Intracellular Compartments and Vesicular Transport

Question 1

Describe the process of transport vesicle cargo selection, budding, transport, targeting and fusion

Answer 1

1) Transport Vesicle Cargo Selection: This is done via receptors in the ER lumen for example that bind to completely folded proteins are target them for export. The protein binds to an exit signal on a receptor in the ER lumen. Then Sar1-GDP is converted to Sar1-GTP via a Sar1-GEF. The Sar1-GTP can then associate with the membrane. This then allows for the COPII to bind to the tail of the cargo receptor and the Sar1-GTP to form a COPII coat that signals the vesicle to leave.
For COPI, which is transport back to the ER from the Golgi, the same practically occurs in which a KDEL (Lysine, Aspartic acid, Glutamic Acid, Leucine) sequence on the incorrect proteins that should be in the ER but are now in the golgi, is recognized by a receptor. Then a COPI coat forms by binding to this receptor and transport back to the ER occurs.
For transport from the cell membrane or from the trans-golgi out, a clathrin coat is formed. Cargo binds to the cargo receptor which then allows adaptor proteins to bind. This then allows for the clathrin coat to form.

2) Budding: This process involved a Dynamin protein that uses the energy from ATP to pinch the budding membrane that formed from the coats off completely
3) Vesicle Transport: Vesicles have an associated Rab-GTP and Rab effector with a motor protein associated with it. The motor protein then binds to the cytoskeleton to move the vesicle to its destined location
4) Targeting and fusion is determined by other Rab proteins and phosphoinositols that “paint” it. The Rab recruits Rab effectors (v-SNARES) that will bind to Rab effectors on the target membrane called t-SNARES. This helps to tether the vesicle to the target membrane and induce fusion.

Question 2

Explain how coat proteins and selective vesicular transport help maintain the specialized character of various membrane-enclosed compartments within a eukaryotic cell.

Answer 2

First off, different coat proteins target the vesicle for a specific location. For example, COPII targets an ER vesicle to the golgi, COPI targets a golgi vesicle to the ER, and Clathrin moves exo- and endocytotic vesicles between the cell membrane and the trans-golgi network. This is a very specialized function. Also, there are other features that “paint” the vesicle for a specific location in the cell or to a certain target via Rab-GTP and Rab effectors as well as through phosphoinositols.

Question 3

Identify Sar as a GTP binding protein that participates in the assembly of protein coats

Answer 3

Sar1-GTP is a classic “coat recruitment” GTPase. It gets activated for membrane binding by a Sar1-GEF, which exchanges GDP for GTP, which exposes hydrophobicity, and marks the transitional ER for COPII binding. Sar1-GTP sticks into the cytosolic face of the ER membrane, and forms a site to which COPII coat subunits bind (these are the outer shell parts of the vesicle). This vesicle is just about ready to bud out from the transitional ER, along with its selected cargo, and once budded, will be on its way to the cis-Golgi.

Question 4

Explain how Sar1-GTP is recruited to the cytosolic surfaces of membranes

Answer 4

The Sar1-GTP is originally in the form of a Sar1-GDP that cannot bind to the membrane. However, in a budding membrane there is a Sar1-GEF that will exchange the GDP for a GTP and form an active Sar1-GTP that can now bind to the membrane and recruit COPII with the protein binding receptors

Question 5

Describe the function of the auxiliary proteins guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP) in coatomer coat assembly and disassembly

Answer 5

The GEF will specifically exchange the GDP on Sar1-GDP to form the Sar1-GTP. This forms an active protein that will bind to the membrane and then bind the COPII in this case. Sar1-GEF will have a specific function to dephosphorylate the Sar1-GTP to get Sar1-GDP that will then dissociate from the membrane and disassemble the COPII coat.

Question 6

What sequence is on the proteins destined for the ER Lumen?

Answer 6

KDEL sequence (Lysine, Aspartic Acid, Glutamic Acid and Leucine)

Question 7

Describe the role of the Golgi Apparatus in the retention of ER resident proteins by the ER.

Answer 7

The Golgi has receptor proteins that bind to the KDEL sequence of these ER Lumen resident proteins that were accidentally incorporated in the transport vesicle. In this case, the receptors will bind the proteins and thus then recruit COPI which marks it to move back to the ER. This continues to occur throughout the golgi complex to lead to a very specific concentration of the desired proteins.

Question 8

COPII

Answer 8

mediate protein transport from the ER to the Golgi

Question 9

COPI

Answer 9

Participate in retrograde transport of proteins from the golgi back to the ER and between golgi compartments

Question 10

Clathrin

Answer 10

Part of endo- and exocytotic vesicles that move between the plasma membrane and the Trans-Golgi network

Question 11

Describe the role of COPI in retrograde transport

Answer 11

COPI is the coat that will surround the vesicles that contain the ER lumen proteins that will then send them back to the ER.

Question 12

Describe the Clathrin structure, assembly, and its role in vesicle formation

Answer 12

The clathrins have three-fold symmetry and are called “triskelions”. The triskelions have homotypic assembly activities, they actually assemble into geometric shapes called icosahedrons (you can see icosahedrons in playgrounds, as jungle-gym setups). The shells, as depicted in EM, have a honeycomb structure and thus the vesicles are more or less similar in volume.

• Clathrin is an interesting protein because it is a triskelion with three-fold symmetry. It is capable of assembling into a network and forms a honeycomb like structure
• It contributes to the bending of a membrane to form a vesicle.

Because of the structure of clathrin, the vesicles are a predetermined volume.

Part of endo- and exocytotic vesicles that move between the plasma membrane and the Trans-Golgi network

Question 13

Describe the function of adaptor proteins (AP Proteins) in concentrating receptors and their cargos in clathrin coated membrane domain and membrane vesicles

Answer 13

The adaptor proteins associate with the cargo receptor proteins, concentrating the amount of cargo receptor proteins and thus cargo. Furthermore, the adaptor proteins are what recruit the clathrin, which binds to the adaptor proteins and begins to form the vesicle. If the substrate binding receptors cannot associate with the adaptor proteins, then the clathrin will not coat the vesicle and there will ultimately be no formation of a vesicle and thus no protein transport

Question 14

Describe the role of dynamin in the formation of clathrin coated vesicles

Answer 14

Dynamin is a snake-like protein that is crucial at the final stage of vesicle formation to finally “pinch” the vesicle off and get vesicle formation. ATP hydrolysis drives the tightening of the dynamin coils to pinch the membranes together to form a vesicle. If the Dynamin function is blocked, you will not get complete budding.

Question 15

Describe how defects in LDL receptors cause atherosclerotic disease

Answer 15

If the LDL receptors are defective in the sense that the cannot bind to the adaptor proteins, then the LDL will bind to the receptor in the extracellular space, but the adaptor proteins will then not be able to associate with the LDL receptor and thus the clathrin will not be able to bind to bring the LDL into the cell. Thus, this will result in the LDL remaining outside the cell and building up, causing disease.

Question 16

Identify the various compartments of the Golgi Apparatus

Answer 16

ER

1) Cis golgi network
2) Golgi Stack
1) cis cisterna
2) medial cisterna
3) trans cisterna
3) trans golgi network

Question 17

Explain the relation between the Golgi compartmentalization and oligosaccharide processing

Answer 17

Each Golgi stack has particular enzymatic functions that operate primarily on the N-glycans, trimming the glycans down and rebuilding them in different ways. This is one reason why glyco-biology is so amazingly complex. In the end, the proteins get sorted into vesicles at the trans golgi network. These vesicles go either to lysosomes, plasma membrane or stay as secretory vesicles (bottom).

Vesicles come in from the transitional ER to the Golgi

Each golgi stack performs a different function and each is a metabolic entity that is processing the cargo into different forms and now it is mature cargo

Question 18

Compare and contrast constitutive and regulated exocytosis

Answer 18

• Some cargo makes it out into the trans-golgi network.
• The trans-golgi has to separate cargo into these forms:

1) Unregulated or Constitutive pathway: The vesicle fuses with plasma membrane and out goes the cargo. Cells constantly need albumins and antibodies so these would be secreted in an unregulated or constitutive fashion. It is not regulated and constantly occurring
2) Regulated Pathway: Some cargo has to build up and wait for a signal such as neurotransmitters and insulin. These has to be accumulated specifically in secretory vesicles and stay there until it receives a signal. Once it receives a signal it will fuse with the plasma membrane and release the cargo. This is regulated and only occurs when there is a signal.

Question 19

Explain how bidirectional vesicular transport can concentrate cargo into secretory vesicles

Answer 19

Finally, it is worth remembering that there is vesicle traffic both to and from secretory vesicles as they mature (as in (A) above), very similar to the bidirectional vesicle transport between ER and Golgi. The forward transport brings the secretory cargo into the vesicles and the reverse transport takes away unintended interlopers. The result is an ever increasing concentration of the desired secretory cargo in the vesicles. This is the same sort of “distillation” process that was alluded to in previous slides. Thus these secretory vesicles become filled up with homogenous material, and show up as very electron dense organelles in electron micrographs (as in the rightmost and bottom images). Signal-mediated release of this cargo generates biological responses. We are also getting the removal of membrane too which confines the proteins that are being transported to a smaller volume and thus further concentrating them

Of note, some secretory cargo molecules are very small, for example, enkephalins and endorphins are only a few amino acids long. Secretory cargo of this sort has to be cotranslationally synthesized into the ER as a much longer protein (recall the previous lecture on cotranslational transport into the ER). The mature secretory cargo is then produced by proteolysis of the signal peptide in the ER, and then by additional proteolysis of the pre-protein in the TGN or the secretory vesicle itself, to generate the smaller cleaved active form. There is no slide for this final note, but it is an important point that is valuable to your understanding of hormone and cytokine secretion.

Question 20

Explain how and extracellular signal can stimulate regulated extracellular exocytosis

Answer 20

The extracellular signal can create an intracellular response that will eventually cause the membrane to be “allowed” to fuse with the cell membrane and release its contents into the extracellular space. However, there are a multitude of cell signals that can cause this response

A classic example of this is with neurons. Extracellular signals may cause an influx of calcium, which stimulates dissociation of “clamp” type proteins that regulate fusion of vesicles with the plasma membrane. Once the clamps are released, there is membrane fusion, and the components of the secretory vesicle are released. This is far different than constitutive release of proteins such as albumins or antibodies, where the vesicle fusions are temporally unregulated

Question 21

What are the functions of the phosphoinositides and the Rab proteins?

Answer 21

To help ensure that newly formed transport vesicles dock to and fuse with the correct target membrane

Question 22

Explain how phosphoinositides can be used to identify distinct organelles

Answer 22

The phosphoinositides “paint” the cytosolic side of the organelle’s membrane and the transport proteins. They contain an inositol sugar with 5 hydroxyl groups that are capable of being phosphorylated in many different combinations. This allows for distinct “marks” on certain membranes that specific proteins can recognize. These are very specific markings that each organelle and vesicle gets a build up of.

Question 23

Describe how Rab proteins and their effectors (Rab effectors) are thought to function to tether transport vesicles to their correct target membrane

Answer 23

The Rab-GTP proteins can bind to a membrane and then recruit Rab effectors such as the SNARES. The Rab effectors are critical in identifying other Rabs on the membrane of their target cell. The Rab-GTP on the target membrane will bind these Rab effector fibrils on the vesicle and effectively tether the vesicle to the target membrane. The v-SNARES and t-SNARES can then intertwine and get effective fusion of the two membranes.

Question 24

Explain how Rab proteins function to determine the specificity of vesicle transport

Answer 24

There are many different Rabs that function and each organelle and vesicle has a specific Rab-GTP and Rab effectors that help facilitate this specificity

Question 25

Compare the activation of Rab proteins to that of other GTP binding proteins such as Sar

Answer 25

They use a both GEF that will substitute the GDP for a GTP and thus activating it. The GAP will dephosphorylate it and thus inactivate it

Question 26

Describe the contribution of v-SNAREs and t-SNAREs to vesicle-membrane fusion

Answer 26

The v-SNAREs and t-SNAREs form a coiled-coil with one another to successively bring the vesicle close to the membrane and cause the fusion of the two.

When the vesicle gets close to the target membrane, via Rab-GTP binding to Rab effector fibrils, then the v- and t-SNAREs can intertwine in ways that get the vesicle so close that it spontaneously fuses. The cargo is then available for release into the target organelle lumen.

• The Rabs and Rab effector will help the vesicle tether to their correct target membrane.
• Then, SNARES promote fusion of the vesicle with their target
• There are v-SNARES (vesicular) and t-SNARES (target)
• The v-SNARE is very important in the final stages of vesicle binding and fusion
• The Rab effectors allow for appropriate docking of the vesicle to the target organelle
• The target organelle itself also has associated Rab proteins and they are associated with t-SNARES.
• The v-SNARES and the t-SNARES with interweave in a coiled-coil alpha helix structure
• These clamp down to pull the vesicle so close to the target that it will fuse.
• You are then leftover with the Rab effectors or SNARES all on the target membrane and have to get unwound and each piece moved back to the appropriate place to be reused

Question 27

Describe the function of NSF and ATP in the recycling (re-use) of v- and t-SNAREs

Answer 27

The NSF with accessory proteins will help pull the SNAREs apart from one another. This requires ATP to pull them apart.

Since some neurons have to “fire”, through neurotransmitter release, up to 1000 times per second, the SNAREs mediating the fusion process have to be re-used many times. To do this, they have to be pried apart and each part sent back to its v- or t- location. The prying apart is accomplished by accessory proteins and NSF. What matters here is that ATP energy is required to get the SNAREs apart from each other.

• These SNARES have to be taken apart to be reused for another round of membrane fusion.
• You could just make new SNARES but that would very very energetically inefficient and take too much time
• The SNARES are taken apart by accessory proteins and NSF protein and REQUIRES ATP AND A LOT OF ENERGY because the coiled-coil is very stable. The SNARES then dissociate. It is not yet known how the v-SNARE goes back to a new vesicle.

Question 28

Identify how vesicle fusion can be targeted with therapeutic drugs to achieve cosmetic effects and alleviate migraine headaches

Answer 28

Depicted here are neuromuscular junctions, where the SNAREs that fuse acetylcholine-filled vesicles are releasing acetylcholine to muscle cells. Of note, BOTOX, which is repurposed botulism toxin, can bind to neurons at these junction points, transfer a toxin protease across the neuronal cell membrane, which then cleaves the SNAREs. The result is paralysis, as Ach vesicles cannot fuse.

If there was a neurotoxin like Botox that cleaves these Rab effector SNARES, then the vesicles could not fuse to their target site –> It would paralyze this region now