Molecular mechanisms of vesicular transport Seibenhener Spring 17

Bulk shipping method to get things from one compartment to another

vesicular transport

What gives us specificity in vesicular transport?

Each biomembrane has a separate phospholipid make up to help differentiate one compartment to another

Look at individual proteins and how they relate to compartments in the cell (every compartment In the cell has a specific protein that can help them be recognized)

coat porteins assembling at the membrane force what to occur?

force the lipid bilayer to begin to bend

As they gather at the membrane, coat proteins also do what?

may select what proteins is packaged into the forming vesicle

As more coat proteins are added they shape what?

they shape the surrounding membrane into an enclosed sphere

What are some of the functions of the protein coat around a vesicle?

1. Localize all the cargo, pulls all the cargo together that needs to be transported

2. Once it gets all the cargo in one area, it comes in and aids in packaging and the formation of the vesicle

Transport from the trans golgi network to endosomes and endocytic transport from the plasma membrane

Clathrin coated vesicles

transport from the golgi back to the ER and probably from one golgi cisternae to the previous one

COP-1 coated vesicles

transport form the ER to the golgi

COP-2 coated vesicles

describe the structure of clathrin coated vesicles

○ 3 legged protein

Has a foot and a central area

That formation is called a triskelion

These proteins come together and overlap (interaction) to bend a form a sphere looking thing

What 2 major processes can clathrin coated vesicles perform?

○ Protein that will coat outside of the vesicles that comes from plasma membrane into the cell, endocytosis

Can go from trans golgi network to the membrane via exocytosis

T or F. Cop1 and Cop2 have same functions but move in different directions

TRue

7 subunits that end up forming the same type of structure that clathrin does, soccer ball looking

COP1

Dimer of two SCC proteins

COP2

Both of these interact with another dimer of SCC proteins called adaptor molecules SCC23 and SCC24 under them

COp2

What forms the legs and feet of the clathrin coated vesicle?

3 heavy chains

What do the clathrin coat's 3 light chain do?

○ 3 heavy chains that form the legs and feet of the protein molecule

3 light chains that hold it all together in the middle

introduces curvature to the membrane

coat assembly and cargo selection

adoption binds both to clathrin trsieklions and the membrane bound cargo receptors

bud formation

selective recruiting of both membrane and cargo molecules and pinching off of the bud by Dynamic to form the CCV

vesicle formation

release of clathrin and adoption from transport vesicle

uncoating

what do cargo receptors generally bind to?

membrane bound receptors

§ Binds to cargo receptor proteins

Connector to Clathrin

Adaptin

What type of protein is Dynamic?

GTPAse

§ Comes in and hydrolyzes ATP

§ GTPase protein

§ Hydrolysis of ATP causes vesicle to kink on one side a little bit and when this happens it brings the inner membranes close together and they fuse

§ Membranes fuse then blank eventually pinch it off

Dyanmin

§ Allows uncoating to occur

Knocks off the clathrin and it becomes available to come back and coat another vesicle

another member of the HSP70 family

what is the control mechanism of coat assembly?

coat reg. GTPase

responsible for clathrin and cop1 assembly

ARF proteins (ADP ribosylation factor)

responsible for COP 2 assembly at the ER

Sar1 protein (ADP ribosylation factor)

exchanges GDP for GTP

GEF

hydrolyzes GTP to GDP

GAP

SAR1 when bound to GTP is in what form?

inactive form

what happens when SAR1 interacts with GEF?

When SAR1 interacts with GEF it gets in high energy state and goes thru conformational change that takes the tail end which is hydrophobic that moves to the outer part of the protein where it can incorporate itself into the membrane of the ER (sits there in a GTP bound State)

Recruit these receptor cargo proteins that bind to specific cargos

As this continues to happen we form a bulge in the membrane

SAR1

pulls adapter proteins that bring in the cargo receptors

SAR1

T or F. Sar1 utilizes spontaneous GTP hydrolysis to move itself out of the membrane

TRUE

large protein superfamily consisting of more than 60 members in mammalian cells; their primary role is to mediate the fusion of vesicles with their target membrane bound compartments

SNARE

What is SNARE's primary role

is to mediate the fusion of vesicles with their target membrane bound compartments

T or F. SNAREs are unique to each individual compartment.

FALSE; SNARE proteins not unique to a particular compartments but combos of them can be very unique

Get the package to where its supposed to go (Address Label)

SNARE proteins

What do SNARE proteins form?

an alpha helix

Why are SNAREs are required for vesicle fusion?

1. sub cellular localization

2. formation of extremely stable complexes with other SNAREs

What does vesicle targeting rely on?

SNAREs

T or F. SNARES are present in most vesicles

FASLE present in ALL

T or F. SNAREs are present in all membranes of the cell

TRUE

What can break SNARE protein interactions?

SDS-page

Vesicle snare; on the outside of the vesicle

VSNARE

Target snare; high amount of specificity

TSNARE

A set of proteins that are membrane bound in the ER called the blank that come together to form a pore. Some type of activity/energy (we don't know where the energy comes from) that gets polypeptide into the blank pore.

Derlins (1, 2 and 3) and Derlin pore

SNAREs coming together

Brings vesicle and membrane adjacent to each other

Zippering

the process that forms a TransSNARE

zippering

What does this form: A reaction occurs that makes the two SNAREs fuse and the membranes come together for vesicle to be transported.

Cis-SNARES

- can still differentiate between the vesicle membrane and the target membrane

- SNAREs have interacted but membranes have not

Trans-SNARE complex

- SNARES and membranes have interacted

- Twisting SNAREs together causes dehydration and membrane fusion

Cis-SNARE complex

- Can no longer tell membranes apart because they are one

Cis-SNARE

SNAREs come apart and are available to use again.

- The release of pressure allows singular membranes to form again

unzippering

Large (70) family of monomeric GTPases that regulates many steps of membrane traffic, including vesicle formation, vesicle movement along actin and tubulin networks, and membrane fusion.

Rab proteins

What are all the functions of Rab proteins

Play a role in vesicle formation and membrane fusion

Identification of compartments

More than just membrane/compartment identification

Coat assembly

Cargo specificity

Uncoating proteins

Move microtubules

Tethering to destination

Bind vesicles to cytoskeleton

Recognize/release cargo

What is the end of the secretory pathway?

Golgi

Where is the Golgi relatively located in the cell?

Close to the nucleus and ER

Talk about the order of movement from ER and all the way through the Golgi.

ER-Cis Golgi Network- Cisternae*- Trans Golgi Network

The Cisternae is three parts:

Cis Cisternae Medial Cisternae Trans Cisternae

sends things to the secretory vesicles to be secreted out of the cell

golgi apparatus

Packaging vesicles to be sent to other organelles, to the cell surface, or back to the ER

function of golgi

What are the two main functions of golgi?

transport and post translational modification

How does golgi participate in post-translational modification?

1. Glycosylation of lipids and proteins: synthesis of O-linked Carbohydrates

2. Sulfation of tyrosine residues

3. Prohormone processing

a. Proteins interact and increase their strength to prepare for the dangerous environment outside of the cell

(golgi performs this post-translational type of modification)

sulfation of Tyr residues

How does the golgi participate in pro hormone processing?

a. Proinsulin- proinsulin- insulin

b. Helps in preparing insulin to get in vesicles and be used by cell.

What is leakage?

Cargo slips in the vesicle with out being on a cargo receptor

Free cargo - not on a receptor

It might not supposed to be in the vesicle

A Structure made up of many ER-derived vesicles that are fused together with one another.

Vesicular Tubular Cluster

What are vesicular tubular clusters functions?

It mediates trafficking between the ER and the Golgi and the facilitating and sorting of cargo.

It's a cluster**** of vesicles from the ER that fuses with the Cis Golgi. Everything in this cluster goes into the Golgi Network

vesicular tubular cluster

What is a KDEL?

It's a calreticulan ER residue protein

Looks for the specific KDEL sequence in amino acids and keeps those proteins from leaving the ER fully.

What are the amino acids involved in KDEL?

K: Lysine

D: Aspartic Acid

E: Glutamic Acid

L: Leucine

What is KDELs function?

It recognizes extra cargo that isn't supposed to be there and ER resident proteins that aren't supposed to be there and move them backwards by fusing with COP2 and then retrotranslocation.

What happens when KDEL sequence is recognized?

When the KDEL sequence is recognized, it is sent back to the ER lumen and completely away from the Golgi Apparatus using retrograde transport.

What are the models for cargo moving through the Golgi? Which one is correct?

Cisternal Maturation Model (believed to be the correct model)

Vesicular Transport Model

Describe the cisternal maturation model

# Entire cisternae moves through the process

# The cisternae itself is maturing

# Enzymes involved in each cisternae are different enzymes

# As cisternae moves through, cargo stays the same and moves with it, but the enzymes move backward.

Describe the vesicular transport Model

Cisternae stays the same

Enzymes stay the same

Cargo moves from one step to the next

How do we get things from outside the cell into the cellular environment?

endocytosis

What are the three types of Endocytosis?

phagocytosis, pinocytosis and receptor mediated endocytosis

Is phagocytosis specific or non specific or both?

both

Which endocytosis types are coated and which are not?

phagocytosis- uncoated

pinocytosis- coated

receptor mediated- coated

molecules brought into the cell via vesicles with receptors to the molecule

receptor mediated endocytosis

Is pinocytosis specific or non specific or both?

non-specific

Is receptor mediated endocytosis specific or non specific or both?

specific

What are two examples of Receptor Mediated Endocytosis?

Clathrin Mediated Endocytosis

Calveolae Mediated Endocytosis

What are the two kinds of Clathrin Mediated Endocytosis?

constitutive and stimulated

describe constitutive clathrin mediated endocytosis

endocytosis occurs constantly

Transferren receptor that brings iron in the cell

Zinc and Copper

describe stimulated clathrin mediated endocytosis

involves signaling pathways; receptor on plasma membrane pointing out and brings in whatever its supposed to into the cell until the cell turns the signaling pathway off to stop bringing in whatever was coming through that endocytic pathway.

What are the five processes for Clathrin Mediated Endocysotis?

1. Nucleation - first stage of vesicle budding; triggering of pit formation

2. Cargo Selection

3. Coat Assembly

4. Scission

5. Uncoating

What proteins are involved in the Nucleation phase of Clathrin Mediated Endocytosis?

(FCHO domain) proteins

EGFR pathway substrate 15

Intersectins

bind into plasma membrane and put kink; lift enough to start invagination; makes it easier to be picked up. involved in the nucleation phase of clathrin mediated endocytosis.

FCH Domain Only (FCHO domain) proteins

associated with FCHO proteins and binds to low curvature membranes

F-BAR domain

another protein that associates with F-BAR proteins.

EGFR pathway substrate 15 (EPS15)

Describe intersectins' role in the nucleation phase of clathrin mediated endocytosis

involved with attaching where the vesicle is going to form with the cytoskeleton. Picture a rope thrown from the cytoskeleton to the bud and then the cytoskeleton pulls on it to help with invagination

What proteins are involved in the Cargo Selection phase of Clathrin Mediated Endocytosis?

1. CLASPS - cargo receptors

2. Clathrin - structure

3. AP2 Complex - most abundant component of the Clathrin Coated Vesicle interacting with both clathrin and the bilayer

How does the AP2 complex function?

a. Functions similarly to Adaptin but not the same

b. The presence of AP2 is only in the plasma membrane; Clathrin from the Golgi wont have AP2

What proteins are involved in the Coat Assembly phase of Clathrin Mediated Endocytosis? What do they do?

1. Clathrin - structure involving transkelions overlapping

2. EPS15 - helps form invagination

3. Epsin - super charges EPS15

what proteins are involved in the scission phase of Clathrin mediated endocytosis?

BAR proteins - locate the Neck of vesicle

Dynamin - cuts neck using GTP hydrolysis

has a rate limiting function in the process of endocytosis

Seems to tell the cell theyre done needing the receptor and that they don't need to bring anything else in

Dynamin