Membrane Traffic

Question 1

What is membrane vesicular trafficking and what are the 2 major players of it?

Answer 1

Vesicular trafficking moves between topologically equivalent spaces. The two major players in vesicle trafficking are Phosphoinositols/phosphoinositides and
GTPases

Question 2

What is inositol?

Answer 2

Inositol is a 6 carbon. sugar that is an isomer of glucose.

• Adding one phosphate group to the first carbon: Inositol-1-phosphate
• Adding three to 1st, 4th, and 5th carbon: inositol triphosphate

Question 3

What are PIP kinases and PIP phosphatase?

Answer 3

PIP Kinases adds a phosphate to inositol phosphates while PIP phosphatase removes phosphate from an inositol phosphate

Question 4

What are GTPase and what are the two types?

Answer 4

GTPases are a family of proteins that act as a molecular switch. The two types are:

1) GAP: GTPase activating protein - converts GDP to GTP to turn on process
2) GEF: Guanine Exchange Protein - Removes phosphate from GTP to for GDP to turn off process

Question 5

What are the 3 endosomal system pathways in a cel?

Answer 5

1) secretory pathway: ER to Golgi, endosomes and plasma membrane
2) endocytic pathway: plasma membrane, endosomes, lysosomes
3) retrieval pathway: returning to originating compartment

Question 6

What is the secretory pathway?

Answer 6

secretory pathway: ER–> Vesicles–> Golgi–> Vesicles–> Plasma membrane

Question 7

What is the endocytic pathway?

Answer 7

endocytic pathway: Plasma membrane–> vesicles–> early endosomes–> late endosomes–> endolysosomes–> lysosomes

Question 8

Why is the retrieval pathway important for the cell?

Answer 8

In the transport of vesicles, there may be ER cargo that has been accidentally removed by the er by vesicles
This pathway is for cargo to go back to where they belong

Question 9

What are the three vesicle “coats” that mediate budding

Answer 9

Coating proteins concentrate in part of the membrane
Outer coat shapes membrane
Inner coat determines cargo

Question 10

What are the 2 main outer coats?

Answer 10

Clathrin (clathrin-coated vesicles)- from PM, from Golgi > endosomes, from endosomes 
COP I (coat protein 1 coated vesicle)– within Golgi and Golgi -> ER
COP II (COP coated vesicle) – ER -> Golgi

Question 11

What does each clathrin triskelaton consist of?

Answer 11

• 3 large heavy polypeptide chains

- 3 small light polypeptide chains

Question 12

Is it easy for clathrin triskelatons to form light and heavy chains?

Answer 12

Yes; because they are at a lower energy form confirmation (getting it to a none-cage requires energy)

Question 13

How are vesicles formed by the coats?

Answer 13

Cargo receptors bind to cargo from the cytoplasmic side. Inner coat proteins start to bind to the cargo receptors. As we get more and more receptors and cargo binding to these inner coat proteins, they start to form a spontaneous reaction and start to bind the outer coat (clathrin). Then they start to form the cage
-The more and more clathrin we get, it starts to form the membrane and form a proper vesicle

Question 14

How does the vesicle pinch off the membrane?

Answer 14

GTPase dynamins and other proteins wrap around the neck of the vesicle and pinch it off. While the actual formation of the vesicle does not require energy, the pinching off of it does (GTP hydrolysis)

Question 15

How is vesicle formation regulated?

Answer 15

Adaptor proteins can determine what cargo receptors will bind with what coat protein. Binding of adaptors and other proteins involved in vesicle formation is regulated by interactions with other membrane components:

1. Lipid markers
2. Coat Recruitment GTPases

Question 16

What are lipid markers in the regulation of vesicle formation?

Answer 16

PI/PIP kinases and PIP phosphates to form different phosphoinositols. Different PIPs bind and recruit different proteins.

Question 17

How do Adaptor proteins select cargo into Clathrin-coated vesicles using PIPs?

Answer 17

Adaptin proteins start in a closed confirmation, so cargo receptors are unable to bind them. If adaptin is able to bind PIP2, is when it becomes active and open for cargo receptors to bind to.
-This is called cooperative assembly; when we get a gethering in one area)

Question 18

How is the clathrin coat lost after budding (2 ways)?

Answer 18

1) PIP phosphates: depletes PIP2

2) Hsp70: chaperon ATPase helps remove clathrin (takes energy)

Question 19

What are Coat recruitment GTPase in vesicle formation?

Answer 19

Coat recruitment GTPase regulate COPI and COPII assembly: Sar-1 has an amphiphilic alpha helix attatched to it which will bind to the membrane when Sar-1 is activated. When GDP is attached to Sar-1, it is inactive and will have to bind to Sar1-GEF in order to bind GTP for activation (GEF will stimulate release of GDP for it to convert to GTP). Inner coat. adaptor proteins will only be recruited if Sar1-Gef is in the area.
-Eventually, GTP will hydrolyzed to form GDP again and leave the membrane

Question 20

How is membrane fusion mediated? What are the types?

Answer 20

SNARE Proteins. There are two types of SNAREs:
1) V - (vesicle) SNARE
2) T - (target) SNARE
SNAREs are found on both the vesicle that is going to fuse, and the target membrane that the vesicle is going to fuse with. The interactions and winding between these SNAREs alpha helices will bring the membrane vesicle down to the target membrane so they can come together and create the fusion.

Question 21

How do vesicles identify the correct target membrane for fusion?

Answer 21

RABs

Question 22

What are compartment doners and compartment targets?

Question 23

What is the difference between V-SNAREs and T-SNAREs?

Answer 23

V SNARES are typically single polypeptide.

T SNARES are typically 3 polypeptides that are on the target membrane

Question 24

How are RAB monomeric G proteins involved in vesicle fusion?

Answer 24

Rab proteins on vesicles and target membrane guide transport vesicles to their target membrane
- Rab effectors include motor proteins, docking/tethering proteins

Question 25

What is the process of vesicle fusion using RAB?

Answer 25

When RAB needs to be turned on, it will activate by interacting with RAB-GEF to give up GDP for GTP. The vesicle with RAB GTP will become close enough to the target membrane where RAB can bind its partner protein (a RAB effector). These effectors help RAB capture the vesicle to bring it close enough to mediate SNARES to attach to each other -Once fusion happens, RAB is not needed

Question 26

Can SNAREs be reused?

Answer 26

Yes. Though ATP is needed to unwrap the SNAREs and separate them before being functional again

Question 27

How does the COPII Coated Vesicles leave ER from special ER exit sites(sites lacking ribosomes)?

Answer 27

ER-Golgi Intermediate Compartment (ERGIC) - After being released, COPII coated vesicles will uncoat, fuse together and form an intermediate compartment. The intermediate compartment starts to change in its content because it's picking up stuff and generating new vesicles

Question 28

What are 4 materials that have been returned back to the ER

Answer 28

1) ER membrane proteins 2) ER soluble proteins 3) Cargo receptors 4) SNARE proteins

Question 29

What are the two major functions of the Golgi compartments?

Answer 29

1. Carbohydrate Synthesis | 2. Sorting and Transfer Station

Question 30

What is the principle site for intracellular digestion/degradation?

Answer 30

Lysosomes --> they contain hydrolytic enzymes that are very acidic The environment in here gets acidic due to a proton pump; this pump drops pH --The type of pump is the V-type pump: Lysosomes are generating products by break down, that need to get back to the cytoplasm

Question 31

How does the cell know to dump the hydrolases and the vesicles to go to the golgi but not the plasma membrane?

Answer 31

Golgi-Lysosomes: The golgi carries enzymes that will attach a sugar molecule (manose-6-phosphate) on lysosomal dydrolase which will result in the formation of a vesicle that will be targeted to the golgi. The sugar is recognized by receptors that will bind inner coat proteins.

Question 32

Where does mannose-6-phosphate sort lysosomal hydrolase?

Answer 32

Trans Golgi Network

Question 33

Once the coat comes off, What do proteins like SNAREs and RABS help vesicles fuse with?

Answer 33

Late endosomes and lysosomes

Question 34

Why does the manose-6-phosphate receptor disattach from the vesicle?

Answer 34

As vesicles move through the endosomal system, the pH drops, increasing acidity. As pH drops, there is a conformational change between the receptor or hydrolases themselves and they release form each other, leaving hydrolases behind

Question 35

What are two pathways that deliver material to the endosomal system?

Answer 35

1) Endocytosis - -phagocytosis - -pinocytosis (ex. Receptor mediated endocytosis) 2) Autophagy

Question 36

What is endocytosis?

Answer 36

A type of pathway to bring material to the endosomal system: Things come in from outside of the cell wrapped in membrane from the plasma membrane. When big materials such a bacteria comes in, phagocytosis is required. Pinocytosis on the other hand bring in smaller materials like fluid and solute -This may involve cytoskeleton for larger molecules (acting and MTs)

Question 37

What is autophagy(self-eating)?

Answer 37

Autophagy (self-eating) is another pathway for materials to be brought to the endosomal system. This degrades and recycles items: When there is nothing coming into the cell and it stops to function, sometimes it will undergo autophagy and start to eat up its own internal membranes and organelles to digest and generate basic products -This pathway is also used for organelles that are not working as good anymore

Question 38

Why are recycling endosomes important?

Answer 38

The endosome serves as a storage and space for things the plasma membrane may need (like GLUT4 Transporters) Let's say you work out your muscles in the morning. Your muscle cells will start to really need glucose and transporters will start to bring in glucose. Once your muscle cells don’t need the glucose anymore, they will recycle the transporters and bring them in through endocytosis but instead of destroying them in the lysosome, they are rather trafficked into the recycling endosome. - Part of transcytosis pathway

Question 39

What are the two types of pathways for the Secretory pathway?

Answer 39

1. Constitutive secretory pathway - ---Continuous and is happening all the time in the cell 2. Regulated secretory pathway - ---Exocytosis does not happen immediately and occurs when a particular signal reaches the cell

Question 40

Where do Secretory vesicles bud from?

Answer 40

trans golgi network

Question 41

What does the signal for secretion typically involve?

Answer 41

The signal for secretion typically involves a change in calcium concentration in the cytoplasm - Ex; in the pancreatic cells that make insulin, insulin is packaged up by being held at the cell surface, - When the signal comes into the pancreatic cells to increase insulin, then there is fusion

Question 42

For many axons that use a neurotransmitter, how do the transmitters get there and how are they regulated?

Answer 42

We've got the formation of vesicles from the golgi, that have to be transferred all the way down to the axon and down to the nerve terminus The vesicles are filled with neurotransmitters once they reach the nerve ending (neurotransmitters are not released until they are told to by the axon) -Being told to means depolarization along the axon, the opening of voltage gated channels -As voltage gated sodium channels change as they are depolarized, and voltage gated calcium channels will be triggered to open and they will pull calcium across the membrane rapidly -The fast rise of calcium in the cytoplasm will cause the vesicles to fuse

Question 43

what is needed for rapid exocytosis for synaptic vesicles?

Answer 43

Synaptic vesicles are primed at the presynaptic plasma membrane. This involves the formation of the trans SNAREs complex. The two types of SNAREs will wrap around each other to mediate vesicle fusion but then stop about halfway.

Question 44

How are neurotransmitters released?

Answer 44

- The vesicles are first pulled down to the cell membrane and docks on to it - The initial portion of the interaction starts off and begins to fuze the membrane when SNAREs begin to interact - Full fusion is blocked by the activity of a protein called complexin that is going to prevent the SNAREs from completing that fusion activity by binding to SNAREs - Complexin is pulled of of SNAREs by another protein called synaptotagmin, so that vesicle fusion can be completed

Question 45

What is Synaptotagmin?

Answer 45

Synaptotagmin is a that is a calcium binder when calcium levels increase in the cytoplasm. This protein is also what removes complexin for vesicle fusion for neurotransmitters.