M4L3 - Co-translational Targeting

Secretory Pathway

• Proteins transported to ER is co-translational instead of post-translational

• Secreted pathways are synthesized in ER before vesicle-based transport carries them out of the cell

RER Characteristics

• Bound by single membrane (it’s also continous with outer nucleus membrane 

• No genome (RER proteins all encoded in nuclear genome

• Site of protein synthesis and modification 

• First destination for secreted proteins

TEM of ER

• RER is continous with nucleus membrane 

• Punctate black dots is membrane-bound ribosomes 

  • They synthesize secreted proteins 

TEM of RER

• ER associated ribosomes are on cytosolic side of the ER membrane

• At any given time, there will be free and ER-associated ribosomes 

• Both are translationally active and come from the same common pool of ribosomes 

• They just cycle on and off the ER membrane 

Proteins Targeted while Translation is Active Experiment

• Microsomes used to mimic ER membrane

  • Made by disrupting the ER membrane by homogenization

  • They spontaneously assemble into spheres mimicing normal ER behaviour 

• Two experiments were conducted 

1. Treat microsomes with detergent

• This releases the proteins inside

• Protease is added to digest the proteins

• This shows that the protease can digest proteins

2. Protease added with intact microsome

• The newly synthesized proteins are protected inside microsome

Conclusion: Newly synthesized proteins move directly into the microsome without being exposed to external env

Translation and Translocation Co-occurance Experiment

• Can protein transport to ER occur post-translationally? 

• Hypothesis: If proteins have completed translation, import won’t occur into ER 

Control: Figure b

• ER-targeted proteins are synthesized in vitro in presence of microsomes

• Mature proteins are found inside microsomes 

Experiment: Figure a

• ER proteins synthesized in vitro in absence of microsomes

• The translated proteins are added to a solution with microsomes

• They’re unable to enter after full translation

Conclusion: Import must occur co-translationally 

Co-Translational Protein Transport Rules

1. Signal sequence

2. Receptor for signal sequence

3. Translocation channels 

4. E requirement (GTP hydrolysis) 

5. Method of targeting proteins to different organelle locations 

Rule 1: ER protein Transport

• Domain of 16-30 amino acids needed

• Sequence located at N-terminus carrying 

  • A short (+) domain 

  • Hydrophobic domain 

  • Polar domain 

• Signal Sequence is produced first bc N-terminus 

  • First part of nascent protein translated 

• 2 roles: 

  • Targets protein to ER 

  • Guides ribosome translating the protein to ER membrane 

Rule 2: ER protein Transport

• Signal Recognition Particle (SRP) 

• Made of 6 proteins and functional RNA (300 nucleotides long) 

• It binds to 

  • N terminal ER sequence

  • Large subunit of Ribosome

• This pauses translation for a bit

• If the sequence was on the C-terminus, translation would be complete before SRP binding

  • No ribosome = no transport bc this is co-translational

• The SRP receptor has hydrophobic bindng groove that recognizes the hydrophobic domain of ER signal sequence

SRP Cytosolic Complex

• Requires:

  • SRP 

  • SRP receptor on ER membrane 

• SRP Receptor

  • transmembrane dimer with an alpha and beta subunit

  • SRP and alpha subunit of SRP receptor are GTP-binding 

• SRP receptor is associated with ER translocon 

Components Associated with ER Membrane Surface During Co-Translational Protein Transport 

• Ribosome

• mRNA

• N-terminus of nascent protein 

• SRP 

• SRP receptor

• closed translocon

Rule 3: ER protein Transport

• The translocon is initially closed

• Opens as the nascent peptide is transferred to interior 

• SRP dissociates from ER signal sequence during translation 

  • Translation then continues 

  • Ribosome remains associated with ER membrane

• As translation continues, the nascent peptide is pushed through translocon 

Rule 4: ER protein Transport

• SRP and SRP receptor have intrinsic GTPase activity 

• Both hydrolyze GTP for E 

• Powers the transfer of nascent peptide into translocon and opens it 

Ribosome-Translocon Complex: Co-Translational Protein Transport 

• The large ribosomal subunit directly interacts with translocon

• Minimal space exposes the nascent polypeptide emerging from ribosome to cytosol

  • So not susceptible to protease in external env

• This close association gives the rough ER speckled appearance 

What Happens when the N-terminus of the nascent protein reaches the lumen of RER

• ER signal sequence is cleaved by signal peptidase

• Peptide is pushed through translocon as translation continues 

• After translation, it’s released into the lumen where folding occurs 

• Translocon closes and ribosome dissociates 

• Depending on the mRNA the ribosome binds to next, it can be a free ribosome or bound 

Co-Translational Protein Transport Translocon Structure

• Comprised of many transmembrane a-helices forming the wall 

• Inner circle is the inner diameter of the translocon 

• When no ribosome present, the opening is narrow (15Å) 

• When ribosome is attached, the open conformation is adopted

  • Nascent protein goes through 

  • opening widens to 50Å in diameter

Rule 5: ER protein Transport

• The proteins here are soluble 

• They either

  • Remain in ER 

  • Transported via secretory vesciles to GA

  • Transported to lysosomes 

  • Transported out of the cell 

• Needs a second signal sequence 

Topogenic Sequence

• Embeds proteins in the ER membrane during co-translational transport

• 25 amino acid long sequence forming a hydrophobic a-helix with ability to embed in membrane

• Determines topology (# of times the protein crosses membrane and orientation of it)

• There are 4 classes of proteins based on their topology and sequences used 

  • Type I / II / III: Span the membrane once

  • Type IV: Span the membrane multiple times 

Type 1 Proteins

• Same N-terminal signal sequence in soluble ER proteins

• STA: Stop-Transfer-Anchor internal togogenic sequence 

  • Forms hydrophobic alpha helix embedding the protein in the ER membrane 

  • Stops translocation through translocon to transfer the protein to membrane 

  • Anchors protein in place 

• The protein maintains this topology even when moving to other locations via vesicle transport 

  • N-terminus faces exoplasmic space (inside ER or GA lumen)

  • C terminus always faces cytosolic space 

Transport and Insertion of Type I Protein

• N terminal sequence recognized by SRP and brought to SRP receptor 

• N-terminal sequence threaded into translocon 

• Protein synethesis pushes the protein through 

• When STA sequence is translated, it folds into an a-helix

  • Recognized by interior wall of translocon 

  • Stops translocon and causes it to open laterally 

  • Allows topogenic sequence to diffuse into surrounding membrane

  • Keeps protein anchored in place

• After translation/translocation is complete the C and N terminal domains fold within their envs

Sec61 Complex

• Part of traanslocon in co-transloation protein transport

• Red a-helix is the plug swinging down to open the pore during translocation 

• Blue helix illustrates the lateral opening of the translocon 

• This complex was determined by x-ray crystallography 

Type II / III Proteins

• Single-pass proteins 

• Lack N-terminal signal sequence 

• They have a single signal sequence 

  • SA Sequence: Signal Anchor 

  • Signal for both SRP and topogenic sequence 

Type II:

• Oriented with N-terminus on cytosolic side 

• C-terminus on luminal side 

Type III:

• Oriented same way as Type I

• N terminus on luminal side

• C terminus on cytosolic side 

• Have very short N-terminus (allows it to be threaded through)

Type II Membrane Protein Transport

• The SA sequence (red) is recognized by SRP to bring the protein and ribosome to ER membrane 

• SA sequence is then transferred to translocon 

• (+) residues prevent transfer of N-terminal portion of protein into translocon 

• Translocon opens laterally to allow diffusion of SA sequence into membrane 

• C-terminus pushed through translocon into ER lumen 

Type III Membrane Protein Transport

• SA sequence (red) is recognized by SRP to bring to ER membrane 

• A short N-terminal sequence threaded into translocon 

• SA sequence enters allowing translocon to open laterally

• (+) residues ensure the sequences neighbouring SA sequence stay in cytosol

• Newly synthesized protein is pushed away from ribosome-translocon complex while being translated

• The C-terminus stays on cytosol side and folds there

Type IV Integral Membrane Protein 

• Pass through multiple times 

• May be even/Odd # of topogenic sequences

• Every time it passes through, there must be a sequence 

Type IV Integral Membrane Protein: 2 Examples

• They differ at N-terminus 

• After N-terminus location decided, the rest is threaded through the membrane by alternating between STA sequence (Type I protein) and SA-II Sequence

Type IV-A

• N-terminus is on cytosolic side 

• SA-II Sequence keeps N-terminus on cytosolic side 

Type IV-B

• N-terminus is on luminal side 

• SA-III Sequence keeps N-terminus on luminal side 

Hydropathy Profile

• Graphic representation of all amino acids on polypeptide

• Left —> Right on X axis 

  • Amino acids from N to C-terminus

• y axis: hydrophobicity of each amino acid 

• Cluster of hydrophobic residues (peaks) represent topogenic sequences

Type I

• Hydrophobic peak at N-terminus

• Typical for an ER signal sequence 

• Middle is another strong hydrophobic peak

  • Represents STA sequence 

Type II

• No hydrophobic sequence at N-terminus

• Single hydrophobic peak representing SA sequence 

Differentiating between Type II / III

• Type II have longer N-terminus

• Amino acids before SA sequence is also an indicator

  • (+) on N-terminal side suggest type II 

  • (+) on C-terminal side suggests type III

Multipass Protein 

• Multiple hydrophobic peaks throughout

• Suggests multiple topogenic sequences

• Amount of peaks = amount of sequences = amount of passes through membrane 

Overview Diagram