leads to the opening of a calcium channel and influx of calcium into the cytosol.
Role of beta cells
Insulin is secreted by beta cells in the pancreas
When does prohormone processing occur?
Prohormone processing occurs during vesicle formation and maturation
Which coat protein is involved in the maturation process (recycling of prohormone convertases)?
Clathrin
What do beta cells measure?
Blood glucose concentration (should be about 3 mM at a fasting concentration)
Series of events that occurs after glucose enters the cell.
- The mitochondria will make ATP
- The ATP will bind to the K ATP channel, causing it to close.
- The membrane potential will fall.
- Depolarization of the membrane.
- Calcium enters the cell
- This stimulates exocytosis of insulin into the bloodstream.
What type of cellular structures are required for the final step of secretion (exocytosis)?
This process requires tethers, Rabs, and SNAREs.
What do regulated vesicles typically require to drive SNARE-dependent fusion?
- Regulated vesicles typically require a calcium influx to drive SNARE-dependent fusion.
- Constitutive vesicles also use SNAREs to drive membrane fusion but do not need calcium.
What process does synaptotagmin play a role in?
The regulation of synaptic vesicle fusion by calcium and synaptotagmin
Two routes that proteins can take to get to the apical surface
- Direct sorting of membrane proteins in the trans Golgi network
- Indirect sorting via endosomes
Rafts
Cholesterol-sphingolipid rich microdomains within the lipid bilayer.
How do rafts move within the cell?
They are presumably formed in the Golgi and preferentially enter vesicles at the TGN that are targeted to the apical membrane.
The raft hypothesis
The raft hypothesis proposes that certain proteins tend to partition preferentially into the cholesterol-sphingolipid rich raft domains. For example, proteins with longer transmembrane domains may prefer to stay in parts of the membrane that are thicker because of the abundance of cholesterol and sphingolipid. Sphingolipids tend to have longer (20 - 24 carbon) acyl chains that are typically saturated.
Signaling and processes associated with the ER
- Cleavable signal sequence
- SRP, translocon
- Folding, N-glycosylation
- Sorting into COPII vesicles
Signaling and processes associated with the Golgi
- Mannose-6-phosphate (M6P)
- Cisternal maturation
- M6P receptor
- Clathrin-coated vesicle
Signaling and processes associated with the late endosome
- Dissociation from receptor
- Recycling of receptor
Signaling and processes associated with the lysosome
Fusion with late endosome
Three different kinds of vesicles being generated from the TGN
- Signal-mediated diversion to lysosomes
- Constitutive secretory pathway
- Signal-mediated diversion to secretory vesicles (for regulated secretion)
What does recognition of a signal patch common to multiple lysosomal enzymes lead to?
Their modification with mannose-6-phsophate.
I-cell disease
I-cell disease is a severe lysosomal storage disorder caused by deficiency of the GlcNAc phosphotransferase. Many soluble lysosomal enzymes are inappropriately secreted in these patients.
Where is the GlcNAc phosphotransferase localized to? What happens as a result?
The cis-Golgi. Lysosomal enzymes receive Man-6-P instead of complex N-linked oligosaccharides.
Pompe disease
Pompe disease is a deficiency in acid alpha-glucosidase, an enzyme needed to break down glycogen in the lysosome.
What brings the clathrin vesicle together?
The C-terminus of the clathrin heavy chain has a trimerization domain that brings the three subunits together.
Terminal domain of clathrin
The N-terminus of clathrin is called the terminal domain and has binding sites for adaptors.
Where do clathrin-coated vesicles bud from?
- TGN
- Plasma membrane
- Endosomes (early endosomes)
- Immature secretory granules
Structure of clathrin triskelia
Each triskelion has three clathrin heavy chains and three clathrin light chains.
How does clathrin bind cargo proteins?
Clathrin does not bind cargo proteins directly. Clathrin adaptors (adaptins) link cargo/receptor to the clathrin lattice.
Role of dynamin in clathrin coating.
The large GTPase dynamin helps the scission event needed to release the vesicle from the membrane. Dynamin may be a “pinchase”.
Drosophila shibire mutant
The Drosophila shibire mutant has a temperature-sensitive mutation in dynamin.
Adaptins
Subunits of adaptor protein (AP) complexes. Adaptins link integral membrane cargo to clathrin.
Where is AP-2 found?
The plasma membrane
Where are AP-1, -3, -4, and GGA found?
The TGN and endosomes.
Why does clathrin have more flexibility in function than other coat proteins?
The ability to interact with different adaptors at different membranes gives the clathrin coat greater flexibility in function.
Role of phosphoinositides
Phosphoinositides are critical signposts in the TGN / endosomal / plasma membrane system.
What do AP-1, AP-3 and AP-4 depend on?
AP-1, AP-3 and AP-4 all depend on Arf-GTP (same small G protein that recruits COPI) and PIPs
Role of PI4,5P2
PI4,5P2 marks the plasma membrane. It’s an important lipid molecule that helps identify the plasma membrane.
Role of PI4P
PI4P marks the TGN.
Role of PI3P
PI3P marks endosomes.
Role of cytosolic tail of the Man-6-P receptor
The cytosolic tail of the Man-6-P receptor has sorting signals that bind to GGA and/or AP-1.
What gets lysosomal enzymes into the secretory pathway?
To get into the secretory pathway to begin with, the lysosomal enzymes have a cleavable signal sequence recognized by SRP.
Before lysosomal enzymes move to the Golgi in COPII-coated vesicles, what happens?
N-glycosylation and disulfide bond formation.
6 steps for the sorting of lysosomal enzymes from the Golgi to the late endosome.
- Recruitment of AP-1/GGA by Arf and PI(4)P
- Assembly of a clathrin bud.
- Capture of Mann-6-P receptor by adaptor/clathrin
- Budding of vesicle (Golgi dynamin may help)
- SNARE-mediated fusion with endosome
- Lysosomal enzyme dissociation from receptor (pH)
5 steps for the retrieval of the Mann-6-P receptor from the late endosome back to the TGN
- Recruitment of retromer by ? and PI(3)P
- Assembly of retromer-coated bud.
- Capture of Mann-6-P receptor by coat
- Budding of vesicle.
- SNARE-mediated fusion with TGN
What is the cause of Alzheimer’s and Parkinson’s hypothesized to be?
Hypothesized that Alzheimer’s and Parkinson’s are caused by defects in the endosomal protein trafficking pathway.
If the lysosome ruptures, what will happen to the cell?
The cell will die. Degradative enzymes like acid hydrolases are stored in the lysosome.
Lysosomal storage disorders
Genetic diseases caused by a deficiency of an acid hydrolase (Tay-Sachs, Sandhoff, mucopolysaccharidosis)
What type of protein mediates phagocytosis?
Actin
Three nonbiosynthetic pathways for delivery of material to the lysosome
- Autophagy
- Phagocytosis
- Endocytosis
Autophagy
The way the cell degrades entire components or organelles. Will have to fuse to the lysosome and “dump” contents.
Autophagy is important in what diseases?
Aging, cancer, and neurodegenerative diseases.
How do autophagosomes use their two phospholipid bilayers?
As these two layers close, they can capture things in the cytosol (such as ribosomes, glycogen granules, and organelles)
Effect of nutrient deprivation on lifespan
Nutrient deprivation will extend lifespan because it induces this pathway to recycle cellular components. When you’re well-fed, you accumulate damaged cellular components.
Role of dynamin in endocytosis
Dynamin is thought to play a role in pinching off the neck of the vesicle so it can be internalized.
3 fates for endocytosed proteins
- Recycling
- Transcytosis
- Lysosome
Unique endosomal organelles of the endocytic pathway
- Early endosomes (Rab5)
- Recycling endosomes (Rab11)
- Late endosomes (Rab7) (multivesicular bodies)
Primary function of endosomes
The primary function of endosomes is protein sorting. They play a major role in defining the specific protein composition of the plasma membrane.
Four trafficking destinations for clathrin-dependent endocytosis
- Plasma membrane
- Lysosome for degeradation
- Cytosol
- Synaptic vesicles that carry neurotransmitter
