Intracellular protein trafficking

Question 1

What are the two types of sorting signal?

Where are nuclear proteins synthesised?

What is a nuclear pore?

BY protein size, how can proteins travel through the nuclear pore?

How do folded proteins with a signal sequence enter the nucleus? 3 steps

What kind of protein is a receptor?

How does a protein exit the nucleus? 2 steps

Why is the nuclear export sequence less defined?

Answer 1

Signal sequence - amino acids at end of polypeptide chain
Signal patch - amino acids separate until protein folds into 3D structure

Cytosol

Complex made up of nucleoporins which allows protein to pass through into the nucleus - aqueous pores

Small <5kDa can diffuse, 5-60 kDa diffuse but slower, >60 kDa need active transport

1. Importin protein recognises signal sequence on protein & binds to it
2. Importin recognises FG repeats (binding site for receptor) on the nucleoporins & binds
3. Importin & cargo taken into nucleus
4. Ran-GTP binds to importin - displacing protein & allows for importin to return to cytosol

Cytosolic soluble protein

1. Protein recognised by exportin-Ran-GTP & binds to signal sequence (string of leucine residues)
2. Exportin interacts with FG repeats on nucleoporins - bringing protein out of nucleus
3. Ran-GTP hydrolysed by Ran-GEF allowing ternary complex to dissociate & for exportin to re-enter the nucleus

String of lysine residues

Question 2

What molecule drives directional transport through nuclear pores?

What are the 4 steps of nuclear import?

What are the 4 steps of nuclear export?

What are the concentrations of Ran-GDP/GTP in which areas of the cell and why?

What kind of import is this?

Of what family does the receptor belong to?

Answer 2

Ran-GTPase

1. Importin recognises signal sequence & binds to protein forming cargo-protein complex
2. Complex enters nucleus through interaction with FG repeats on nucleoporins
3. Ran-GTP in nucleus binds to importin to displace cargo protein
4. Importin with Ran-GTP taken out through nucleoporin to release importin back in cytosol (where it is hydrolysed in the cytosol back to GDP)

Exportin recognises signal sequence of protein & binds to it with Ran-GTP (tertiary complex)
2. Complex exits the nucleus via interaction with FG repeats on nucleoporins
3. Ran-GTP is hydrolysed by Ran-GEF into Ran-GDP & protein dissociates from complex
4. Exportin moves back into nucleus for further rounds

High conc Ran-GTP in nucleus to export cargo out into the cytosol. High conc Ran-GDP in cytosol

Post-translational: proteins folded before import

Karyopherin

Question 3

What is an example of a typical mitochondrial targeting sequence?

Where does translocation occur in mitochondria?

What is the key difference between the state of the protein prior to mitochondria import compared to nuclear import?

What maintains this state?

What are the steps of protein import into the mitochondrial matrix - 5 steps?

What is ATP hydrolysis required for?

Answer 3

Amphiphatic alpha helix

Between specific sites where inner & outer membrane are close together

Protein is unfolded

Cystolic chaperones & hydrolysis of ATP

1. Signal sequence on protein interacts with receptor TOM complex
2. Protein threaded through TOM complex
3. Signal sequence recognised by TIM23 complex & translocates through inner membrane into matrix
4. Signal sequence cleaved by signal peptidase
5. Protein folds into final 3D structure

Maintain unfolded
Pull through TIM23 complex (with use of chaperones)

Question 4

What is required for transport across the TIM23 complex?

How does a redox potential influence the protein after transport through TOM complex in intermembrane space?

How do proteins insert into the outer mitochondrial membrane? 4 steps

How do proteins insert into the inner membrane? 3 steps

Answer 4

Membrane potential

Interacts with Mia40 protein in intermembrane space & allow formation of disulphide bonds - allow fold protein in intermembrane space so cannot go through TIM23 complex - nothing to do with reduction (breaks)

1. Protein translocates through TOM complex
2. Bound by chaperones in IMS
3. Inserted into SAM complex (outer membrane) & translocates through until reach hydrophobic area (interactions between protein & hydrophobic tails of membrane space) such that the protein is stuck in the membrane
4. Build up multi-span of membrane proteins

Protein translocated through TOM & into TIM 23 complexes
Signal sequence cleaved in matrix space to reveal hydrophobic region of protein whilst still in TIM23 complex
Protein moves sideways into inner membrane

Question 5

How does a protein with an internal signal sequence become an inner membrane protein? 3 steps

How does a protein synthesised in the mitochondrial matrix become an inner membrane protein? 2 steps

How does a protein become an inner membrane protein from outside the mitochondria using the OXA complex? 3 steps

Answer 5

1. Protein translocates through TOM & is bound by chaperones
2. Internal sequence allows insertion of protein into TIM22 complex using membrane potential as an energy source
3. Protein moves sideways to form inner membrane protein

Protein inserts into OXA complex from the matrix
2. Hydrophobic region on protein becomes stuck in OXA complex & moves sideways to form inner membrane protein

Protein enters through TOM & TIM23 so signal sequence is cleaved in matrix
2. Signal cleavage causes exposure of 2nd signal sequence - which inserts into OXA complex
3. Hydrophobicity imbeds into inner membrane & moves sideways to become inner membrane protein

Question 6

What are the differences between receptors for nuclear & mitochondrial import?

Receptors are proteins - but what is the 1 exception?

What is a protein without a signal sequence?

What does a typical ER signal sequence look like?

Answer 6

Nuclear = soluble
Mitochondrial = membrane

SRP

Cytosolic

has 1+ positively charged amino acids followed by a stretch of 6-12 hydrophobic acids followed by signal peptidase cleavage sequence after entered into lumen of ER

Question 7

For ER protein import, in what state is the protein?

What are the steps for the protein import into the ER? 4 steps

What is it powered by?

What is SRP made up of?

What is the cycle of ribosomes?

What feature does the signal sequence have?

Answer 7

Nascent protein/polypeptide chain being synthesised from the ribosome in the cytosol

1. N-terminus of nascent protein is recognised by SRP which binds to the signal sequence & the ribosome pausing translation
2. Complex is translocated to ER membrane where SRP interacts with SRP receptor
3. Signal sequence is transferred from SRP receptor to Sec61 complex so polypeptide directly synthesised into the ER
4. SRP & SRP receptor dissociate & recycled

GTP hydrolysis

6 proteins & 1 small RNA molecule

Ribosomal subunits & components constantly cycled either stay in cytosol or recruited to ER membrane

Hydrophobic

Question 8

What are the steps for the translocation of a soluble protein into the ER lumen? 4 steps

What happens to the cleaved signal sequence?

Answer 8

1. Protein bound by SRP & ribosome & binds to SRP receptor through its signal sequence
2. Protein synthesised into ER lumen via Sec61 complex
3. Signal sequence cleaved off in lumen by signal peptidase if contains signal peptidase sequence
4. Mature polypeptide folded in ER lumen by chaperones

Hydrophobic so remains in membrane until degraded

Question 9

How does a transmembrane polypeptide enter the ER lumen? 4 steps

What are the 2 types of transmembrane protein?

Answer 9

1. Hydrophobic region of membrane acts as the signal sequence & is inserted through Sec61 complex by SRP & SRP receptor
2. Hydrophobic region remains in the membrane
3. Depending on the orientation of how the peptide enters the translocater, end up with type 1 or type 2 transmembrane protein
4. Protein moves sideways in membrane to build up multi spanning region

1 = C-terminus in cytosol
2 = N-terminus in cytosol

Question 10

How is a protein firstly glycosylated (core/N-linked) into ER on synthesis/entry? 3 steps

What is protein glycosylation used for? 4

Answer 10

1. Oligosaccharyl transferase (ER membrane protein) interacts with Sec61 & ribosome
2. OT recognises asparagine residues N-X-S/T on peptide entry into ER
3. Transfers lipid-linked oligosaccharide to the asparagine side chain

Tag/marking protein folding - if protein spent time in ER it is misfolded so recognised for degradation
2. Make proteins resistant to proteases
3. Protection against pathogens - cell signalling
4. transport signal

Question 11

What are the features of the SRP pathway? 4

How is the Sec62/63 pathway different with components required?

How does the protein need to be in this pathway?

What are the differences between this and the mitochondrial import?

What are the features of the TRC40 pathway? (hint think opposite to SRP)

Answer 11

(SRP, SRP Receptor & Sec61) Co-translational, N-terminal signal sequence, GTP & SRP dependent

All of above but: ATP dependent, can be co or post-translational, requires SEC complex translocator & BiP ATPase

Protein needs to be unfolded in cytosol if post-translational & pulled through SEC complex by BiP ATPase

ER is single membrane/mitochondria is double, and requires different translocator (TOM/TIM vs SEC)

SRP independent, requires C-terminal domain or glycosylphosphatidylinositol anchor, post-translation BUT ATP-dependent (like Sec62/63)

Question 12

What happens inside the ER regarding the imported peptides?

What else do both of these do?

What is the ER site for? How come?

What is ERAD? What does it do?

Answer 12

Glycosylated & helped to fold by chaperones

Mark proteins for degradation

Formation of disulphide bonds to stabilise tertiary & quarternary structures of proteins - lumen is oxidising environment whereas cytosol reducing

ER associated degradation - system to mark proteins as misfolded if they remain in the ER longer than they should

Question 13

In 5 steps, how does ERAD recognise & degrade misfolded proteins in the ER?

Answer 13

1. Lectin recognises sugar on protein marked by glycosylation
2. Protein translocator complex threads protein out of ER lumen into cytosol
3. Protein recognised by ubiquitin ligase complex in cytosol & ubiquitin added in form of polyubiquitin chain using ATP
4. Sugar removed from protein with N-glycanase
5. Protein recognised & degraded proteasome - but ubiquitin removed prior to degradation for reuse

Question 14

What is the transfection method to studying protein translocation across membrane?

What is the problem?

What happens if there is no signal?

What is the biochemical approach method to studying protein translocation across membrane?

What is the genetic approach method to studying protein translocation across membrane?

Answer 14

Adding amino acids/signal sequences to the a terminus of cDNA encoding GFP to observe where it goes

Putting signal sequence at wrong terminus

Cytosolic protein

Seeing whether a protein with co-purify from an organelle e.g if protein is concentrated in the ER then can you purify it from the ER?

Use yeast as a model organism as used to look at protein trafficking in studies & observe - easy to grow, cheap, exist in haploid (1 copy each gene)

Question 15

If ER is the starting place for protein trafficking, what happens to the proteins after glycosylation & folding?

What signals can tell protein destination from the ER?

How can a protein move to other organelles via vesicular transport?

Direct fusion is a way of transport of proteins without requiring vesicles- how does it do this & what is an example?

Answer 15

Enter different pathways via golgi, endosomes, lysosomes, secretory vesicles & plasma membrane etc

Amino acid sequences & protein modifications

Budding of protein into vesicle from donor compartment to target compartment where the vesicle fuses

Donor compartment & target compartment content mix (kiss & run) and then fuse to form hybrid organelle with a content exchange. Late endosome to lysosome

Question 16

What are the 2 different vesicle coats for?

What is a coated vesicle budding made up of?

What are the 2 roles of vesicles?

What are the steps for clathrin coated vesicle assembly? 5 steps

Answer 16

COPI - ER to golgi
COPII - golgi to ER
Clathrin - to endosomes

GTP-binding protein (assemble/disassemble coat), v-SNARE protein for fusion, coat proteins, soluble cargo protein or membrane cargo protein

Structural & selective

1. Cargo molecules bind to receptors
2. Adaptor protein AP2 recognises endocytosis signal (endosomes) & starts assembling coat
3. Clathrin forms cage structure around budding vesicle
4. When vesicle almost formed - think neck attached to plasma membrane containing fission proteins
5. GTPase dinimine severs the neck
6. Clathrin & adaptor protein dissociate from vesicle due to Hsp70 protein leaving naked transport vesicle for the endosome

Question 17

What are the limitations of dinimine?

How does the uncoated vesicle fuse with the target membrane? in 3 steps

How is the trans-snare complex broken?

What is the fusion specificity determined by?

Answer 17

Don’t work at high temperatures

1. Rab effector binds to Rab-GTP(ase) associated on vesicle which brings vesicle closer to membrane
2. 3 t-snares alpha helices & the v-snare alpha helix interact to form trans-snare complex across the membrane
3. This brings vesicles very close to target membrane for membrane fusion involving the hydrolysis of Ran-GTP & removal of Ran-GDP

Broken by NSF, accessory proteins & ATPase using ATP hydrolysis to break apart complex so v-snare can be retrieved to original membrane
2. Toxins cleave snares & prevent neurotransmitter release (need membrane fusion for this)

combination of v-snare & t-snare proteins and Rabs

Question 18

What do phosphoinositides do?

How are they regulated?

How would proteins be sorted that are soluble ER resident proteins (need to be in ER for function)? 4 steps

How would transmembrane ER resident proteins be sorted? 2 steps

Answer 18

Proteins that mark organelles & membrane domains to recruit proteins to specific organelles

By kinases & phosphotases as can be phosphorylated at 3’, 4’ and 5’ positions

1. Soluble ER protein with KDEL sequence (c-terminal aa preventing secretion) with KDEL receptor at ER membrane & secreted protein - COPII coat assembles around vesicle (encapsulating both)
2. pH in golgi is lower than in ER - so ER soluble protein binds to KDEL receptor which recognises the KDEL signal on the soluble protein
3. KDEL is an extreme C-terminus retrieval signal
4. KDEL receptor-ER complex interacts with COPI coat & buds to transport soluble protein-KDEL back into ER

kk

1. Transmembrane protein incorporates into COPII vesicles with ER soluble protein, KDEL receptor & bud from ER
2. KKXX c-terminal retrieval sequence of transmembrane ER protein recognised by COPI coat - as well as ER soluble-KDEL receptor so binds & buds back to ER

Question 19

How would a plasma membrane protein & secreted protein be sorted via secretion/exocytosis?

How would a hormone be secreted?

What happens in each golgi stack?

What happens to the sugars from glycosylation on the proteins?

How can you observe how far along a protein is in the golgi?

What is O linked glycosylation?

Answer 19

Both associate with COPII & enter trans-sack of golgi & fuse with the plasma membrane

Via regulated secretory pathway to golgi before packaged into a regulated secretory vesicle to fuse with the plasma membrane ONLY with the addition/response of a signal

Proteins undergo different modifications with different enzymes/substrates in each stack

Further modified progressing through the golgi stack producing a complex/modified oligosaccharide

After certain point modifications make protein resistant to Endo H - expose protein to it to see how far it is

When sugars are added to OH groups of serine & threonine catalysed by glycosyl transferases in golgi

Question 20

What is the vesicular transport model for how proteins transport through golgi/stack?

What is the basis of cisternal maturation?

What is a lysosome?

Why does it have a low pH?

What does it fuse with in the secretory pathway?

Answer 20

Vesicles bud off each golgi stack & fuse with next & this transport is balanced with vesicular transport in the opposite direction

Accumulation of proteins/enzymes of transgolgi in opposite directions allows for each golgi department to gradually mature into the next department up

Organelle containing acid hydrolases, proteases & lipases to degrade proteins/lipids

Has a H pump so proteins can be degraded within the lysosome

Endosome

Question 21

How are soluble lysosomal resident proteins/hydrolase (lysosomal enzymes) sorted to the lysosome? 6 steps

Answer 21

1. Synthesised in ER & acquire mannose 6 phosphate to sugar in golgi
2. This signal is recognised by mannose 6 phosphate receptor & binds to allow proteins to enter transport vesicle
3. Vesicle is coated, transports & fuses to endosome
4. Mannose 6 receptor & lysosomal hydrolase dissociate due to the low pH in the endosome
5. The lysosomal enzyme/lysozyme translocates to lysosome
6. M6P receptor recognises retromer coat (endosome) & fuses back into golgi via retrieval sequence back to ER

Question 22

How does protein sorting of endocytosis of LDL receptors work? 4 steps (from plasma membrane)

How was LDL receptor endocytosis signal discovered?

Answer 22

1. LDL binds to LDL receptors inducing an endocytosis signal FXNPXY - so adapter proteins also bind to LDL receptor
2. LDL incorporates into coated vesicles & then uncoats & fuses into early endosome
3. Low pH in endosome causes LDL to dissociate from receptor so receptor is returned by retrieval to bind to more LDL in plasma membrane
4. LDL translocates to late endosome & finally endolysosome where degraded & released as free cholesterol

Patients with familial hypercholesterolaemia had amino acid change from NPVY to NVPC - meaning LDL bound to mutant receptor but failed to internalised so LDL didn’t clear from blood

Question 23

How does protein sorting of endocytosis of growth factors work (Epidermal Growth Factor)? 6 steps (plasma membrane)

Why is it important that the signal is switched off?

When does the signal stop initiating?

Answer 23

1. EGF binds to EGF receptor at plasma membrane resulting in receptor to dimerise
2. Ligand-receptor complex is endocytosed by signal on EGF receptor YXXØ
3. EGF & receptor transported to early endosome where vesicles fuse
4. Ubiquitin tag added to receptor-EGF complex allowing for initiation of signalling cascade at plasma membrane
5. Ubiquitin recognised by late endosome & EGF-receptor fuses to intralumenal vesicles & bud inwards
6. Late endosome fuses with lysosome causing EGF & receptor to degrade - switching off the signal

EGF stimulates signalling cascade in cells resulting in cell proliferation - want it to be transient as cells will keep proliferating

When EGF & receptor away from cytosol - can no longer be induced by phosphorylated enzymes

Question 24

What is ubiquitin a signal for?

What happens once the signal is recognised by an escort?

What escort is involved in pinching of vesicles?

What else is the formation of multi vesicular bodies involved?

How do viruses exploit protein trafficking pathways?

What characteristic of protein trafficking pathways make it great to track a protein?

Answer 24

Protein sorting at the late endosomes

Protein & receptor directed onto inner membrane of multivesicular bodies

Escort 3

Cell division, virus buddying of particles etc

Enveloped virus particles are endocytosed & fuse across the endosome membranes

Saturable - over-expressing a protein can look at where it goes (tag with GFP)