Lecture 8 Flashcards
All proteins of the secretory pathway are synthesized in?
The ER
Why are all proteins synthesized in the cytosol inserted into the ER?
The ribosomes sit on the ER and translate the proteins and insert them into the ER which transports them into further compartments. THe inner/outer membranes of the compartments are continuous with the ER membrane
Rough ER vs Smooth ER?
ROugh : Many attached ribosomes, secretory protein synthesis
Smooth : No ribosomes, lipid synthesis
Targeting signals?
Sequence of amino acids within a protein that specify its organelle localization
-Often independnt sequences from the functional/structural sequences
-Often cleaved
-Have a pattern not typically a specific sequence(ex. 8 polar AA then 2 non polar)
Targeting steps?
- Recognize the targeting signal
- Bring the peptide to connect the protein to the ER membrane
3.Translocate the protein into the ER membrane/lumen
Why are proteins larger than their mature form?
Target signals get cleaved off
What happens if we mix the cytosol and ribosomes but no organelles?
Protein will be translated but not inserted into the ER
What happens if we mix the cytosol, ribosomes and ER?
Protein will be translated and inserted into the ER and the signal is cut off (smaller mature protein)
Where do polypeptide chains exit the ribosome?
Through a tunnel at the 60S subunit
How does the ribosome insert chain into the ER?
Signal sequence will interact with a translopore in the ER membrane and the ribosome will also interact with the translopore
What does it mean when we say secretory signal peptides direct proteins to the ER co translationally?
As the protein is being synthesized by the ribosome the nascent chain is also being pushed into the ER
How do proteins get from ER to other organelles?
-Can have motifs
-Can have PTMs
Organelles not in the secretory pathway have their own targeting signals
Signal Peptide characteristics?
Often found at the N-terminus, 8-16 hydrophobic amino acids
Signal Anchors?
Signal peptides that also become TM helices(beta strands/alpha-helices)
- Not cleaved
- Can be anywhere in the protein
-Larger than signal peptides
Signal peptide targeting steps?
- Ribosome translates the new polypeptide with a signal.
- Signal is recognized by Signal Recognition Particle(SRP), which binds the signal and ribosome
- SRP is in the ER membrane and links the ribosome to the translocon pore in the ER
- Energy of translation on the ribosome helps drive the polypeptide through the translocon pore
Free Ribosome Cycle?
Ribosomes are not attached to the ER and translate proteins into the cytosol
Membrane Bound Ribosome Cycle?
Ribosome bound to the ER membrane by SRP. Then SRP receptor in the ER membrane binds the SRP and brings the ribosome to the membrane. GTP hydrolysis occurs to keep the SRP bound to the SRP-R
What kind of molecule is SRP?
Ribonucleoprotein , made of 6 protein subunits + RNA
Domains of SRP and what they do?
Signal sequence binding pocket: recognizes the target signal in the nascent polypeptide
Translation pause domain: Interacts with the ribosome to stop translation in the cytosol
Can also GTP and stimulate GTPase activity
How does SRP know to bind a ribosome?
- SRP samples all nascent polypeptides being translated
2.When a signal peptide is recognized, SRP attaches tightly to both the signal and the ribosome - SRP pauses translation and binds GTP
SRP-ribosome to the translocon?
- The ribosome-SRP complex binds to the SRP-R on the ER
- GTP hydrolysis occurs and the SRP and SRP-R will be recycled
- We are left with the ribosome attached to the translocon together with the nascent peptide
- Translation will now push the nascent peptide into the ER lumen
How are SRP-R and SRP recycled ?
- SRP bound to the ribosome in GTP bound state
- SRP-R is a GTPase
- When SRP binds to SRP-R hydrolysis occurs which dissociates the two molecules and recycles them
Another name for the ER translocon?
Sec 61 complex
Inactive vs Active ER translocon?
Inactive: pore is plugged
Active : por eis opened and tightly sealed to the ribosome
Er translocon pore?
-neutral
-polar
How is a protein with a TM helix and signal peptide transported across the translocon?
- Synthesize the hydrophobic region of the helix
- When the translocon senses this type of sequence (18-24 hydrophobic amino acids) it stops transport of the protein into the lumen
- The rest of the translation occurs in the cytosol
- Then the translocon will open and the hydrophobic region of the protein will be inserted into the membrane
- N-terminus in lumen and C-terminus in the cytosol
Type 1 TM Proteins ?
-N-terminus in lumen and C-terminus in the cytosol
How do we get N-terminus in the cytosol and C-terminus in the lumen?
N-terminus will stay in the cytosol and the C-terminus will be pushed into the lumen
Type 2 TM protein
Positive vs Negative charges in terms of the cytosol + lumen?
Positive: go into the cytosol
Negative : go into the lumen
Multi pass TM proteins?
Combinations of signalling anchors and TM helices causes alternating orientation
Hydrophobicity?
Indicates the number of TM helices
Charge distribution?
Indicates the orientation of the protein in the membrane
N-linked Glycosylation
All proteins of the secretory pathway recieve the same PTM as they enter the ER
What amino acid recieves the N-linked Glycosylation and what is the sequence?
Asparagine
-Asn-x-ser/thr
x= any aa
What is the glycan added to all secretory proteins?
-Two N-acetylglucosamine
- Mannose
- 3 glucose
Purpose of the N-linked Glycosylation?
-Stabalizes native state
-Protects against proteases
What enzyme adds the N-linked glycosylation?
Oligosaccarhyl Transferase (OST)
Steps of Glycosylation?
- Oligosaccharides are synthesized attached to a specialized lipid in the ER
- OST(oligosaccharide transferase) attaches the glycan during translocation to the protein
- State of glycan is used as a signal in nER quality control of folding
4.Glycan is modified in the Golgi after exit from ER
Where are secretory proteins folded?
The ER
Misfolded Secretory Proteins ?
Degraded at the ER by the ubiquitin protease system or by lysosomes
Protein Quality control checkpoint for all organelles?
ER
