Transporting proteins into Membranes

Question 1

Signal Based targeting

Answer 1

Major Protein sorting pathway:

• targets newly synthesised proteins from cytosol to an organelle (during translation or following synthesis)
• Soluble proteins are translocated across membrane into aqueous organelle interior

Question 2

The Rough Endoplasmic Reticulum (RER) in protein synthesis and trafficking

Answer 2

ONLY proteins destined for the cytosol, nucleus, mitochondria or peroxisomes DON’T enter the RER:
- most other proteins are cotranslationally translocated into the RER (otherwise they undergo post-translational translocation: enter the RER following translation by cytosolic ribosomes)

Question 3

Targeting proteins to the RER

Answer 3

Soluble proteins: mRNA’s N-terminal sequence directs protein/ribosome complex to the ER for co-translational translocation to occur.
- N-terminal sequence: hydrophobic, variable, 16-30 residues long (6-12 hydrophobic amino acids), attracted to ER membrane

Question 4

Experimental evidence of simultaneous translation and translocation using microsomes (and Pulse Chase)

Answer 4

Producing microsomes in vitro from homogenized eukaryotic cells.

Those with ribosomes bound were isolated using differential and sucrose density-gradient centrifugation

Pulse chase can be carried out using these

Question 5

Pulse Chase

Answer 5

Tracking radiolabelled proteins inside the RER:

1) incubate cells in radiolabelled amino acids (newly synthesised = labelled)
2) Microsomes treated with protease to remove any protein NOT protected by the ER (radiolabelled ones are safe)
3) Add detergent to dissolve the ER membrane, no longer protecting the newly synthesised proteins - they get lysed - fluorescence observed

Proof of newly synthesised entering RER

Question 6

Proteins invloved in initialising co-translational translocation

Answer 6

1) Signal Sequence Recognition protein
- mechanism that targets secretory protein to ER

2) Signal Recognition Particle (SRP) and its receptor
- Bind to ER signal of nascent protein and large ribosomal subunit
- SRP = 6 proteins bound to 300nt RNA (ribonucleoprotein)
- Its p54 subunit binds the hydrophobic residues of Signal Sequence
- SRPR binds SRP: release upon GTP hydrolysis

Question 7

Translocon

Answer 7

Binds signal sequence once it’s released by the SRP and facilitates the insertion of polypeptide into ER membrane

• forms channel through which the polypeptide is passed
• formed from Sec (secretory) proteins with a central translocation channel and various other components including a signal peptidase to cleave the SRP

Question 8

Signal Peptidase

Answer 8

A serine protease in ER lumen, associated with translocon

- active site endoproteolytically cleaves signal sequence located at the extracytoplasmic site of the membrane

Question 9

Unidirectional Movement across ER

Sec63/BiP interaction

Answer 9

Protein chaperones (Sec63 complex/BiP) bind to and stabilise the unfolded protein
- The Heat Shock Protein (HSP) BiP contains a peptide binding domain and an ATPase domain

1) Sec63 hydrolyses BiP: ATP promotes conformational change
2) BiP-ADP binds to the protein as translocation continues, preventing the protein from ‘sliding back’ and stabilising it
3) BiP releases upon maturation and binds to ATP - the cycle repeats

Question 10

Oligosaccharyltransferase (OST)

Answer 10

Catalyses N-linked glycosylation during co-translation

• uses energy from the cleavage of pyrophosphate bond between the dolichol-glycan molecule
• attaches glycan to nitrogen atom (aspargine) of protein: attachment requires consensus sequence (Asn-X-Ser/Thr)

Question 11

Membrane Protein classes

Answer 11

Type 1 (e.g. LDL receptor)

• Cleaved N-terminal ER sequence
• N-terminal = lumenal; C-terminal = cytosolic

Type 2 (e.g. Transferin receptor)

• No cleavable N-terminus
• N-terminal = cytosolic; C-terminal = luminal

Type 3 (e.g. Cytochrome 450)

• Same orientation as Type 1 but no cleavable N-terminus
• Different position of positive charges to type 2 (it’s on the C-terminal side)

Question 12

Stop-Transfer Anchor Sequence (STA)

Answer 12

Embedded into the membrane, following which translation continues to occur but growing into the cytosol

Question 13

Type 1 - Integral Membrane Protein

Answer 13

Signal Peptidase cleaves Signal Sequence and translation continues
- When hydrophobic transmembrane domains (TMDs) enter translocon, translocation stops, the translocon opens laterally and the TMD integrates into ER membrane

Question 14

Type 2 - Transmembrane Protein Insertion

Answer 14

No N-terminal Sequence: instead internal sequence in polypeptide recognised by SRP

• inserted into translocon pore, the signal acts as a TMD
• released by the translocon, it embeds into the ER bilayer (a.k.a. Signal Anchor (SA) sequence)

Question 15

Type 3 membrane protein

Answer 15

Several transmembrane sequences (STAs and SAs)
- each time sequence enters translocon it integrates (different orientations depending on signals and sequences, hence different classified types)

Question 16

Tail Anchored Proteins

Answer 16

Type of Single Pass protein

• TA proteins are post-translationally targeted to ER
• Involves the ‘Get3’ ATPase rather than SRP which binds to the C-terminal hydrophobic fragment of protein
• Get3/protein complex recruited by Get1/Get2
• ATP is hydrolysed and the hydrophobic C-terminus gets embedded into the membrane

Question 17

Using Hydropathy profiling to determine protein’s type and function

Answer 17

By analysing the protein sequence the number of hydrophobic amino acids can be deducted as well as their distribution patterns

• Long hydrophobic sequences will mean there’s N-terminal sequences, STA and SA sequences
• Positions of hydrophobicity help determine type of integral membrane protein

Question 18

GPI-linked protein

Answer 18

Glycosylphosphatidylinositol (GPI) is a covalent amphipathic molecule

• Amino acid sequence near N-terminus recognised by a GPI transamidase which cleaves off original Stop Anchor Sequence and transfers ER luminal portion to a pre-formed GPI membrane anchor
• GPI anchor proteins tend to cluster in lipid rafts