BICD 110 - Midterm

Proteins traffic through the ___ to enter/exit the nucleus

nuclear pore complex (NPC)

Are nuclear proteins transported before or after they have been folded?

after

The NPC spans

both the inner and outer nuclear membranes

proteins < 40 kDa through the NPC

can freely diffuse

proteins > 40 kDa through the NPC

require a nuclear localization or nuclear export signal

The nuclear pore is composed of

• structural nucleoporins

• membrane nucleoporins

• FG-nucleoporins

FG-nucleoporins

line the pore and form a gel-like matrix that allows small molecules to diffuse but prohibits free diffusion of proteins >40kDa

FG-nucleoporins are critical for the ___ of the NPC

selectivity

Nuclear localization signals (NLS)

direct proteins to the nucleus

NLS structure

no strict motif but rich in basic amino acids

NLS experiment goal

To test whether an NLS is sufficient to target the protein to the nucleus

NLS experiment

• GFP-tagged normal pyruvate kinase is cytosolic

• Localization of GFP-tagged chimeric pyruvate kinase where an NLS was fused on pyruvate kinase

Mechanism for nuclear import of proteins

1. Importin binds to an NLS of a cargo protein

2. The importin-cargo complex diffuses through the NPC by interacting with the FG-nucleoporins

3. In the nucleus, a Ran-GEF interacts with Ran, causing it exchange GDP for GTP, and Ran-GTP interacts with the importin, displacing the cargo

4. The Ran-importin complex diffuses back to the cytosol

5. A Ran-GTP interacts with Ran, causing it to hydrolyze GTP to GDP, lowering its affinity for importins

Ran-GAP is localized in

the cyotosol

Ran-GEF is localized in

the nucleus

Nuclear Export Signals (NES)

direct proteins out of the nucleus

NES structure

no strict motif but rich in hydrophobic amino acids

Mechanism for Protein Export from Nucleus

1. Ran-GTP in the nucleus binds to exportin 1, which induces a conformational change that increases its affinity for NES-containing cargo

2. Exportin-Ran-GTP-cargo complex diffuses into the cytosol

3. Ran-GAP interacts with Ran-GTP, causing it to hydrolyze GTP into GDP, and Ran-GDP dissociates from the cargo and diffuses back to the nucleus

Destinations of the secretory pathway

• lysosome

• secretory proteins

• plasma membrane

Forward Genetics

isolate individuals with a phenotype of interest and identify causative mutations

Reverse Genetics

alter the expression of specific genes and look for a phenotype

Traditional forwards genetic screen

1. Mutagenize a population = create random DNA damage

2. Conduct phenotypic screen that will identify genes in your pathway of interest

3. Figure out which gene is perturbed in your mutant

Problem with traditional genetic screens

if a mutation is lethal, you will never be able to find this gene through this screening strategy because you cannot recover these individuals

Temperature-sensitive alleles

missense mutations where the encoded protein is functional at the permissive temperature but is not functional at the restrictive temperature

Basic Principles of Vesicular Trafficking

• Membrane faces are conserved during budding and fusing

• Specific proteins are required to initiate coat formation and select cargo

• Coat proteins deform the membrane and select cargo

• The protein coat falls off once the vesicle has budded off from a membrane

• Proteins are required for fusion

ER-Golgi trafficking is __ (consider direction)

bidirectional

Retrograde trafficking involves

COPI vesicles

Anterograde trafficking involves

COPII vesicles

COPII Coat Assembly

1. Sar1-GTP inserts in the ER membrane

2. COPII Coat Assembly

3. Sec23 GAP activity stimulates Sar1 GTP hydrolysis, inducing a conformational change in Sar1

4. Sar1-GDP releases from the vesicle membrane, triggering coat disassembly

COPII Coat Assembly - Sar1 Membrane Insertion

1. Sar1-GDP interacts with Sec12

2. Sec12 is a GEF → stimulates Sar1 to exchange its GDP for GTP

3. Sar1-GTP undergoes a conformational change and inserts its N-terminal amphipathic helix into the outer leaflet of the ER membrane

COPII Coat Assembly - Sec23/24 Recruitment

• Sar1-GTP recruits coat proteins Sec23/24

• Sec23/24 binds to specific sorting membranes in the cytosolic domains of transmembrane cargo proteins

• Sec13/31 assemble into coat, completing coat assembly

• Once the coat assembles, the COPII vesicle pinches off from the ER membrane

ER exit sites (ERES) are major sites of

cargo packing and COPII vesicle formation

Which proteins recognizes sorting signals for sorting into COPII vesicles?

Sec24

COPII sorting signals

• diacidic signal = Asp-X-Glu (DxE)

• dihydrophobic signal = Phe-Phe (FF)

How is cargo that lacks a transmembrane domain sorted into vesicles?

luminal cargo is packed via their interactions with transmembrane proteins that are also being sorted

Luminal Cargo Packing Mechanism

• Proteins with N-glycan modifications are recognized and bound by ERGIC-53

• In the neutral ER lumen, binding between ERGIC-53 and the bound cargo is enhanced

• In the acidic cis-Golgi, binding between ERGIC-53 and the bound cargo is reduced, allowing the cargo to release once vesicles fuse at the cis-Golgi

How are uncoated vesicles targeted to a specific membrane?

• Cytosolic Rab-GDP is attached to an uncoated protein’s membrane via its isoprenoid anchor

• Different Rabs will be targeted to specific vesicles

• A GEF in the membrane converts Rab-GDP to Rab-GTP

Vesicle Targeting and Fusion

1. Rab-GTP binds to a Rab effector on the target membrane, docking the vesicle to the membrane

2. Once the vesicle is docked, v-SNAREs and t-SNAREs form stable coiled-coil interactions, bringing the membranes close enough to fuse

3. After fusion is complete, NSF and alpha SNAP use ATP to separate the SNARE complex

v-SNARE

integral membrane proteins found on the vesicle membrane

t-SNARE

integral membrane proteins found on the target membrane

COPI Coat Assembly

1. Insert Arf1-GTP into the outer leaflet of the cis-Golgi membrane

2. Coat assembly and cargo sorting

3. ArfGAP activity simulates Arf1 GTP hydrolysis

4. Arf1-GDP falls off from the vesicle membrane, causing disassembly of the coat

Arf1 Membrane Insertion

• Arf1-GDP interacts with p23/p24

• GBF1 GEF activity simulates Arf1 to exchange its GDP to GTP

• Arf1-GTP undergoes a conformational change and integrates its N-terminal amphipathic helix into the membrane outer leaflet

Coatomer Interactions

1. Arf1-GTP recruits heptameric coatomer coat protein complex

2. Coatomer proteins bind to specific sorting signals

3. Coat assembly disforms the membrane until the COPI vesicle pinches off from the cis-Golgi

Sorting Signals for COPI vesicles

• Lys-Lys-X-X (KKXX)

• Di-arginine (X-Arg-Arg-X)

KDEL sorting signals

targets ER proteins that are non-specifically packed into COPII vesicles back to the ER

KDEL Targeting to the ER

• KDEL receptor in the cis-Golgi membrane bind to KDEL sequences in the acidic cis-Golgi

• KDEL receptor contains a KKXX sorting signal that binds to COPI coatomer subunits

• KDEL receptor dissociates from the KDEL sequences in the neutral ER lumen following vesicle fusion

The Golgi Complex is composed of

cisternae

The Golgi Complex is divided into three regions

cis, medial and trans

Cisternal Maturation Model

retrograde trafficking moves enzymes to the previous compartment, causing it to mature

N-glycans are remodeled in the Golgi complex

N-glycans undergo additional modifications during cisternae maturation → specific enzymes in found in different cisternae modify N-glycans in a defined sequence

O-glycosylation occurs in the Golgi Complex

carbohydrate chains can be attached to oxygen in serine and threonine resides