Section 4 Endomembrane pt 2

What is a proteosome

Barrel-shaped protein degrading machines

What do proteosomes degrade?

Only proteins that have been tagged for destruction by ubiquitins

What parts make up a proteosome

• Cap

• Half a dozen proteases (to degrade proteins)

• Several ATPases (to drive the process by hydrolyzing ATP)

What are the three enzymes required to add ubiquitin (small peptide)

• E1 and E2 (ubiquitin carriers)

• E3

What does the ubiqutin enzyme E3 do?

it is Ubiquitin ligase and recognizes misfolded proteins to transfer ubiquitin’s from E1 and E2 to the misfolded protein to mark it for destruction

Once a misfolded protein becomes polyubiquinated, where does it bind?

To the cap of the proteasome

What type of tag is for Lysosomes to know which to destroy?

M6P tag

What does BiP normally do?

Normally binds to misfolded proteins plus some BiP stays in the ER bound to sensors

What happens if there are many unfolded proteins

BiP will be recruited from the membrane and they will abandon their sensors causing the sensors to become active

When BiP leaves their sensors in the ER because there is too many unfolded proteins, what do some sensors do?

Some dimerize and add a phosphate to elF2alpha which will bind to the small subunit of ribosome and reduce protein synthesis

When BiP leaves their sensors in the ER because there is too many unfolded proteins, what do other sensors do?

Undergo Proteolytic cleavage and their cytosolic portion now acts as a transcription factor to make more proteins that alleviate stress

Which side of the golgi faces the ER

Cis golgi network

Who discovered the Golgi complex?

Camillo Golgi

What is the vesicular transport model of golgi transport?

The cisternae are stable compartments and cargo gets shipped from compartment to compartment and eventually leaves through the trans face

What is the cisternal maturation model of golgi transport (more widely accepted)

Cis cisternae are formed by fusion of vesicles from the ER and they mature as they move towards the trans face carrying cargo

What is proof #1 of the cisternal maturation model for the golgi

Cargo proteins were not found in vesicles

What is proof #2 of the cisternal maturation model for the golgi

Golgi resident proteins were seen in vesicles moving in the retrograde direction

What was proof #3 of the cisternal maturation model for the golgi

A yeast Sec mutant with a mutation in the ER vesicle formation had the golgi disappear when heated up

What are the three types of coats for vesicles?

• COPII

• COPI

• Clathrin

What do coat proteins of vesicles act like?

a mechanical device that assemble to produce a force which will then curve the membrane until it forms a bubble

What is the origin, destination, and direction of COPII vesicles

originate from the ER and move towards the golgi; anterograde

What are the three types of coat proteins

• Monomeric G protein (GTPase)

• Adaptor proteins

• Outer coat proteins

What is the first step of COPII vesicle formation

Sar1 (a GPTase) binds a GPT and becomes activated. Hydrophobic tail swings out of Sar1 and enters the lipid bilayer. This starts to curve the membrane

What is the monomeric G protein (GTPase) of COPII vesicles

Sar1

COPII vesicles: once Sar1 binds GTP and starts to bend the membrane, what happens?

It recruits two adaptor polypeptides (Sec23 and Sec24) to form a heterodimer which binds the cytoplasmic tails of transmembrane cargo receptors

COPII vesicles: after the adaptor polypeptides (Sec23 and Sec24) forma heterodrimes and bind the cytopalsmic tails fo the transmembrane cargo receptors, what happens?

Two COPII outer coat polypeptides (Sec13 and Sec 31) join complex to form an outer coat

What do the adaptor polypeptides of COPII bind to in total (Sec23 and Sec24)?

• Tails of transmembrane cargo receptors

• Sar1-GTP

• Outer coat polypeptides

When do coat proteins of COPII vesicles disassemble?

When Sar1-GTP is hydrolyzed

What type of movement are COPI coated vesicles used for?

Retrograde movement:

• Golgi to the ER

• Trans golgi to the cis golgi

What monomeric G protein do COPI coated vesicles use

Arf1

What coat proteins do COPI coated vesicles use?

7 different proteins that form a coatamer which has a triskelion shape

How does the cell know which proteins to ship back to the ER?

KDEL which is a retrieval sequence

What is the vesicle formation of Clathrin Coated?

Triskelions with 3 heavy and 3 light chains that can join together to form a hexagon or pentagon

What can Clathrin Coated vesicles form?

Lattices

Which locations are Clathrin coated vesciles?

• From trans golgi to endosomes/lysosomes—anterograde

• From plasma membrane to endosomes/lysosomes—retrograde

How does clathrin coated vesicle formation often start?

Arf1 bending an alpha helix into membrane to initiate bending and recruitment of other adaptor proteins

What are the two adaptor proteins that can help recruit clathrin ?

• GGAs from the trans golgi

• AP2 from the plasma membrane

What is Dynamin and what does it do?

It is a monomeric G protein that uses GTP hydrolysis to help clathrin coated vesicles to leave the membrane

What happens when Dynamin is fed nonhydrolyzable GTPs

It will make a strand of dynamins without cutting off the vesicle

What is the optimal environment for the enzymes in lysosomes and what do they need to be tagged with to know to go to lysosomes?

acidic pH; Mannose 6 phosphate (M6P)

When a lysosomal enzyme is synthesized, what happens?

a carbohydrate is added as well as a phosphate which then binds to a M6P receptor and packaged in a clathrin coated vesicle until it comes into contact with the low pH of the late endosome

What are all the things GGA adaptor protein binds?

• Mannose 6 phosphate receptor

• Arf1 (GTPase)

• Clathrin

What are the two ways you could break a monomeric G protein?

• _{Nonhydrolyzable GTPs were used}

• _{Temperature sensitive sec mutants}

What are Rabs?

Small GTP binding proteins that specify vesicle destination

In targeting vesicles to specific compartments, what does the targeting and tethering?

Rabs

In targeting vesicles to specific compartments, what does the docking?

SNAREs

What are SNAREs

membrane proteins that mediate vesicle fusion

What are the two type of SNARE proteins?

• t-SNAREs = on the target membrane

• v-SNAREs = on the transport vesicle membrane

How do Rabs associate with membranes?

Via a lipid anchor

How does Rab help vesicles combine into their specific compartments?

It stays attached to the vesicle via a lipid anchor and recruits tethering proteins to hold the vesicle in place so the two SNAREs can form a coil and pull the vesicle close to the target membrane

Once a vesicle fuses with the targeted membrane, what happens?

NSF uses ATP to untwist the SNAREs

What is receptor mediated endocytosis? (like yolk proteins taken up by a chicken oocyte)

Cell brings in extracellular materials after they bind to receptors on the plasma membrane (use clathrin coated vesicles)

How does the cell endocytose ligands?

Receptor mediated endocytosis. They bind to receptors on the surface of the plasma membrane and the more that bind, the more curved it gets until dynamin cuts it off and an endosome is created.

In receptor mediated endocytosis, after dynamin hydrolyzes GTP and causes the vesicle to pinch off of the plasma membrane, what happens?

Uncoating ATPase hydrolyzes ATP causing AP2 and Clathrin to be recycled

When a receptor mediated vesicle becomes an early endosome, whats the pH and what happens?

5.9-6.5; receptors are freed

When an early endosome becomes a late endosome, what is the pH and what happens?

5.5-6.0; can no longer fuse with endocytic vesicles

What are the two methods for acidification of receptor mediated endocytosis?

• ATP-dependent proton pump

• Transfer material to existing lysosome

What is receptor mediated endocytosis necessary for?

The uptake of housekeeping materials e.g., cholesterol uptake via LDL receptors

What is autophagy?

Destruction of organelles by isolation in a double-membraned vesicle followed by fusion with a lysosome

What did Yoshinori Ohsumi win the nobel prize for?

Discovering autophagy

What is the first step for how proteins get proteins into the mitochondria?

They have a transit sequence added Hsp70 and Hsp90 deliver proteins to the mitochondria in an unfolded state and they then go through TOM (transport outer membrane complex) in the outer membrane

Once proteins get through the TOM complex of the mitochondria, what does it do if it wants to lodge in teh inner membrane?

Goes into TIM22

Once proteins get through the TOM complex of the mitochondria, what does it do if it wants to get into the mitochondrial matrix?

It moves through the TIM23 complex with Mitochondrial Transit Peptidase cleaving the transit sequence while Hsp60 is added as a chaperone