Cell Biology # 5

Front 1

How does budding happen?

Back 1

- membranes of lumen side pull away and get so close together that the lipids start to interact

Front 2

What are endosomes?

Back 2

- sorting compartments

Front 3

What is the lumen of each membrane-enclosed compartment topologically equivalent do?

Back 3

- the outside of the cell

Front 4

Why is it difficult to bud off from the plasma membrane?

Back 4

- because cholesterol is so stiff

Front 5

What are the three main pathways of intracellular vesicular traffic?

Back 5

1. Secretory pathway - proteins from ER to plasma membrane or lysosomes
2. Endocytic pathway - molecules from plasma membrane to endosomes or lysosomes
3. Retrieval pathway - molecules retrieved from vesicles and endosomes and taken back to the plasma membrane, ER or Golgi

Front 6

What are the three main characteristics of vesicular traffic?

Back 6

1. Highly organized
2. Balanced - between using ER, Golgi and plasma membrane for vesicles
3. Very selective - about what goes in vesicles

Front 7

How do inositol phospholipids work?

Back 7

- can undergo rapid cycles of phosphorylation and de-phosphorylation at the 3', 4' and 5' positions of their inositol sugar head groups to produce phosphoinositides (PIPs)
- can be used to control protein binding to a membrane and these PIP-binding proteins in turn help regulate vesicle formations

Front 8

How are PIPs named?

Back 8

PIPs are named according to the ring position and number of phosphate groups.

Ex. PI(3,4)P2 means that there are two phosphates on the 3' and 4' positions

Front 9

What are coated vesicles?

Back 9

- vesicles with a distinctive cage of proteins covering their cytosolic surface

Front 10

What are the two main functions of coated proteins?

Back 10

1. Selectivity of specific proteins
2. Helps to mold the membrane into a round shape

Front 11

What are the three types of coated vesicles? And where do they transport?

Back 11

1. Clathrin-coated - transport from Golgi and plasma membrane
2. COPI-coated - transport from ER and Golgi
3. COPII-coated - transport from ER and Golgi

Front 12

What is the make up of clathrin?

Back 12

- 3 large and 3 small polypeptide chains forming a triskelion
- spontaneously assemble to form a spherical structure

Front 13

What are adaptor proteins and what do they do?

Back 13

- form a second layer of the coat, positioned between the clathrin cage and the membrane
- trap transmembrane receptors, cargo receptors, that in turn capture soluble cargo molecules inside the vesicle
- each adaptor protein is specific for a certain protein
- can either be single chain or 4 subunits

Front 14

What is the process of creating a clathrin-coated vesicle

Back 14

1. Coat assembly and cargo selection
2. Bud formation
3. Vesicle formation
4. Uncoating - coat is really only important for forming shape and selecting proteins, need to get rid of coat so proteins can fuse with target

Front 15

What is the enzyme for budding off a vesicle?

Back 15

Dynamin

Front 16

What chaperone functions as an uncoating ATPase for clathrin-coated vesicles?

Back 16

Hsp70

Front 17

What are the two coat recruitment proteins?

Back 17

1. Arf 1 protein - for COPI and clathrin coat assembly at Golgi
2. Sar 1 protein - for COPII coat assembly at ER
- these protein's signals are activated by the addition of a phosphate group

Front 18

How does a COPII coated vesicle form?

Back 18

- Sar 1 protein is the coat-recruitment GTPase

- inactive soluble Sar1-GDP binds to a Sar-1 GEF in the ER membrane causing Sar1 to release its GDP and bind to GTP
- a GTP-triggered conformational change in Sar1-GTP exposes an amphipathic helix which inserts into the ER membrane initiating membrane curvature
- Sar1-GTP recruits COPII subunies to the membrane to form a bud (Sec 23/24 for the inner coat and Sec 13/31 for the outer coat)
- a membrane fusion event then pinches off the coated vesicle

Front 19

What are Rab proteins and what do they do?

Back 19

- monomeric GTPase
- largest family, more than 60 members
- direct the vesicle to specific spots on the correct target membrane
- when Rab is activated by GTP is associates with the membrane of a vesicle and bind to other proteins called Rab effectors which facilitate vesicle transport and tethering

Front 20

What are SNARE proteins and what do they do?

Back 20

- mediate the fusion of the lipid bilayers in vesicle formation

Front 21

What are v-SNAREs?

Back 21

- found on vesicle membranes
- made of 1 polypeptide

Front 22

What are t-SNAREs?

Back 22

- found on target membrane
- made of 2-3 polypeptides

Front 23

What is the trans-SNARE complex? What does it do?

Back 23

- formed by v-SNAREs and t-SNAREs wrapping around each other to form a stable four helix bundle
- this forces lipid bilayers close together and expels water molecules from between them so that lipid molecules can fuse together

Front 24

What happens after SNAREs have mediated the fusion of a transport vesicle to a target membrane?

Back 24

- the NSF protein binds to the SNARE complex and hydrolyzes ATP to pull the SNAREs apart

Front 25

How does the tetanus toxin work?

Back 25

- it cleaves SNAREs in nerve terminus so vesicles cannot fuse
- this blocks nerve transmission over synaptic clefts

Front 26

What is homotypic fusion?

Back 26

- fusion of membranes from the same compartment

Front 27

What is heterotypic fusion?

Back 27

- a membrane from one compartment fuses with the membrane of a different compartment

Front 28

What is the KDEL receptor?

Back 28

- present in vesicular tubular clusters and the Golgi
- it captures the soluble ER proteins and carries them in COPI coated transport vesicles back to the ER

Front 29

What is the Golgi apparatus? What are the two faces?

Back 29

- 4-6 stacks of membrane enclosed compartments
- cis-FACE faces the ER and receives arriving vesicles
- trans-FACE faces the outside and vesicles leave from here

Front 30

What are the main jobs of the Golgi?

Back 30

- carbohydrate synthesis (modify oligosaccharides in a certain sequence of steps)
- sorting of products from the ER
- proteins in Golgi are usually membrane bound ( in ER they are soluble)

Front 31

What are complex oligosaccarides?

Back 31

- generated when the original N-linked oligosaccaride added in the ER is trimmed and further sugars are added
- occurs if position on protein is accessible

Front 32

what are high-mannose oligosaccarides?

Back 32

- are trimmed in the Golgi but no new species are added
- happens when position is not-accesible

Front 33

What is the core region that remains in all oligosaccharides regardless of Golgi modifications?

Back 33

2 N-acetylglucosamine and 3 mannose

Front 34

What are the four purposes of glycosylation?

Back 34

1. Resistant to digestion by proteolytic enzymes
2. Proper protein folding
3. regulatory roles (ex. cell cycle)
4. Protects against pathogens

Front 35

What is a lysosome?

Back 35

- membrane enclosed organelles filled with hydrolytic enzymes that digest macromolecules in the cell
- pH inside is about 5.0

Front 36

How do lysosomes maintain a low pH (5.0) compared to the cytosol (7.2)?

Back 36

- use a v-type ATPase to pump protons into lysosome

Front 37

How are lysosomal membrane proteins protected from the hydrolases within the lysosome?

Back 37

They are highly glycosylated?

Front 38

What are three pathways to deliver to lysosomes?

Back 38

1. Pinocytosis (small molecules)
2. Phagocytosis (large molecules)
Both of the above two are examples of endocytosis of macromolecules from the ECF
3. Autophagy - recycling of molecules inside the cell

Front 39

Which vesicles are involved in endocytosis?

Back 39

clathrin-coated vesicles

Front 40

What are the two functions of phagocytosis?

Back 40

1. Defense - ingest invading microorganisms
2. Recycling - ingest dead cells

Front 41

What is the process of phagocytosis?

Back 41

- initiated by Rho-GTPases which turn on local PI kinases by phosphorylating them
- pseudopod formation and phagosome formation are driven by actin polymerization and reorganization

Front 42

What are the two main types of pinocytosis?

Back 42

1. Constitutive process - occurs constantly regardless of cell need
2. Receptor mediated endocytosis - more specific process to get certain molecules from ECF into the cell

Front 43

How does LDL pinocytosis work?

Back 43

LDL (low density lipoprotein) receptors bind to a clathrin-coated pit in the plasma membrane of a normal cell and attract LDL
- LDL is made of cholesteryl ester molecules which will then be hydrolyzed into cholesterol
- on clathrin coated pit can uptake up to 1000 receptors

Front 44

What are three possible fates of transmembrane receptor proteins?

Back 44

1. Recycling - retrieved receptors are returned to the same plasma membrane domain from which they came
2. Transcytosis - retrieved receptors are transported to a different domain of the plasma membrane
3. Degradation - by lysosomes

Front 45

What are the two pathways for exocytosis?

Back 45

1. Constitutive Secretory Pathway - operates in all cells, many soluble proteins are continually secreted from the cell by this path, also suplies the plasma membrane with new lipids and proteins, etc
2. Regulated secretory pathway - only in specialized cells, can secrete hormones, digestive enzymes or neurotransmitters etc
- signal is required to start secretion

Front 46

What three networks must proteins be separated into before they leave the trans-Golgi?

Back 46

1. Signal mediated diversion to lysosome
2. Signal mediated diversion to secretory vesicles
3. Constitutive secretory pathway - default pathway

Front 47

Why does the inside of vesicles become more acidic as they mature?

Back 47

- due to increasing ATP-driven proton pumping into the vesicles

Front 48

Why are proteins synthesized as inactive precursors?

Back 48

- so they don't mature until they are inside the vesicle in order to avoid harmful damage to the cell

Front 49

How does synaptic vesicle recycling process work?

Back 49

1. Delivery of synaptic vesicle components to plasma membrane
2. Endocytosis of synaptic vesicle components to form new synaptic vesicles
3. Endocytosis of synaptic vesicle components and delivery to endosome
4. Budding of synaptic vesicle from endosome
5. Loading of neurotransmitter into synaptic vesicle
6. Secretion of neurotransmitter by exocytosis

Front 50

Usually we want to maintain a constant size, but sometimes exocytosis is regulated in order to enlarge a plasma membrane. What three ways does this happen in?

Back 50

1. cleavage furrow - need more surface area when cells divide after cytokinesis
2. phagocytosis - increase in surface area of plasma membrane (vesicles from lysosome)
3. wound repair - vesicles come from lysosome to repair membrane