BS2092 - Intracellular Compartments and Protein Sorting

Define co-translational insertion

the process where ER-bound ribosomes synthesise proteins and simultaneously translocate them into the ER

What is required for protein sorting to the correct membrane?

• a signal, intrinsic to the protein

• a receptor that recognises the signal and directs it to the correct membrane

• translocation machinery

• energy to transfer the protein to its new place

Signal for nucleus importation

a sequence of 5 consecutive positively charged amino acids anywhere in the protein

Signal for mitochondria importation

positively charged amino acids spread out by 2-3 other amino acids

Signal for ER importation

a stretch of between 5-12 hydrophobic amino acids present at the N-terminus of the protein (within the N-terminal 16-35 residues)

What are the 3 positively charged amino acids

• Lysine (Lys/K)

• Arginine (Arg/R)

• Histidine (His/H)

What is TOM (translocase outer membrane) complex

the receptor that recognises the matrix protein

What does TOM do

• binds to the signal sequence

• delivers protein through a protein conducting channel in the complex

What is the TIM23 (Translocase of the Inner Membrane 23) complex

a mitochondrial protein machine that transports proteins across the inner membrane into the matrix

What happens to the signal sequence after the protein has been successfully delivered into the matrix

it is cleaved

Role of cytosolic hsp70 chaperone proteins

keep the protein unfolded to be delivered through the protein channel into the matrix

Role of mitochondrial hsp70 chaperone proteins

• binds to the protein as it enters the matrix

• causes a conformational change in the import ATPase (with aid of ATP hydrolysis) which helps pull the matrix protein into the matrix

What does NPC stand for

nuclear pore complexes

How many NPCs in a typical cell

3000-4000

What is an NPC

an aqueous pore lined by proteins with extended conformation, which facilitates the bidirectional transport of molecules in and out of the nucleus

Purpose of the proteins lining the pore

they act as a sieve/meshwork to block diffusion of large molecules

How do larger molecules move across the nuclear envelope

active transport

How does nuclear-cytoplasmic transport work

• transported molecules contain sorting signals

• importins and exportins (nuclear transport receptors) recognise the signals and carry the molecules through the pore

• proteins are transported against their concentration gradient using energy from GTP hydrolysis

Examples of proteins which undergo nuclear import

• structural proteins of the nucleus

  • histones, nuclear lamins

• DNA/RNA polymerases

• ribosomal proteins

• gene regulators

  • transcription factors, chromatin modifying enzymes

What regulates nuclear import and export

Ran

Features of Ran

• involved in signal transduction

• acts as on/off molecular switches

• activity is controlled by their GTP/GDP bound status

How does their GTP/GDP bound status control the activity of Ran

• GDP-bound = inactive

• GTP-bound = active

What catalyses the activation of Ran

Guanine nucleotide exchange factor (GEF)

How does GEF catalyse the activation of Ran

• binds to Ran, stimulating the release of GDP

• GTP binds in place of GDP, due to a higher concentration of GTP in the nucleus

Ran-GTPase intrinsic activity

long enough time allows it to hydrolyse GTP to GDP itself

The effect of GAP (GTPase activating protein) on Ran-GTPase intrinsic activity

accelerates the hydrolysis of GTP to GDP to switch off Ran

Which types of Ran are more abundant in the nucleus and in the cytoplasm

• Ran-GTP is more abundant in the nucleus

• Ran-GDP is more abundant in the cytoplasm

How does Ran allow importins/exportins to sense whether they are in the nucleus or cytoplasm

• Ran-GAP and Ran-GEF are tethered in the cytoplasm and nucleus, respectively

• creating a gradient so that Ran-GDP is predominantly in the cytoplasm and Ran-GTP is predominantly in the nucleus

What is Ran-GAP tethered to in the cytoplasm?

cytoplasmic fibrils

What is Ran-GEF tethered to in the nucleus

chromatin

How does Ran act as a molecular switch for importins

• Ran-GTP binds to importins, changing their confirmation to allow cargo release

• Ran-GDP dissociated from importins, changing their conformation to cause cargo binding

Why can importins change their conformation

they consist of multiple stacked α-helices that act like a flexible spring-like structure

How does Ran-GTP binding allow for cargo release

binding of Ran-GTP moves a loop, forcing the release of cargo

Nuclear import summary in cytoplasm

• Ran-GDP predominates

• Ran-GDP does not bind importin

• Free importin can bind cargo

Nuclear import summary in nucleus

• Ran-GTP predominates

• Ran-GTP binds to importin

• Conformational change displaces cargo

What is the SRP (signal recognition particle)

a multidomain riboprotein that mediates a 3-way association with its receptor in the ER, the ribosome and the signal peptide

Pathway of SRP-mediated delivery of ribosomes to ER membrane

1. as the protein is synthesised it is immediately recognised by the SRP

2. this stops further translation occuring so that the protein is not produced within the cytosol

3. SRP then binds to its receptor in the ER (this receptor is in close association with the protein translocator in the ER membrane)

4. Once the SRP binds to its receptor, together with its ribosome, ER protein synthesis occurs

Pathway of SRP-mediated delivery of ribosomes to ER membrane (part 2)

5. After SRP is bound to its receptor, the ribosome is placed on top of the protein translocator, causing it to open

6. GTP hydrolysis occurs from both subunits of the SRP receptor to provide the energy required

7. protein synthesis carries on, proteins are translocated through the translocator until the fully synthesized protein is delivered into the ER lumen

8. the signal peptide is cleaved when it protrudes through the membrane by the signal peptidase

Comparison of Targeting to different organelles

Organelle:

• ER

• Mitochondrial matrix

• Nucleus

Signal:

• Hydrophobic signal sequence (N-term)

• amphipathic helix (N-term)

• short stretch Arg/Lys (anywhere)

Signal removed?

• Yes

• Yes

• No

Receptor:

• SRP/SRP Receptor

• TOM Complex receptor

• importin

Translation machinery:

• Sec61 protein translocator

• TIM/TOM complex

• nuclear pore complex

Energy source:

• GTP hydrolysis

• ATP hydrolysis and membrane potential

• GTP hydrolysis

What types of ER membrane proteins insertion involves SRP

Types I-IV

Type I ER membrane protein

• N-terminus in ER lumen

• C-terminus in cytosol

• N-terminus signal sequence is cleaved during insertion

Type II ER membrane protein

• N-terminus in cytosol

• C-terminus in ER lumen

• anchored by a signal-anchor sequence close to the N-terminus

Type III ER membrane protein

• N-terminus in ER lumen

• C-terminus in cytosol

• anchored by a signal-anchor sequence close to the N-terminus

Type IV ER membrane protein

• multi-pass proteins which span the membrane multiple times

• divided into subtypes IV-A (N-terminus in cytosol/C-terminus in ER lumen) and IV-B (N-terminus in ER lumen/C-terminus in cytosol)

• contains multiple signal-anchor/stop-transfer sequences that allow them to span the bilayer several times

Mechanism of synthesis of Type I membrane protein at the ER

• similar pathway to the soluble example until a 2nd hydrophobic sequence is reached

• when the 2nd hydrophobic sequence is reached, translocation is stopped in the lumen and the protein is anchored into the membrane

• the rest of the protein synthesis continues in the cytosol until a STOP codon is reached to form the C-terminal

Insertion of Type II membrane protein into ER membrane

• SRP recognises an internal hydrophobic stretch of amino acid

• protein is delivered into membrane by SRP

• protein synthesis continues and the protein is inserted such that its N-terminus is in the cytosol and C-terminus in the ER lumen