Dr Priget Lectures

name organelles in eukaryotic cells?

• Nucleus

• ER

• Golgi apparatus

• Lysosomes

• Mitochondria

• Plasma Membrane

• Ribosomes

• Vesicles

• Cytoskeleton

Where do the proteins get made?

all proteins start in the cytosol made by ribosomes

• some stay in cytosol

• others go to the

-nucleus

-mitochondria

-ER → golgi → membrane or secreted out of cell

Types of ribosomes and how they sort proteins?

ER -bound ribosomes

• deliver proteins for membranes or secretory pathways via co-translational insertion

Cytosolic Ribosomes

• make proteins destined for the cytosol, mitochondria, nucleus or peroxisomes

What are the requirements for protein targeting?

1. signal address → encoded in amino acid sequence so it knows where its going

2. Receptor → recognises the signal (SRP)

3. Energy → ATP or GTP ( transfer the protein to new place)

4. Translocation Machinery → moves protein across or into membrane

Role of ATP in protein translocation

helps molecular chaperones that pull or push the proteins through the protein channel

Role of GTP in protein targeting

helps the timing and specificity of receptor binding and the release of the cargo

What are the different types of signal sequences?

• ER signal Sequence

• Mitochondrial signal

• Nuclear localisation signal (NLS)

ER signal sequence

• the signal is found in the N terminus (read while protein is being made and sent straight to the ER membrane)

• Type: hydrophobic amino acids

• cleaved off after import

Mitochondrial Signal Sequence

• mitochondria have their own DNA but still import cytosol proteins

• found in the N terminus

• Type: Amphipathic alpha-helix (1 side = + charged, other side hydrophobic)

• used by TCA cycle enzymes and matrix proteins

• cleaved off after import by matric peptidase

Mitochondrial Matrix Protein

Nuclear Localisation Signal Sequence

• proteins imported into the nucleus

• signal sequence can be found anywhere on the protein

• Type: short sequence with many Lysine (K) and Arginine (R)

• not cleaved after import because needed for mitosis

How does a protein travel across the ER membrane?

Co-Translational Import

Co-Translational Import Process

1. ribosomes translate protein in cytosol

2. ribonucleoprotein SRP (Signal Recog Particle) pauses translation by binding to signal sequence

3. SRP guide ribosome to RER = bind to SRP receptor

4. The closed Sec61 channel is next to it and then receives the ribosome

5. channel opens and inserts signal peptide

6. SRP leaves protein using GTP energy

7. protein unpaused → ribosome translates → goes into the ER lumen

8. signal peptidase cleaves off signal peptide = protein free in ER.

ER Secretory Pathway

once its in the ER it has own transport network

• stay in the vesicle and fuse with the golgi apparatus

• golgi travel to final destination (Plasma membrane, Lysosome. Outside cell)

Pulse Chase Experiment

• Prove Linear path of ER secretory pathway

• Cells exposed to radiolabelled amino acids for 3 minutes → any proteins made in 3 mins had radioactivity especially in the RER

• RA amino acids were washed and replaced with normal amino acids → no new RA protein made so RA could be observed → RA moved out of ER into golgi then secretory vesicles and out of cell

Mitochondrial Protein Import Process

Post - Translational = protein is done before sent to mitochondria

1. mitochondria has double membrane → to get to central matrix protein has to pass 2 translocase complexes (TOM & TIM)

2. chaperone proteins (Hsp70) help keep protein unfolded so it can fit TOM/TIM thin channels

3. moving unfolded protein needs ATP

4. ATP hydrolysis used by chaperones to pull proteins in

5. Membrane potential used in TIM complex

Nuclear Import Transport Process

nucleus wrapped in double membrane nuclear envelope but uses NPCs

NPCs acts like sieve:

• proteins <40kDa can go through via passive diffusion w/o energy

• proteins >40kDa are too big and need Active transport

• to move

• to move large proteins use importins and exportins

against conc gradient using GTP hydrolysis energy

what are importins?

specialised escort proteins that bind using to the nuclear localisation signal (NLS) of cytosolic protein and move it into protein

What are exportins?

specialised escort proteins that bind to a nuclear export signal (NES) to carry molecules (i.e. RNA) out of the nucleus

Compare the different types of Protein Import

Feature	ER Import	Mitochondrial Import	Nuclear Import
Timing	Co-translational	Post-translational	Post-translational
Protein Structure	Threaded while being made	Must be Unfolded	Can be Fully Folded
Gateways Used	Sec61 Translocon	TOM & TIM complexes	Nuclear Pore Complex (NPC)
Energy Source	Translation / GTP	ATP + Membrane Potential	GTP (via a molecular switch called Ran)

examples of proteins using nuclear import?

• Structural nucleus proteins (Histones, Nuclear lamins)

• DNA/RNA Polymerase

• ribosomal proteins

What is GTPase Ran

• regulates nuclear import and export

• acts as on/off switches

What is GTPase?

• specialised enzyme that binds to GTP (Guanosine Triphosphate) = importins/exportins to function

• can remove own phosphate group = GDP (Guanosine Diphosphate) = turn off importins/exportins function

What are the specific locations and functions of Ran-GAP?

Ran-GAP

• located in the cytoplasm

• hydrolyses GTP into GDP

What are the specific locations and functions of Ran-GEF?

• located in the nucleus

• makes Ran to release GDP do it can bind to fresh GTP

How do transport receptors know where they are?

• Ran-GDP → cytoplasm

• Ran-GTP → nucleus

Nuclear Export Process?

• Exportin, Ran-GTP and the cargo with the NES signal all bind together in the nucleus

• complex move through the NPC into cytoplasm

• Ran-GTP stimulates Ran = GTP → GDP.

• GDP removes self and complex disassembles = cargo released

Nuclear Import Process?

• Importin, Ran-GTP and the cargo with the NLS signal all bind together to form complex

• the complex moves through the NPC int the nucleus

• In the Nucleus the Ran-GEF stimulates Ran to release GDP do it can bind to fresh GTP = cargo released.

How does Ran-GTP affect cargo binding differently for importin and exportin?

• Importin → Ran-GTP displaces cargo in the nucleus

• Exportin → stabilises cargo and binding in the nucleus

Are proteins functional when first made?

NO

• must be modified, folded in the ER and the golgi

Function of ER in the secretory pathway?

Modification, Folding and Quality Control

• handles co-translational transport

• signal peptide cleaved

• N-Linked glycosylation

• disulfide bon formations

Function of golgi in the secretory pathway?

Modification and Sorting

• Co-translational transport

• modifies carbs and add targeting signals

Function of secretory vesicles in the secretory pathway?

• final transport and secretion

What is glycosylation?

where carbs (glycans) are added to proteins or lipids = glycoproteins/ glycolipids

Why does glycosylation happen to proteins?

Prevents Aggregation

• hydrophobic regions of unfolded proteins stick together

• carbs = hydrophilic → make folding intermediate soluble

Helps Chaperon Binding

• helps the chaperone proteins to bind and hold the sugar which help the protein fold

Help Protein stability

• carbs block the extracellular proteases from reaching protein backbone = increases extracellular stability.

Mucus & Pathogen Protection

• highly glycosylated proteins create thick protective layer = trap/ward off pathogens

Oligosaccharides on some cell surface glycoproteins have a role cell-cell adhesion

Types of glycosylation?

• N - Linked

• O - Linked

N - Linked glycolsylation process?

1. before protein translated cell builds large branched carb (14 sugars)

• 14 sugar precursor is made on the ER membrane called the Dolichol

2. Polypeptide chain goes through a translocon and membrane-bound enzyme called Oligosaccharyl Transferase scans chain

3. enzyme is looking for N-X-S/T (concensus motif)

• N → Asparagine

• X → any amino acid but NOT proline

• S/T → Serine or Theorine

4. N-X-S/T passes into ER lumen and Oligosaccharyl Transferase cleaves off the 14 sugar and add it to the target protein on the -NH group

5. glycosidases start trimming sugars to monitor its folding correctly

6. moves via transport vesicles to the golgi apparatus

Why doesn’t Proline work in N-Linked Glycosylation?

• Proline has a rigid structure and physically distorts the chain → blocks the enzyme

• Oligosaccharyl Transferase is blocked form binding

• protein = not glycosylated

Compare N and O - Linked glycosylation?

What does the drug Tunicamycin do?

• drug inhibitor

• blocks the synthesis of the dolichol-linked oligosaccharide precursor

• proteins not glycosylated

BiP function?

• chaperone protein

• binds exposed hydrophobic regions

• prevents misfolding

• prevent exit of incorrect proteins

• binding immunoglobin protein

Calnexin + Calreticulin

• chaperone protein

• binds glycosylated proteins

• ensure proper folding

• binds to oligosaccharides on incomplete folded proteins

What is a disulfide bond?

• covalent bond between thiol -SH groups of two Cysteine amino acids

• Oxidation reaction

• stabilise tertiary and quaternary protein structures

where are disulfide bonds made?

• oxidising environments like ER where enzymes like Protein disulfids isomerases (PDI) help correct bond formation.

Why are disulfide bonds important?

structurally stabilises the proteins final 3D shape = no unfold when outside the cell where drastic environmental changes

PDI function?

1. ER enzyme catalyses the chemical reaction and creates the disulfide bond

2. breaks incorrect covalent bonds formed

3. helps rearrange mismatched cysteine pairings

4. if folding protein mismatched/incorrect PDI cleaves incorrect bond = allow protein chain to rotate until the ideal native configuration is achieved.

Why do cytosolic proteins rarely contain disulfide bonds?

• Cytosol highly reducing environment = prevents oxidation needed for disulfide bonds to form

• oxidising environment is in the lumen of the ER

What is Protein Disulfide Isomerase?

PDI

• enzyme that catalyses the oxidation reaction to make disulfide bonds

proline isomerisation?

Proline can exist in cis/trans forms

• proline struggles to rotate and gets stuck switching between cis/trans

• conversion is slow

• a rate-limiting barrier to efficient protein folding

Peptidyl-prolyl isomerases?

PPIases

• enzymes that speed up proline cis/trans conversion

• this is needed for correct protein folding

• acts as a chaperone protein

What if the protein is folded incorrectly?

Normal Process:

• protein folded

• checked by chaperone

• exit the ER

Misfolded Protein:

• Retained in ER

• Refolded

• OR destroyed

Name how the ER quality Control checks proteins?

• Glycosylation-Based Quality control

• Unfolded Protein Response (UPR)

• ER - associated Degradation (ERAD)

Glycosylation-Based Quality control

• glucosidase chop off two glucose molecules = 1 left

• 1 glucose is a tag for chaperone proteins Calnexin and Calreticulin → prevent clumping so it can fold

• final glucose removed = chaperones removed

• Sensor enzyme Glucosyltransferase evaluates released proteins

Folded correctly = exits ER

Folded Incorrectly

• detects exposed hydrophobic patches

• adds a UDP- glucose to the sugar backbone

• protein goes through the Calnexin/Calreticulin stage for refold attempt

Unfolded Protein Response

emergency stress signal for when misfolded proteins build up in ER faster than cell can repair.

• chaperone enzyme BiP binds to 3 transmembrane sensors (IRE1, PERK, ATF6) = turned off

• misfolded proteins arrive → BiP leaves sensors and attaches to misfolded proteins = sensors turn on

• Once activated IRE1 unmasks endoribonuclease domain → domain targets Xbp1 pre-mRNA in cytosol = cutting off intron

• two remaining exons join together = frameshift mutation

• unspliced Xbp1-u makes unstable protein

• spliced Xbp1-S makes stable protein

• Xbp1-s moves to nucleus → turn on genes that make more ER chaperones and folding catalysts

ER - Associated Degradation (ERAD)

If protein is still misfolded then destroyed

• misfolded protein moved out of ER lumen back into cytosol via translocator complex

• In cytosol, enzyme N-glycanase strips off sugar tags

• E3 Ubiquitin Ligase tags the polypeptide with a polyubiquitin chain

• Polyubiquitin chain is a marker for Proteasome which digests the protein = amino acids to reuse

Successfully folded protein go to…

transport via vesicles to golgi apparatus

Different compartments in the Golgi?

• cis-Golgi (Entry)

• medial - Golgi

• Trans - Golgi (Exit)

each region has specific enzyme

Modification steps in the Golgi stacks after protein folding?

Golgi stacks remodel the sugar trees added to the protein in the ER

1. protein leaves ER as high-mannose oligosaccharide = not functional

2. Get modified:

• Golgi Mannosidase removes extra mannose residue = makes space for new sugar to be added

• N-acetyleglucosamine (GlcNAc) transferase I added a new GlcNAc sugar = make it complex glycoprotein

• Golgi Mannosidase II remove more mannose residual

3. Golgi adds more sugars

• GlcNAc

• Galactose

• Sialic Acid

4. results on complexed branched oligosaccharide

Lysosome function

digest proteins, DNA, lipids

can lead to cell damage/ death

How does the cell tell lysosomal enzymes and secreted enzymes apart?

GlcNAc - Phosphotransferase recognises signal patch and adds sugar = M6P

• formed after protein folding

Where is the M6P tag found?

Cis Golgi Network

How does the M6P signal patch work?

• M6P receptor binds to tagged enzyme

• Clathrin coat forms - vesicle formed

• Protein is packaged into vesicle

• Vesicle goes to the endosome then lysosome

• when in lysosome low pH = enzyme detach from receptor

• receptor goes back to golgi

What happens if M6P is defective?

• GlcNAc- phosphototransferase is defective

• no Mp6 tag is added

• lysosomal enzymes no recognised

• get secreted outside the cell

• lysosomes lack enzymes

• waste builds up = cell damage

Inclusion Cell Disease

Inclusion Cell Disease

mutation in the GlcNac-phosphototransferase = cannot add M6P

Normal:

1. Lysosome enzyme made in ER → folded → signal patch forms → in Cis Golgi M6P tag added → in Trans Golgi M6P receptor binds → sent to lysosome

Disease:

1. no M6P

2. Enzyme is not recognised

3. enters default pathway → secreted out

Diagnostic Profile for Inclusion Cell Disease?

• patients blood has high level of lysosomal enzymes

• cellular lysosomes empty and non-functional

• cells have build up debris

Diseases of disposal (Loss-of-Function)

ER quality control is too strict

Mechanism:

• Protein fold slightly incorrect

• chaperones detect defect

• sent to ERAD

• destroyed in cytosol

Example of Diseases of Disposal

Cystic Fibrosis

• mutation in $\Delta\text{F508}$ = slight misfolding

• ER detects → destroys via ERAD

• no CTFR = no chloride channels

• extra mucus made = lung disease

Toxic Gain of Function (Diseases of retention)

Misfolded proteins not removed efficiently = accumulate in cell

Mechanism:

• Proteins misfold

• Cannot exit ER

• aggregates accumulates

• ER become stressed = cell damage

• Toxic

Toxic Gain of Function (Diseases of retention) Example

alpha ₁ Antitrypsin Deficiency (Liver Failure)

• misfolded protein accumulates in the ER

• Forms aggregates that accumulates

• kills Liver hepatocytes = widespread tissue destructions

Discuss how defects in protein processing and targeting in the secretory pathway can lead to disease?

• Secretory Pathway important for protein structure and localisation

• involves protein folding and modification in ER

• further processing in Golgi

• Explain what happens in ER

1. folding assisted with chaperone BiP, Calnexin, Calreticulin

2. additional modification = N-Linked Glycosylation and Disulfide bonds

3. Correctly folded leave ER

• misfolded go ERAD pathway

• ERAD may lead to diseases.

1. Liver Disease

2. Cystic Fibrosis

3. I- Cell diseases

Function of the Vesicle Coats?

• bends the membrane = spherical bubble

• Selectively capture and packages cargo proteins

Types of Vesicle Coats?

COPII

COPI

Clathrin

COPII

Anterogade

• moves fresh cargo forward → ER to Golgi

• Sar1 GTPase needed

COPI

Retrograde

• Receives protein backwards → Golgi to ER

• Arf1 GTPase needed

Clathrin

moves cargo between TGN, endosomes and plasma membrane

Describe the mechanism of COPII vesicle formation?

1. In ER membrane, Sec12 acts as Sar1-GEF protein and activates Sar1

2. Sar1-GDP to Sar1-GTP

3. Binding to GTP = exposes hydrophobic helix at th N-Terminus

4. Helix inserts into ER membrane

5. Sar1-GTP recruits

• Sec23 → binds Sar1-GTP and slowly converts to Sar1-GDP via hydrolysis

• Sec24 → binds sorting signals on cargo receptors

6. inner section pinned down → recruits outer structural framework

• Sec13/Sec31 → quickens Sec23 GAP activity = Sar1GTP to Sar1GDP

7. Sar1-GDP loses structure, loses amphipathic helix and pulls off membrane

8. inner coat detaches

9. outer coat collapses

10. vesicle becomes uncoated

11. ready to fuse vesicle

What are RAB proteins?

• small monomeric GTPases

• Attached to membrane via lipid

• Two types of RAB

What are the types of RAB proteins?

Rab-GTP → active

Rab-GDP → inactive

Examples of Rab

• Rab1 - cisGolgi targeting

• Rab5 - Early endosome

• Rab7 - Late endosome

What do RAB proteins do?

• acts as identifying markers for vesicles

• ensure vesicles dock at correct membrane

What would happen without RAB proteins?

Vesicles would fuse randomly

cell organisation would collapse

How does RAB work as identifying markers?

Membrane Docking Sequence

1. Vesicle carries RAB-GTP

2. The target membrane has a RAB effector

• large protein complex

3. Effector reaches across cytosol to the specific RAB-GTP on the incoming vesicle → pulls it towards membrane = tethering

4. RAB effector also binds to specific PIP

5. Vesicle can dock when correct RAB and PIP

6. SNARE proteins interact and fuse Vesicle with membrane.

What are PIPs?

• lipids in membrane

• helps RABs with vesicle localisation

How do PIPs helps RAB?

• Mark the type of membrane that the Vesicle will dock with lipid identity code

• The target membrane for the vesicle will have the correct RAB and PIP

Why can’t vesicles fuse without a SNARE protein?

Lipid Bilayer are surrounded with water and strongly resist fusion

What are SNARE proteins?

• specialised molecular machine

• Helps with vesicle fusion to mebrane

• 2 types

What are the types of SNARE Proteins

v - SNARE (vesicle SNAREs)

• on vesicle membrane

• e.g. Synaptobrevin

t- SNARE (target SNAREs)

• on the target membrane

• e.g. Syntaxin

What is the problem SNAREs must solve?

• lipid bilayers naturally repel each other

• fusion between vesicles and membranes need energy

How do the SNARE proteins work?

Zipping Mechanism

1. RAB proteins bring the vesicle close to the membrane → Tethering

2. v-SNARE on the vesicle meets matching t-SNARE on target membrane

3. v-SNARE helix interlocks with t-SNARE and wrap around each other

4. this interlocking = stable, 4 alpha helix bundle called trans-SNARE complex or SNAREpin

5. SNAREs continue interlocking = more energy released and membranes pulled closer and water molecules pushed out.

6. vesicle membrane and target membrane and outer lipid touch = Hemifusion

7. The membrane continue to open = channel appears between = fusion pore

8. fusion pore = cargo released from vesicle into target cell

After Fusion what happens to the the SNARE?

• V and T SNAREs in the same membrane = cis-SNARE complex

• Cell has to recycle SNAREs and cant dismantle coz its stable

1. protein called alpha SNAP wraps around cis-SNARE bundle

2. recruits big hexameric AAA+ ATPase enzyme called NSF

3. NSF binds to alpha SNAP

4. NSF uses energy from ATP hydrolysis to unravel the helixes

5. Individual SNARE proteins released

Why do SNARE proteins need ATP?

• SNARE interlocking provides the energy needed for the membrane fusions

• When recycling the SNARE, NSF uses this ATP to separate SNAREs after fusion

NSF full name?

N-ethylmaleimide Sensitive Factor

Vesicle Sequence Steps?

1. Vesicle uncoating

2. RAB targeting with PIPs

3. Tethering by RAB

4. v-SNARE bind to to t SNARE

5. Trans-SNARE complex forms

6. Interlocking helices = membranes together

7. Hemifusion

8. Fusion Pore opens

9. Cargo delivered

10. Cis SNARE complex remains in cell

11. alpha SNAP binds

12. NSF hydrolyses ATP

13. ATP break cis SNAP complex

14. SNARE recycled

How do vesicles release neurotransmitters?

• RAB targeting

• SNARE interlocking

• Membrane fusion

• cargo release in fusion pore

• cargo is neurotransmitter

What is Botulinum Toxin?

• bacterial protease that is a neurotoxin

• AKA Botox

What does Botulinum Toxin target?

• SNAP-25

• Syntaxin

What does Botulinum Toxin do?

1. cleave SNARE complex

2. no trans-SNARE complex forms

3. no hemifusion of membranes

4. no fusion pore

5. No ACh released

6. muscles don’t receive stimulation

7. muscles become weak and relaxed

What does Tetanus Toxin do?

• cleaves SNARE proteins

• No fusion of membranes

• no fusion pore = no cargo released

• no inhibitory neurons released = motor neurons fire = muscle is rigid and contracted.

What are the mutations that affect cargo accumulation?

• Sec61

• SAR1

• RAB1

• v-SNARE

cargo accumulation: Sec61 mutation

• Sec61 is in the ER channel

• prevent translocation into ER

• Secretory proteins stay in the cytosol

Cargo accumulation: Sar1 or Sec12

• Sec12 = GEF for Sar1

• Sar1= starts COPII coat assembly

• mutation prevents COP11 coat assembling and vesicle budding

• Secretory proteins accumulate in ER lumen