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Flashcards in 8.31.16 Lecture Deck (52):
1

Proteins made in the ER are transported in vesicles comprising the ___ pathway. Proteins from the cell exterior are transported in vesicles comprising the ___ pathway.

Exocytic; endocytic

2

Vesicle trafficking is ___, often ___, and highly specific.

Continuous; two-way

3

What are the three types of vesicle trafficking?

Anterograde, retrograde, endocytosis

4

What is anterograde vesicle trafficking?

Movement of vesicles from the ER to the Golgi, and then from the Golgi to either the plasma membrane (directly or via a secretory vesicle) or to the lysosome (via the early and/or late endosome)

5

What is retrograde vesicle trafficking?

Movement of vesicles to the ER from the late endosome, early endosome, and pre-secretory vesicle (can also go from early endosome to plasma membrane)

6

All of the vesicle interiors, known as the ___, are topologically the same as the extracellular space. What does this mean?

Lumen; When a protein is co-translationally inserted into the lumen of the ER, it is going to an environment similar to the cell exterior.

7

What are the 5 general steps to vesicular transport? Note that these steps are the same for exocytosis and endocytosis.

1. Cargo selection/loading from the donor compartment
2. Vesicle budding (pinch off via fission)
3. Vesicle transport
4. Vesicle targeting (recognition of target compartment)
5. Vesicle fusion/cargo delivery

8

Three different ___ are used at different steps in the secretory and endocytic pathways to mediate cargo selection and vesicle budding. What are they?

Coat proteins; Clathrin, COPI, COPII

9

What does clathrin do?

Moves exocytic and endocytic vesicles between the TGN, endosomes, and membrane

10

What does COPI do?

Move vesicles in the retrograde direction

11

What does COPII do?

Move vesicles in the anterograde direction (ER to Golgi)

12

What does assembly of a COPII coat at the ER membrane do?

Regulates the content of ER-derived transport vesicles

13

Describe the process of COPII formation.

1. ER chaperones hold onto unfolded proteins, keeping them from premature transport. Folded ER proteins are bound by cargo receptors.
2. Cargo receptors have cytoplasmic tails that bind with subunits of the COPII coat.
3. Sar1-GTP, a Rab-GTP-like protein, sticks into the cytosolic face of the ER membrane and forms a site to which COPII coat subunits bind.
4. Vesicle is ready to bud and move to the cis-Golgi.

14

What is Sar1-GTP? Describe the Sar1-GTP cycle.

A coat recruitment GTPase; Sar1-GDP is inactive. Sar1-GEF exchanges GDP for GTP and activates Sar1-GTP (binds to the membrane via an amphipathic helix)

15

Assembly of a COP1 coat at the Golgi regulates the return of ___-containing proteins back to the ___.

KDEL; ER

16

Why are proteins returned to the ER?

Some proteins accidentally escape the ER.

17

Assembly of a clathrin coat at the trans-Golgi and plasma membranes regulates ___ and ___.

Cargo selection; budding

18

Describe the process of clathrin coat formation.

Receptors have cargo-binding proteins sticking into the vesicle lumen. These pick up cargo, which change their cytoplasmic tails to allow for the binding of adaptor proteins. The clathrin coat proteins then stick to the adaptors and deform the vesicle membrane into the vesicle. Clathrin is shed once the bud forms.

19

Integral membrane proteins can be selected on the basis of what three things?

Phosphorylation, amino acid motifs (instrinsic signals), and ubiquitination - the cytoplasmic tails of the receptors are marked in these ways.

20

Low density lipoprotein (LDL) receptors are required to endocytose LDL, a lipoprotein complex that transports cholesterol between cells. Describe this process.

1. LDL binds to LDL receptors, which stimulate formation of clathrin coated pits.
2. Endocytosis occurs.
3. LDL dissociates from receptors in acidic endosomes
4. Once in lysosomes, LDL gets hydrolyzed and cholesterol is made available to the cell.
5. Receptors are recycled.

21

What happens when LDL receptors are defective and cannot associate in clathrin-coated pits?

Hypercholesterolemia

22

___ drive most of the vesicle morphogenesis.

Coat proteins

23

Clathrin molecules have three-fold symmetry and are known as ___. These assemble into geometric shapes called ___.

Triskelions; icosahedrons

24

Dynamin assists with the membrane fission process. How?

While coats often have enough membrane bending force to form a complete vesicle, dynamin can assist. ATP hydrolysis drives tightening of dynamin coils, bringing the membranes so close that they fuse together.

25

Vesicle transport requires what two things and why?

Cytoskeleton and motor protein; large transport vesicles cannot flow freely through the goopy cytoplasm.

26

Cytoskeletal rails and motors such as ________ transport vesicles.

Myosin on actin, dynein and kinesin on microtubles

27

___ and ___ connect transport vesicles to motors, which are then attached to the cytoskeleton.

Rab; Rab effectors

28

The Golgi Apparatus is a major vesicle sorting station and is characterized by membranous ___, flanked by numerous small ___, and is often found near the ___.

Stacks; vesicles; nucleus

29

Describe the layers of the Golgi Apparatus.

1. Cis Golgi Network (CGN)
2. Cis cisterna
3. Medial cisterna
4. Trans cisterna
5. Trans Golgi Network (TGN)

30

What does the CGN do?

Phosphorylation of oligosaccharides on lysosomal proteins

31

What do the cis cisterna do?

Remove Man

32

What do the medial cisterna do?

Remove Man, add GlcNac

33

What do trans cisterna do?

Add Gal, NANA

34

What does the TGN do?

Sulfation of tyrosines and carbohydrates

35

The stacks of the Golgi operate primarily on ___.

N-glycans

36

Protein secretion occurs by both a ___ and ___ pathway.

Regulated; non-regulated

37

Describe the regulated pathway of protein secretion.

Receptors bind to the cargo destined for secretory vesicles, and partition this cargo from proteins secreting constitutively. Secretory vesicles fuse with the plasma membrane in response to extracellular signals.

38

There is vesicle traffic both to and from secretory vesicles as they mature. What does forward transport do? Reverse transport?

Forward transport brings the cargo to the vesicles. Reverse transport takes away unintended interlopers. This increases the concentration of the desired cargo in vesicles.

39

Some secretory cargo molecules are very small and have to be co-translationaly synthesized into the ER as a longer protein. What happens next?

The mature cargo is produced by proteolysis of the signal peptide in the ER and additional proteolysis in the TGN or vesicle.

40

When are coat proteins removed?

After budding

41

What identify specific organelles or membrane domains for vesicle targeting? How?

Specific phosphoinositol-containing phospholipids; PIs paint the cytoplasmic side of membranous organelles and transport vesicles that bud from them.

42

___ of inositols can get phosphorylated by specific kinases.

Hydroxyls

43

Vesicle targeting and recognition is facilitated by ___ and ___.

Rab proteins; PIs

44

Each organelle membrane has specific ___.

Rab-GEFs

45

PI markings work in concert with Rab markings. Describe the positive reinforcement loop that can arise.

PI(3)P can recruit more Rab5-GEF, which causes an increase in the deposit of Rab5-GTP on membranes, which binds more Rab effectors. These are sometimes filamentous tethering proteins that link vesicles to the cytoskeletal motors and are sometimes membrane fusion proteins.

46

Rabs help tether vesicles to their correct target membrane; ___ promote fusion of the vesicles with their target.

SNAREs

47

Vesicles have ___; target membranes have ___.

v-SNAREs; t-SNAREs

48

When the vesicle gets close to a target membrane via Rab-GTP binding to the Rab effector fibrils...

...v- and t-SNAREs can intertwine in ways that get the vesicle close enough to spontaneously fuse.

49

SNARE proteins contribute to ___ in vesicle targeting and promote vesicle ___ to its target.

Specificity; fusion

50

v- and t-SNARES have ends that go all the way through...

...each membrane.

51

SNARES have to be recycled. How does this occur?

Accessory proteins and NSF (with ATP) pry them apart; they are then sent back to their origin.

52

___ poison neurons by cleaving SNAREs.

Botulism toxins