Endomembrane Flashcards
What is the function of the endomembranes?
to compartmentalize eukaryotic cells by dividing the cytoplasm
T or F: the endomembrane system is composed of multiple organelles that function as a coordinated unit
True
What are the 6 organelles that make up the endomembrane system?
endoplasmic reticulum Golgi complex endosomes lysosomes vacuoles secretory vesicles
Which 2 organelles are not included in the endomembrane system?
mitochondria
chloroplasts
T or F: the combined surface of endomembranes is always less than the surface area of the surrounding plasma membrane
False
the combined surface of endomembranes can be MUCH larger than the surface of the plasma membrane
T or F: cells with different functions can have vastly different proportions of each endomembrane
true
Approximately how much of an animal cell’s total membrane does the ER constitute? How much of its volume?
~half of its membrane
~10% of its volume
How is the ER divided?
into ‘smooth’ and ‘rough’ ER
T or F: the smooth and rough ER are discrete and enclose two luminal spaces
FALSe FALSE FALSE
They are continuous and enclose a single luminal space
What is the structure of the rough ER referred as?
flattened sheets
What is the structure of the smooth ER referred as?
tubules
Where do the rough and smooth ER extend?
throughout the cytoplasm
Give some examples of the functions of the smooth ER
calcium sequestration
membrane lipid synthesis
detoxification of the cell
steroid hormone synthesis
glycogen storage in liver
Which half of the smooth ER bilayer are newly synthesized phospholipids inserted?
into the half bilayer facing the cytosol
How does the insertion of the phospholipids into the smooth ER bilayer facing the cytosol affect the orientation of the enzyme that synthesizes these lipids?
they are bound to the smooth ER membrane with their active sites facing the cytosol
How are the phospholipids flipped into the opposite leaflet?
by flippases
What are functions of the rough ER?
biosynthesis and processing of proteins
What types of proteins are included in the rough ER?
secretory proteins
membrane-bound proteins
proteins for internal use that require modification (ex. glycosylation, disulfide bridges, etc.)
What is another word for the structure of Rough ER (Sheets)?
cisternae
T or F: almost all proteins are initially synthesized on ribosomes within the cytosol
true
Where are polypeptides that are fully synthesized on ‘free’ ribosomes located?
in the cytosol and they remain in the cytosol
What are 4 proteins that fully synthesize on ‘free’ ribosomes?
cytosolic proteins
peripheral membrane proteins
nuclear proteins
proteins incorporated into chloroplasts, mitochondria, and peroxisomes
Where does a polypeptide with an ER signal sequence move to? How does this movement usually occur?
into the ER cisternal space through a protein-lined pore
this usually occurs co-translationally
Describe post-translational translocation
proteins that are synthesized completely on cytosolic ribosomes and are THEN moved to an organelle (ex. nucleus or peroxisome) AFTER translation
What do almost all proteins produced on membrane-bound ribosomes become?
glycoproteins
Describe glycosylation
the covalent addition of oligosaccharides
When does glycosylation occur?
After a protein is properly folded in the ER lumen
What is the most common type of protein glycosylation that occurs in the ER?
N-linked glycosylation
Describe N-linked glycosylation
the most common type of protein glycosylation that occurs in the ER
adds sugars to an amino group of the asparagine (Asn) amino acid
What does glycosylation begin with?
the addition of an identical 14 sugar core to every glycosylated protein
What is added to every glycosylated protein?
an identical 14 sugar core
T or F: the 14 sugar core added to every glycosylated protein is identical
true
What are the 3 components of the core sugar on glycosylated proteins?
mannose
glucose
N-acetyl-glucosamine
T or F: the core glycosylated protein can be modified to give different oligosaccharides
true
Describe dolichol
A carrier lipid embedded in the ER membrane which binds to sugars in the core one by one
On which side of the ER membrane are sugars INITIALLY added to dolichol? how are they added?
sugars are added enzymatically to the cytosolic side of the ER membrane
What happens to the core dolichol halfway through formation? What is the final product?
it is flipped
the final product has the sugars facing the ER lumen
Describe oligosaccharyl transferase
a membrane bound enzyme that catalyzes the transfer of the core sugar on dolichol to a protein
Where is the enzyme active site for oligosaccharyl transferase?
in the ER lumen
What is the purpose of chaperones in the ER? give an example of a chaperone
they ensure proper folding of glycoproteins
they will attempt to refold any misfolded proteins
ex. calnexin
What happens if chaperones are unsuccessful in refolding misfolded proteins?
misfolded proteins are tagged by a terminal glucose and translocated to the cytosol and degraded
Describe proteasome
A large barrel-shaped protein complex that degrades misfolded glycoproteins in the cytosol
How many key stacks does the Golgi apparatus contain? what are they?
3
Cis
Medial
Trans
T or F: the Golgi is unpolarized
False, it is polarized
Explain why the Golgi is polarized
The cis face is always closest to the ER
the trans face is always closest to the cell surface
Which face of the Golgi is always closest to the ER?
cis
Which face of the Golgi is always closest to the cell surface?
trans
What are the two networks the Golgi also includes?
Interconnected tube and stack structures:
Cis network
Trans network
What does the cis Golgi network include?
fused vesicles arriving from the ER
What is the function of the trans Golgi network?
it separates proteins into different vesicles depending on their final destination
What happens to the core oligosaccharide in the Golgi? What are some examples?
it is modified to produce the unique sugar combinations of glycoproteins
ex. removing mannose
What is another type of glycosylation that occurs in the Golgi?
O-linked glycosylation
Describe O-linked glycosylation
the addition of oligosaccharides to the hydroxyl group of the serine (or threonine) amino acid
How are materials shuttled through the endomembrane system?
in membrane-bound transport vesicles
How are membrane-bound transport vesicles formed?
by budding of the donor membrane
How do transport vesicles release their contents in a different location?
the vesicles fuse with membranes of an acceptor compartment
T or F: Vesicles maintain their orientation as they move through cell components
true
If material was within the lumen of the donor compartment, where will it be relative to the transport vesicle and the target compartment?
it will be within the lumen of the transport vesicle and within the lumen of the target compartment
What are the 3 directional routes for vesicular traffic flow between endomembranes?
- biosynthetic pathway
- secretory pathway
- endocytic pathway
What kind of proteins use the biosynthetic pathway?
proteins destined for certain organelles (ex. lysosomes)
What happens in the biosynthetic pathway?
synthesis, modification, and transport of proteins destined for specific organelles
What proteins use the secretory pathway?
proteins to be discharged from the (secretory proteins)
What are the 2 types of secretory pathway?
constitutive secretion
regulated secretion
What is constitutive secretion?
secretion that is continuous
as soon as the protein is in a vesicle, it can fuse the contents can be released
When does regulated secretion happen?
in response to a stimulus
What experiment helped us understand the endomembrane system? What cells did they do the experiment in?
autoradiographic ‘pulse chase’ experiments in pancreatic cells
Describe the autoradiographic ‘pulse chase’ experiments
experiments run in pancreatic cells
followed a secretory protein through the different endomembrane stages
What are the 2 models of movement through the Golgi?
vesicular transport model
cisternal maturation model
describe how vesicles move in the vesicular transport model
the vesicles move forward through the complex
Describe movement in the Golgi in the Cisternal maturation model
individual cisternae move forward and vesicles transport resident proteins backwards
In the vesicular transport model, how does cargo move? how does cisternae move?
cargo is carried forward by transport vesicles
trick question - cisternae do NOT MOVE, they are stable
In the cisternal maturation model, how does cisternae move?
each cisterna ‘matures’ as it moves from the cis face to the trans face and eventially disperses in the trans golgi network
In the cisternal maturation model, how does cargo move?
transport vesicles carry resident golgi enzymes in the reverse direction after the cisterna matures
In the cisternal maturation model, how are new cis compartments formed?
by membrane vesicles budding and moving forward from the ER
T or F: vesicles bud off membranes randomly
False!!
How do vesicles form?
from specific regions of membrane covered with distinctive coat proteins on their cytosolic surface
Which membranes form vesicles?
membranes covered with coat proteins on their cytosolic surface
Where on the membrane are coat proteins located?
on their cytosolic surface
What are the 3 purposes of coat proteins?
select the components to be carried by vesicle
cause the membrane to curve and form a vesicle
target the vesicle to the appropriate destination (coat proteins shed before vesicle fusion)
What are the 3 types of coated vesicles?
COPII
COPI
Clathrin
Describe COPII
A coated vesicle that move materials from the ER ‘forward’ to the Golgi complex
Describe COPI
A coated vesicle that moves materials from Golgi ‘backward’ to the ER, or from the trans Golgi to the cis Golgi cisternae
Describe clathrin
a coated vesicle that moves materials from the trans Golgi network to endosomes, lysosomes, and plant vacuoles
they are also involved in exo and endocytosis
Describe how COPII functions
it selects certain cargo for transport in vesicles moving forwards
What happens after COPII selects certain cargo for transport in forward moving vesicles?
cargo proteins can then bind to transmembrane cargo receptors, then the receptors will bind to COPII proteins
What is the purpose of transmembrane cargo receptors with cargo proteins binding to the COPII proteins?
it allows specific cargo to concentrate in an area of the lumen that will eventually become a vesicle
How many layers does a COPII protein have?
2
they have an inner and outer layer
What protein is involved in the layers of the COPII protein?
Sar1 protein (a G protein)
How is Sar1 activated?
By a GEF
What is the activated state of Sar1?
Sar1-GTP
What happens when Sar1 is activated?
Sar1-GTP (activated) binds to the ER cytosolic leaflet band to recruit Sec proteins
What is the structure of Sec proteins?
curved
What happens when Sar1-GTP and Sec proteins bind together?
they help the membrane to form a bud
Which proteins are on the outer coat layer?
Sec 13/31
What type of proteins are on the inner coat layer?
Sec 23/24
T or F: sec proteins help form only the inner layer of the COPII coat
False, sec proteins help form both the inner and outer layer of the COPII coat
How is the COPII coat disassembled? When does this happen?
When it reaches its target membrane, hydrolysis of GTP turns Sar1 off and the coat disassembles
What direction of transport does COPI mediate?
backward transport within the golgi or from golgi to ER
Why would we need backward transport in the Golgi?
ER resident proteins or cis-Golgi/medial Golgi proteins can accidentally get packaged into vesicles and sent forwards out of their home compartment
How does the cell identify which proteins might need to be transported backward?
a KDEL sequence that is distinct from the original ER signal sequence
Describe the structure of clathrin subunits
subunits consists of 6 polypeptide chains (3 heavy chains and 3 light chains) that together form a triskelion structure
How do triskelions assemble? What is the final product?
into a series of hexagons and pentagons to make a misshapen sphere
What are clathrin proteins bound to?
an inner layer of adaptor proteins
When bound to clathrin proteins, what else do adaptor proteins bind to? what does this do?
the cargo receptors - this will capture the soluble molecule of interest
what does the clathrin coat induce in the membrane?
induces membrane curvature and then many other proteins help to pinch off the vescile
What happens to the clathrin coat after the transport vesicle is pinched off?
it quickly leaves
Which two coats will shed quickly after the after the transport vesicles form?
clathrin and COPI
What happens to the COPII coats after the vesicles form?
it remains on the vesicle until they dock with the target membrane and Sar1 is inactivated again
What has to happen for the COPII coat to disassemble?
the vesicle has to dock on the target membrane and Sar1 must inactivate by GTP hydrolysis
What prevents a transport vesicle that has budded off the ER and is moving to the Golgi complex from fusing to the wrong compartment?
a tethering and docking process regulated by Rab G-proteins and SNAREs
What regulates the tethering and docking process that prevents transport vesicles from fusing to the wrong compartments?
Rab G-proteins and SNAREs
What is Rab? Where do they reside?
the largest family of monomeric GTPases
they reside on the cytosolic surface of many organelles
What is the function of Rab-GTP?
it is involved in initially tethering an incoming vesicle to the target membrane
What is one way Rab-GTP tethers an incoming vesicle to the target membrane?
a filamentous effector protein on the target membrane extends into the cytosol and binds to Rab-GTP on the vesicle membrane
What is the purpose of Rab effectors?
they ensure the target protein stays on the vesicle
Where are SNARE proteins located?
on both the vesicle and target membranes
What do the SNARE proteins do?
SNAREs on the vesicle bind to SNAREs on the target membrane to dock the vesicle on the membrane and initiate membrane fusion
What does the vesicle docking on the membrane cause?
the initiation of membrane fusion
How do SNARE proteins exist?
as complementary sets = 1 v-SNARe and one organelle specific t-SNARE
What are the SNARE sets composed of?
1 v-SNARE + 1 organelle-specific t-SNARE
T or F: t-SNARES and v-SNARES can exist on their own
FALSE, they will ALWAYS be a pair
What facilitates membrane fusion?
the tight binding of a v-SNARE and t-SNARE pair
What needs to be properly incorporated into budding vesicles for proper endomembrane transport?
cargo proteins
v-SNAREs
How many types of acid hydrolases does the lysosome contain?
more than 40
What pH do the acid hydrolases in the lysosome work best at?
low pH (acidic)
Are acid hydrolases initially active or inactive?
inactive
What do acid hydrolases in the lysosome require to be activated?
proteolytic cleavage
Most resident lysosomal membrane proteins are ____
highly glycosylated
Why are most resident lysosomal membrane proteins highly glycosylated?
to protect them from the hydrolases in the lumen
what establishes the low acidity in the lysosome?
a V-type pump
Are lysosomes heterogenous or homogenous? why?
heterogenous because they receive different cargo from multiple places (Golgi, EC environment, cytosol)
What often matures into lysosomes?
endosomes
How do endosomes mature into lysosomes?
they fuse with each other and their contents degrade
How are newly synthesized acidic hydrolases targeted to the lysosome?
a mannose 6 phosphate ‘tag’
Where is the newly synthesized acidic hydrolase targeted for the lysosome tagged? Where do they transport from?
the M6P tag is added in the cis Golgi
they transport from the trans Golgi network to the lysosome
How can M6P tagged proteins be directed from the trans Golgi network to the lysosome?
a mannose 6 phosphate receptor in the trans Golgi network isolates the tagged proteins into budding vesicles cloated in clathrin
Where is the M6P-bound protein released? What happens to the receptor?
the M6P-bound protein is released at the lower pH of endosomes
the receptor is recycled
What is the default endomembrane pathway for constitutive secretion?
Movement from the Trans Golgi Network to the cell surface
Does directing proteins from the TGN to the plasma membrane require an additional (to the original ER signal sequence) signal? Why?
No because movement from the TGN to the cell surface for constitutive secretion is the DEFAULT endomembrane pathway
What type of compounds would move through the default endomembrane pathway?
Compounds destined for the ECM (ex. proteoglycans)
When is regulated secretion used?
when certain cell types need to produce high volume if products on demand
ex. hormones, neurotransmitters, digestive enzymes
Where are the products of regulated secretion stored? Are they stored in high or low concentrations?
stored in high concentrations in secretory granules
When are the products of regulated secretion released from secretory granules?
when exocytosis is triggered
How is the content of a secretory vesicle concentrated?
clathrin-coated vesicles retrieve excess membrane and excess luminal content so that what is left is just the cargo
How does insulin relate to the secretory pathways? What technique can be used to study this?
beta cells of the pancreas secrete insulin via regulatory secretion from the TGN to the cell surface
anti-clathrin antibodies can be bound to gold particles and visualized (immunogold)
After cargo is transferred to the cell surface in high concentrations in a mature secretory granule, what must they wait for before they are released?
an exocytosis signal
T or F: migration in a secretory granule of a neuron from the TGN to the cell surface is a short migration
FALSE, the secretory granules are packaged in the CELL BODY and must travel down the AXON to wait at the SYNAPSE
this can be more than a meter
Why might it take a long time for the secretory granules of a neuron to reach the cell surface?
because they are packaged in the cell body and must travel down the axon to reach the synapse
T or F: it is always the same signal that triggers exocytosis
false, it can vary (ex. action potential or hormone binding to a receptor)
What does the exocytosis signal usually increase?
intracellular Ca2+
What does Ca2+ control?
the SNARE machinery and initiates exocytosis
