4.3 Vesicular Transport I (771-779)

Question 1

What’s the name of trafficking outbound material b/tween organelles?

Answer 1

Biosynthesis pathway: synthesis & targeting of proteins that are necessary for the function of specific organelles and proteins that will be secreted

Question 2

What is the retreival pathway in charge of?

Answer 2

moving material inward. Cell exterior to early endosome to late endosome

late endosome moves material to lysosome

Question 3

Which two pathways cross the same organelle system in the golgi & ER?

Answer 3

biosynthesis pathway and retrieval pathway

Question 4

What are the three types of coated vesicles? Are they membrane bound?

Answer 4

clathrin
cop II
COP I

All are membrane bound

Question 5

In appearance, what the difference between clathrin, COP I, and COP II?

Answer 5

clathrin has a woven appearance (think of its 3 arm structures)

COP I and II are fuzzy

Question 6

How are clathrin coats formed? and what process are they involved in?

Answer 6

formed on the plasma membrane and are involved in endocytosis

Question 7

COP I coats what kind of vesicles?

Answer 7

surrounds vesicles moving w/in the gogli

Question 8

COP II vesicles form where?

Answer 8

bud off endoplasmic reticulum (ER)

Question 9

what is the importance of the protein coats on vesicles?

Answer 9

coat on vesicle allows SELECTION of cargo and SHAPING of vesicle

Question 10

What is the structure of a clathrin?

Answer 10

Structure is called triskelion

3 large subunits called heavy chains

3 small subunits called light chains

arms have globular ends

Question 11

What is the purpose of the globular ends in triskelion?

Answer 11

interact w/a number of adaptor proteins that links triskelion to the vesicle cargo.

Question 12

How is clathrin linked to cargo proteins?

Answer 12

using adaptor protein , adaptin

Question 13

What does the lateral linkage between clathrin subunits casue?

Answer 13

membrane deformation and creates high local concentration of cargo molecules

Question 14

What has to happen for a clathrin coated vesicle to dock to a target membrane?

Answer 14

the clathrin coat has to be disassembled in the cytoplasm in a step called uncoating

Question 15

What protein is involved for clathrin vesicle to separate (bud off) from the main organelle?

Answer 15

Dynamin - which requires an energy source from GTP

Question 16

What happens when Dynamin is bound to GDP?

Answer 16

it associates w/the vesicle membrane in a diffuse or non localized manner

Question 17

how does dynamin-GDP change to Dynamin-GTP? What happens when dynamin is attached to GTP?

Answer 17

GEF swaps GDP for GTP

Dynamin-GTP state forms a helical ring or collar around the membrane extensions that links the clathrin coated vesicle to the membrane it is budding off.

Question 18

After dynamin-GTP has formed a collar around the membrane extensions and clathrin coated vesicle, How is the vesicle pinched off from membrane?

Answer 18

GTP will be hydrolyzed to GDP + Pi this change releases energy and the energy creates a shape change which pinches off the vesicle.

Question 19

how was the role of dynamin discovered?

Answer 19

mutant fruit flies (Drosophila) that were paralyzed b/c vesicles containing neurotransmitters couldn’t bud off properly at nerve ending.

Question 20

Who are the players in formation of COP II?

Answer 20

Sar1-GDP -inactive form

Sar1-GEF - switches GDP to GTP

Sar1-GTP - active form binds to the membrane

Sec24 + Sec 23 - recruited by Sar1-GTP to bind to Sar1-GTP (sec23) and sec 24 binds to cargo protein.

Sec 13 and Sec 31 - deform the membrane and drives vesicle formation (outer coat of COP II vesicle)

Question 21

Why is it important for sar1-GDP to covert to it’s active form GTP?

Answer 21

Sar1-GTP active form recruits coat proteins to membrane (Sec 23 and 24) and (Sec 13 and 31)

Question 22

What is the difference between the formation of COP II and COP I?

Answer 22

COP II use Sar1 and COP I uses Arf-1 (btw: clathrin also uses Arf1)

Question 23

How does Sar-GTP insert itself into the cytoplasm side of the lipid bilayer?

Answer 23

has an exposed amphipathic helix that is inserted to bilayer

Question 24

What other vesicles can be formed on cells?

Answer 24

Retromers - coats endosomal vesicles that go back to golgi

Caveolae- vesicles are not coated and have same characteristic as lipid raft membranes

Question 25

What is the structure of a retromer?

Answer 25

Retromer is made up of four different proteins, one of which binds to a specific membrane cargo
and others of which insert themselves into curved membranes. So these are membranes that are
already curved. The insertion of retromer into a membrane tends to stabilize the curved shape
which then facilitates the recruitment of additional retromer complexes until a completed vesicle
is formed. Retromer coated vesicles are frequently involved in retrieval of membrane proteins
back into the cell. That is why they are called retromer coated vesicles.

Question 26

Where do retromer proteins bind?

Answer 26

1 protein binds to membrane cargo

3 other proteins bind and insert themselves into the curved membrane.

Question 27

How does insertion of retromer into the membrane help form vesicles?

Answer 27

stabilizes curve shaped membrane.

facilitates the recruitment of additional retromer complexes until vesicle is formed

Question 28

Where are caveolae formed?

Answer 28

small vesicles formed at the cell surface. Formed by protein caveolin (integral membrane protein)

Question 29

What is the difference between retromers and caveolae? what about similarities?

Answer 29

Similarities: both insert themselves into the membrane

Differences: retromers recruit additional retromer complexes until vesicle is formed and; does not bend membrane b/c they are already on a curved membrane

whereas caveolae do not recruit additional proteins to form coats. when they insert themselves on membranes they form a bend which forms the vesicle

Question 30

How do cells determine WHERE to form vesicles?

Answer 30

PIPs and PIP-binding proteins (these proteins are involved in vesicle formation)

PiPs: phosphorylation of membrane lipid phosphatidyl ionositol, which results in a variety of phosphoinositides (PIPs).

PIPS are found in localized areas of membranes which are then recognized by PIP-binding proteins.

Each organelle in cell has different combinations of PIPs in its membrane. And there are many PIP-binding proteins that recognize specific PIPs.

This system allows vesicles to form in cells in a specialized/regulated manner.

Question 31

What mechanism regulates docking & fusion of vesicles to their target membrane?

Answer 31

Ras biniding proteins (Rab) direct vesicles to specific membrane target

Question 32

GTP binding to Rab helps rab do what?

Answer 32

undergo a cyclic association with membranes

Question 33

What role does SNARE play in docking?

Answer 33

V-SNAREs are found on vesicles

T-SNAREs are found on target membrane

Interaction of these two proteins facilitate fusion of membrane between vesicle and membrane

Question 34

What are Rab proteins?

Answer 34

Rab is a GTPase that direct vesicles to correct site. In their GTP bound state they tightly associate with vesicle membranes

Question 35

What is the SNARE hypothesis?

Answer 35

fusion of two lipid bilayers is energetically not favorable. The minimum needed for fusion is trans SNARE complex (4 alpha helices formed between association of v-SNARE and t-SNARE). Water has to be squeezed out as trans-SNARE complex is formed

Question 36

Why is fusion of vesicle and target membrane not energetically favorable?

Answer 36

b/c of the drastic rearrangements of the membrane lipids that is required for the fusion

Question 37

What is needed to make fusion between vesicle and target membrane?

Answer 37

Protein complex composed of SNARE. V-SNARE (1 on vesicle) and T-SNARE (3 on target membrane)

Question 38

How do SNARES help vesicles + target membrane keep their fidelity?

Answer 38

There are many different SNARE proteins and the interaction between them is
specific for particular vesicles and particular target membranes and this increases the fidelity of
vesicle transport by making sure that vesicles only fuse with the correct target membrane.

Question 39

How are SNAREs recycled?

Answer 39

they have to be actively disassembled. Disassemble is mediated by ATP dependent activity of membrane protein called NSF.

Question 40

What does NSF stand for and why?

Answer 40

N-ethylmaleimide sensitive fusion protein.

NSF is highly sensitive to reducing agents such as N-ethylmaleimide.

Question 41

How do vesicles move from the ER to cis face of the golgi?

Answer 41

COP II coated vesicles

Proteins to the ER contain peptide domains called EXPORT SIGNALS. export signals are recognized by receptors embedded in the ER membrane.

The receptors contain domains that facilitate the formation of export vesicles by attaching to COP II coupling system.

Question 42

What happens if the concentration of a protein is very high in the ER?

Answer 42

the protein will get into export vesicles just by passive diffusion.

Question 43

What two things can happens after COP II coated vesicles leave the ER?

Answer 43

First, the vesicles fuse to form larger ones and second, they are transported to and fuse with the cis-face of the Golgi

Question 44

What is needed for two COP II vesicles leaving the ER on their way to the cis gogli to fuse?

Answer 44

vesicles need to have an identical membrane (homotypic fusion). this is done by v-SNARE and T-SNARE found on both vesicle membranes

Question 45

How is the homotypic fused cop II cell guided to the golgi?

Answer 45

Microtubules guide the movement of vesicles toward the cell center where the Golgi apparatus is found. And finally, export vesicles will fuse to the Golgi membrane using a heterotypic interaction, not a homotypic interaction, and they then deliver contents into the golgi

Question 46

Which vesicle helps move proteins from golgi to ER?

Answer 46

COP I

Question 47

What is the mechanisms that deals with proteins that leave the ER in export vesicles but belong in the ER?

Answer 47

Instead of having an “export” signal, proteins that need to be returned to the ER have what’s called a “retrieval” signal.

ER proteins that are found within
the lumen of the ER have the sequence KDEL at their C-terminal end and proteins found in the
ER membrane have KKXX at their C-terminal end, where X is any amino acid. The KDEL signal is recognized by receptors in the Golgi membrane that recruit adaptor proteins and COPI coated proteins. The KKXX signal actually binds directly to COPI coat proteins. In turn, COPI coat proteins get targeted back to the ER membrane because their associated SNARE proteins interact with SNAREs that are on the ER membrane.

Question 48

What sequence do ER proteins that are found w/in ER lumen have?

Answer 48

KDEL at their c-terminal end

Question 49

What sequence to proteins found in the ER membrane have?

Answer 49

KKXX at their c-terminal end where x is any amino acid.

Question 50

KDEL is recognized by which receptors?

Answer 50

receptors in the golgi membrane that recruit adaptor proteins and COP I coated proteins

Question 51

What does the KKXX signal bind to? What does this do?

Answer 51

directly to COPI coated proteins in turn COPI coat proteins get targeted back to the ER membrane b/c their associated SNARE proteins interact with SNARES that are on the ER membrane

Question 52

Why does the cisternal maturation model require that retrograde transport be robust?

Answer 52

so the golgi enzymes moved to the trans side move back to the cis side.

this model requires extensive use of COP I coated retrieval pathways to move the proteins that are typical of the cis-gogli face backwards as the cisternae move forward.

Question 53

How are clathrin vesicles formed?

Answer 53

1) clathrin is linked to cargo proteins using some sort of adaptor protein on a flat membrane surface.

2)These subunits concentrate together and link in some sort of localized membrane region. Good
candidates for what might create such a region would be things like lipid rafts.

3) As increasing numbers of clathrin subunits are recruited to the patch, the membrane begins to deform and eventually the deformation of the membrane leads to the formation of a complete vesicle.

4) Finally, in order for the vesicle to dock to another membrane, the clathrin coat has to be
disassembled in the cytoplasm at some point and that happens in a step that’s called uncoating.

Question 54

What controls when and which secretions and endocytosis occur in a cell?

Answer 54

PI and PIP kinase and PIP phosphatase enzymes control the balance of PIP species.
 A specific PIPs is recognized by a PIP binding protein, which in turn regulates vesicle formation and
transport.
 Thus, controlling of kinase and phosphatase enzyme activities can control when and which secretions and
endocytosis occur in a cell.

Question 55

What helps direct vesicles to their target membrane?

Answer 55

variability in Rab genes.

In humans, there are over 60 different Rab proteins, each of which identifies unique classes of membrane vesicles and there are corresponding Rab effector proteins for every Rab.

Question 56

What are Rab effector proteins?on target membrane recognize specific Rab proteins and facilitate membrane tethering.

Answer 56

Rab effector proteins are found on target membrane and recognize specific Rab proteins to facilitate membrane tethering between vesicle and target membrane