Membrane Trafficking Flashcards
What is a transport vesicle topologically equivalent to?
The extracellular environment
What is a vesicle?
A small membrane-enclosed compartment usually containing cargo
-Leave membrane by exocytosis or fuse with a membrane via endocytosis
What is the Exocytic (secretory) pathway?
The “ default pathway”: ER-> Golgi-> Outside the cell
-uses COP2 coat protein
What is the endocytic pathway?
-performs endocytosis, brings things in a vesicles from outside to inside the cell into endosomes
-“ late” and “ early” endosome differentiated by how far from the membrane/how long they’ve been in the cell
-Uses coat protein Clathrin
What is the retrieval pathway?
-utilizes KDEL signal, returns proteins that were accidentally sent to Golgi back to the ER (where it is meant to stay)
-uses coat protein COP1
What determines a vesicle’s final destination?
The markers on their surface, specifically phosphatidylinositol (PI)
What is phosphatidylinositol?
-what coat proteins bind to to target the vesicles
-the phosphorated patterns on the ring tell you where to go and recruits different proteins that direct vesicles
-“Specifically phosphorated at ring positions by kinases that are unique to each organelle (specific kinases found in specific parts of the cell are what cause each different signal)”
Budding versus fusion?
Budding: the pinching off of a vesicle from the original organelle membrane
Fusion: the fusion/attachment of vesicle to target compartment
What coat proteins are found with each pathway?
Exocytic (secretory) pathway= COP 2
Endocytic pathway = Clathrin
Retrieval pathway = COP 1
What is the physical composition of Clathrin?
- Has three heavy protein chains and three light protein chains “beautiful”
-Naturally forms cytosolic Clathrin pits both in test tubes and on membranes without energy needed
-“ spontaneously assembles into symmetrical basket”
What are the four steps to the assembly of the Clathrin coat?
1: adaptins form inner vesicle coat
-They bind the Clathrin coat to the membrane
2: bud formation
-Clathrin in vaginate the flexible membrane
3: Vesicle formation
-Dynamin wraps around the stem of the vesicle, and uses GTP to squeeze it until it pinches off
4:Clathrin uncoating
-Coach disassembles immediately in two steps
What are the two functions of Adaptins?
-to bind the Clathrin coat to the membrane
-to trap the transmembrane proteins that capture soluble cargo molecules inside the vesicle
What induces the curvature of the vesicle?
The Clathrin coat assembly ( her triskelions force flexible membrane to bulge inward)
What is Dynamin’s and Clathrin’s binding domain?
P1(4,5)P2
-this is how it knows where to go
What is Dynamin?
A GTPase that pinches off vesicles
-wraps itself around tail to be pinched off
-hydrolizes ATP rapidly to tighten/squeeze around tail to pinch off and become budded vesicle
What are the two steps to Clathrin disassembly?
1: PIP phosphatase (inside the vesicle) depletes PI(4,5)P2 from the membrane. This weakens adaptin protein binding
2: Hsp70 chaperone proteins in cytosol act as ATPase and strips off Clathrin coat
Why must vesicles be transferred naked?
-bc they need membrane exposed to be able to do fusion
-also external signals on membrane show it where to gi
What type of vesicle assembly is Sar1 used for?
COP2 (Exocytic/excretory pathway)
What is Sar1’s inactive vs active state?
Inactive: Sar1-GDP. Amphiphilic tail hidden internally, floating in cytoplasm
Active: Sar1-GTP. Amphiphilic tail exposed, ER membrane bound
How does Sar1 “know” to get turned on by only going to the ER membrane?
That’s where it’s Sar1 GEF is, to turn it from Sar-GDP to GTP!
How does Sar1’s Amphiphilic tail orient itself in the membrane?
It is monolayer associated, on the cytosolic side! (When in GTP/active form)
What adaptor proteins does Sar1-GTP recruit?
Sec 23/24
(24 binds to cargo receptors and they both start invaginating the membrane)
What outer coat proteins do Sec 23 and 24 recruit?
Sec 13/31! They form the outer coat and vesicle pinches off by an unknown GTPase
Creates “COP 2-coated vesicle “
How does COP2 Uncoating occur?
COP2 coat stimulates the GTPase activity of Sar1, so that it goes from GTP to GDP.
-once hydrolized, coat then disassembles and Sar1-GDP returns to cytosol in its inactive form
What keeps Sar1 from creating vesicles in places other than the ER?
It’s Sar1 GEF is embedded in the ER membrane, so it only becomes active (GTP form) at the ER membrane and then the Amphiphilic tail is immediately exposed and monolayer associated with the ER membrane
What is an example of a slow GTP-ase?
Sar1-GTP
- a molecular timer that begins when the tail associates with ER membrane
What’s an example of fast GTP-ase?
Dynamin, when it uses GTP to undergoes fast hydrolysis to tighten corkscrew rapidly
Are vesicles always round?
No! Depending on the cargo, the shape of the vesicle may be tubular to accommodate larger cargo (ex: procollagen is packaged this way)
What is the COP 1 (retrieval pathway) for making vesicles?
1: Starts when Arf1-GTP embeds in membrane with Amphiphilic tail (the exact same as SAR, except in Golgi NOT ER membrane!)
2: an unknown GTP-ase pinches off the vesicle
How do proteins get where they’re supposed to go after budding?
Rab proteins!
What are Rab proteins?
Proteins that are GTPases expressed on the surface of membranes
-different organelles have different Rabs
-there are over 60 known Rab proteins, but still unknown how they are incorporated into the correct membranes
What do Rab proteins do?
They guide vesicles!
-they get vesicles to the membrane they are supposed to get to
Where are inactive Rabs (Rab-GDP) found?
In the cytosol!
-their inhibitor GDI keep them from exchanging into GTP
What is GDI-GDP?
GDI= GTPase Dissociation Inhibitor
-keeps Rab-GDP in cytosol from turning into Rab-GTP
How do Rabs get activated into Rab-GTP on the vesicle and target membrane?
Rab-GEFs activate Rabs on vesicle and target membrane!
-active Rab- GTP then binds to membranes
What is a Rab effector?
1: Tethering protein on the target membrane that reaches out to the “grab the vesicle” (shown as green rope)
2: also includes “motor proteins” that move vesicles along cytoskeleton
What do SNAREs do?
Mediate membrane fusion!
-actually “ties” the rope to the “deck”
-act like twisty tie that fuse membranes of vesicle and membrane
What are the complimentary pair components in which Snares exist?
v-SNARE on the vesicle membrane and t-SNARE on the target membrane
-complimentary are attracted to and bind it each other!
-only exist in the cytosol!
How many different SNAREs are in animal cells?
At least 35!
How does the helical bundle nature of SNAREs exclude water?
The helical domains of v- and t- SNAREs wrap so tightly around each other that the water head that is binded to polar head groups is removed and a bundle is formed that is able to lock the membranes together
-asymmetry of outer and inner membrane is maintained in this fusion!
Do disassembly of SNAREs require energy? Why or why not?
Yes! Bc helical bundles are an energetically favorable structure, pulling them apart requires action of NSF protein, an ATPase
How does the NSF protein work?
It undoes the SNAREs helical bundles by “untwisting the twisty ties” using ATP and sends them out to be recycled
Does the acrysomal process use membrane fusion?
Yes!
What is an example of a virus that “hacks” our membrane fusion?
HIV!
Where are SNAREs found?
Only on the cytosolic side of our vesicles and organelles! (AKA inside the cell!)
Do Viruses use our SNAREs or make their own to get into our cells?
Trick question! Since SNAREs are only in cytoplasm in cytosolic side of organelles, viruses use their own mechanism to get into our cells
How are Transmembrane and soluble proteins incorporated into COPII vesicles (ER to golgi and out)?
Transmembrane proteins: directly interact with COPII coat
Soluble proteins: use “exit” ticket to bind to receptor on cargo receptors to be included in vesicle
Where are the first vesicles made in the COPII pathway?
In the smooth ER!
What will most proteins leaving the ER have?
An exit signal! (We don’t know what it is)
What must proteins be before editing the ER via the COPII pathway?
Properly folded!
What is it called when Vesicles from the ER fuse en route to the Golgi?
They form VESICULAR TUBULAR CLUSTERS (VTCs)
-happens when ER vesicles come together on way to the Golgi
What is Retrieval Transport?
When VTCs (vesicular tubular clusters) bud off their own transport vesicles and return escaped proteins and proteins needed only for budding back to the ER
How is the microtubule involved in vesicular transport?
It is the “super highway” of the cell!
-everything that needs to be moved in the cell is moved through a microtubule and bound by a MOTOR PROTEIN
What is a motor protein?
The protein that binds VTCs to microtubules (the “walking” little guy in the video)
What causes the retrieval pathway to occur?
KDEL receptors for leaked ER resident proteins!
How are leaked ER resident proteins retrieved?
-when they accidentally get enclosed in transport vesicles they are bound by KDEL receptors (activated once pH dips after leaving ER)
-leaked proteins then sent back to ER in COPI vesicles derived from VTC
When are proteins sent back to the ER via the retrieval pathway?
Both when in VTCs and from the Golgi
(Along the way and at destination)
Where do KDEL receptors originate?
In the ER!
-they are transmembrane proteins that get sent out in vesicles to screen for leaked proteins
- turned on by dip in pH that occurs when vesicles leave ER, and even more so once in Golgi
What is the Anterograde transport?
The COPII pathway! ER to Golgi and out
What is Retrograde transport?
COPI transport, “retrieval pathway”! From Golgi back to ER bc of KDEL signal
What forms inner vesicle coat in endocytosis?
Adaptins! (External coat is Clathrin)
What is major difference between Arf1 and Sar1?
Arf1 is in Golgi membrane, Sar1 is in ER membrane! (Otherwise works exactly the same)
Rab versus SNAREs?
Rab gets the vesicle to the membrane, the SNAREs actually “tie”/fuse them together!
What are the three parts that make up the Golgi?
1: Cis Face (closest to ER)
2: medial cisterna (proteins get modified as they pass through here)
3: Trans Face (faces outside the e cell, proteins secreted this way)
How do VTCs save energy?
Because it only requires one motor protein to transport it down the microtubule to the Golgi
What are the three places the Golgi can send out to?
1: Lysosomes
2: Plasma Membrane
3: Secretory Vesicles
Does a protein change from the time it enters the Golgi to the time it leaves?
Yes! It gets modified a lot as it passes from Cis FACE to trans FACE
How does a lysosome maintain its low pH?
A lysosome maintains its 5.0 pH bc of the ATPase pump that pumps H+ against its gradient into the lysosome
What is the pH of the lysosome versus the pH of the cytosol?
Lysosome = 5.0
Cytosol = 7.2
Why are internal membrane proteins highly glycosylated in the lysosome?
In order to both protect them from the acid hydrolases inside the lysosome as well as from the high acidity
How do Acid Hydrolases work in the lysosome?
They break stuff down inside the lysosome, confined to there bc they only work in highly acidic environment!
Ex: nucleases, proteases, lipases (don’t memorize)
How are monomers recycled from the lysosome?
Transporter pumps embedded in the membrane recycle these monomers after the macromolecules are broken down
(Ex: nucleotides, amino acids)
What are the 3 sources of Digestive Material that can fuse with Lysosomes?
1: Autophagy (self eating- cellular structures)
2: (Receptor-Mediated) Endocytosis: (Endosomes from cell membrane)
3: Phagocytosis (only some cells can do it, large stuff from outside the cell)
What is one minor way that material can fuse with lysosomes?
Pinocytosis
“Cellular drinking”
-continual pulling of small amounts of extracellular fluids
What occurs in Autophagy?
“Self eating”
Ex: if a mitochondria is not working anymore, it is autophaged and destroyed for its energy
-it is engulfed into a vesicle and then fused to lysosome, lysosome then breaks it down
What occurs in Receptor-Mediated Endocytosis?
-Clathrin mediated process of endocytosis to form a vesicle
-receptors and ligands separate, receptors are sent in a vesicle via exocytosis back to the membrane
-cargo alone in naked vesicle is sent to either the Golgi for sorting or lysosome for destruction/freeing
How is LDL internalized?
Via Receptor-Mediated Endocytosis!
What is HDL?
High Density Lipoprotein
“Good cholesterol”- scavenges LDL from arteries and prevents clogs
-20% cholesterol, 50% protein, 30% monolayer
What is LDL?
Low Density Lipoprotein
“Bad cholesterol”-clogs arteries
(Most common)
-50% cholesterol, 20% protein, 30% monolayer
Why is Cholesterol important to the body?
It is necessary for cell membranes and for making steroid hormones
How is Cholesterol transported?
Through the bloodstream by carriers called lipoproteins! (HDL and LDL)
What is a lipoprotein?
Transports Cholesterol in the form of HDL or LDL!
-lipoproteins = 1.5K esterified cholesterol molecules surrounded by proteins and a lipid monolayer
How does the cell take in Cholesterol?
It inserts LDL receptors into its plasma membrane, which LDL bind to and begin and Clathrin-mediated endocytosis process
-endosome then sent to lysosome, where enzymes convert the cholesterol esters to free cholesterol!
-empty LDL receptors are returned to cell membrane
How many cisternae (flattened sacs) are present in a Golgi?
3-20
How many sets of Golgis can be in a cell?
1-100!
Where is the origin of Clathrin? (Where does it originate)
The plasma membrane!
Where is the origin of Sar1/COP2?(Where does it originate)
ER lumen!
Where is the origin of Arf1/COP1? (Where does it originate)
The Golgi lumen!
Where do we get most of our cholesterol from?
Most is made in our liver! (Only a small percent comes from our food)
What separates receptors and cargo in endocytosis and allows for receptors to be sent back to the plasma membrane?
A low pH in our endosomes!
What is the projected prevalence of Cholesterol and cardiovascular disease in 2035?
About 50% of the population having some form of it
What are healthy cholesterol levels?
Both:
-under 100 mg/dL LDL cholesterol
-over 50mg/dL HDL cholesterol
What is the main “fighter” we have against CVD?
Statin therapy!
How does Statin therapy lower serum cholesterol? What’s an unwanted therapy of this treatment?
It blocks cholesterol biosynthesis in the liver!
-unfortunately, it targets an enzyme way upstream of cholesterol, so stops a bunch of other stuff from being made too. Most severe side effect = skeletal muscle myopathy
How can Statin therapy prevent CVD?
-modulates inflammatory response (reduces inflammation)
-maintains plaque stability
Prevents thrombus formation
-improves endothelial function
Who can perform phagocytosis in our bodies?
Macrophages and neutrophils!
What is Phagocytosis?
A specialized form of endocytosis employed by phagocytic cells of the immune system (macrophages and neutrophils)
What are the general steps to phagocytosis?
-bear hug pulls external organism into phagosome inside the cell
-that phagosome then sent to lysosome!
What are the two types of secretion?
1: Constitutive secretion (common- secretes immediately after production)
2: Regulated secretion (specialized hormones/neurotransmitters-held until signal, then secreted)
What type of secretion is in non-polarized cells?
Constitutive secretion!
-if secretory vesicle is not tagged, it will leave the trans Golgi and fuse with the cell membrane in non polarized cells
What is are two examples of Regulated secretion?
1: Mast cells that make histamines!
-they hold them in vesicles near the PM until signal causes mass release
2: neurons that emit neurotransmitters!
What special pathway do neurons have?
-a special local recycling system for neurotransmitters
-“re-uptake” = goes to endosome, then sent back to wait at PM (don’t need to go back to Golgi or use motor protein/microtubule!
-much faster this way
How is phosphotidylinositol (PI) given different signals?
It is PHOSPHORYLATED at particular ring positions by KINASES unique to each organelle
-coat proteins then specifically bind to these PHOSPHOTIDYLINOSITOL PHOSPHATES to target the vesicles
What does Dynamin force to fuse?
The two inside leaflets of the vesicle membrane!
How does Dynamin regulate the rate of its pinching off?
With its GTPase domain!
What depletes PI(4,5)P2 from the membrane during endocytosis disassembly?
PIP phosphatase!! (
Opposite bc kinase phosphorylated them to turn them on!)
What strips Clathrin coat off of vesicle?
Hsp70 chaperones in the cytosol! They act like ATPase and force Clathrin coat off
Is SAR 1 transmembrane?
No! It is monolayer associated with ER membrane
What is the physical difference between Sar 1-GDP AND Sar1-GTP?
In GDP: the Amphiphilic tail is hidden within Sar1 itself
In GTP: the Amphiphilic tail is out and ready to monolayer associate in the ER membrane
What the heck are rabs doing in their free time?
Rab-GDP is being lazy in the cytoplasm just chillin, it’s bad influence friend GDI is inhibiting it from doing anything
-Rab-GEF turns it into Rab- GTP and telling it to get to work
-Rab GTP then becomes active and binds to membranes
What are the two “groups” of effectors?
1: motor proteins (move vesicles along cytoskeleton)
2: tethering proteins (linking two membranes)
Is assymetry of the membrane maintained during membrane fusion via snares?
Yes!
What is the NSF protein?
An ATPase that pulls apart SNAREs
Do COP I and COP II use VTCs?
Yes!
How many Golgis are in 1 cell?
1-100!
Does the Lysosome’s ATP pump pump protons (H+) into or out of the Lysosome?
Into the lysosome! This is how it maintains its acidic pH
Define autophagosome?
The dead organelle engulfed in the cells vesicle heading to the lysosome
Is constitutive or regulatory secretion the default in lack of “tagging”?
The constitutive is default!
How do rabs go from cytoplasm to the membrane?
They go to their GEFs that are in the vesicle and target membrane to get turned on!
How many mannoses are chewed away?
4! Once these are chewed away you’re done!
What are pips?
Phosphorylated phospholipids! Kinases phosphorylated different carbons on the ring structure to attract different things
