Lecture 11: Co-translational targeting to the ER

Question 1

How does protein synthesis in eukaryotic cells occur, and what is the role of the signal peptide in protein secretion?

Answer 1

• Protein synthesis in eukaryotic cells apart from mitochondria n plastids is a cytosolic affair
  
  • Starts in cytosol on 3 then free cytosolic ribosomes cluster to form polysomes
  • First part of the protein that is extruded is the N-terminus
  • If the protein is destined for secretion: signal peptide
    ○ Targeting device that allows engagement w a translecon in the ER membrane
    ○ Protein extruded thru the membrane n processed
  • ER targeting requires proteins to be made initially in the cytosol

Question 2

How does protein secretion occur?

Answer 2

• Proteins leave the ER in secretory vesicles
  
  • Fuse w golgi -> fuse w outside of the cell
  • Allows protein to leave the cell
  • Alternative destinations for secretory proteins e.g. endesomal system, transgolgi network, lysosome system
    Entry point for secretion is ER

Question 3

“What is the function of the signal peptide in protein synthesis and ER targeting in eukaryotic cells?”

Answer 3

• Signal peptide is part of the primary sequence of the protein
  
  • AUG (start codon) that encodes N terminus of the protein
  • Signal peptide that will fuse into the rest of the ORF
  • Primary product following translation is a protein w an end terminus signal peptide n the rest of the protein
  • When joined the process of ER entry, signal peptide removed by proteolytic cleavage

Question 4

What do ER signal peptides look like?

Answer 4

• Huge range of variation
  
  • 15-30 AA in size
  • No primary sequence conservation
  • So, how can they all be recognized if they all look different?
    ○ Broad similarities
    § Positive charge towards N terminus (Arg, Lys)
    § Downstream there is a hydrophobic stretch that is recognized by signal recognition particle (SRP)
    § Cleavage site usually follows small AA
  • Broad substrate specificity

Question 5

How do signal peptides exhibit variability even among highly related proteins? Provide an example

Answer 5

EXAMPLE:
* Erp2p n Erp4p (proteins required for ER to Golgi transport in the yeast) are derived from a gene duplication event n are 93% similar
* BUT signal peptides are v different in length n AA sequence
* SPs are free to evolve rapidly as long as they retain overall features
○ Positive charge towards N terminus n hydrophobic stretch

Question 6

How does a SP ensure that the translating ribosome binds to the ER membrane?

Answer 6

• SP is recognized by signal recognition particle (SRP) in the cytosol
  
  • This targets SP to the translocon

Question 7

Describe the SRP cycle

Answer 7

• Ribosome starts to make proteins
  
  • SP extruded first
  • SP binds to the SRP
    ○ Bent back into a hook
  • SP complex docks SRP receptor, which is dimeric (alpha n beta subunit)
  • Beta subunit associated w a closed translocon
    ○ Translocon (protein conducting channel)
  • Translation is interrupted
  • Translocon opens -> hook shape inserted into open translocon
  • SRP complex disengages -> SRP recycled for later use
  • Cleavage site presented into ER
  • Available for Spase (signal peptidase), which cleaves the SP from the protein n SP is ejected laterally from the translecon
  • Rest of the protein can be extruded so translation restarts n protein is passed onto ER lumen

Question 8

What does the specificity of the ER targeting depend on?

Answer 8

• Broad substrate specificity in the recognition step
  
  • Folding of the SP into a hook shape by SRP
    ○ Allows SPase to cleave signal within the translocon -> releasing SP into the ER membrane
  • RESULT: problems downstream bc SPs disrupts the ER membrane, requiring their removal by chopping w SPPase altho Saccharomyces lacks SPP

Question 9

What happens in the ER lumen?

Answer 9

• Major site of protein folding
  ○ This requires ER chaperones
  
  • 2 major ER modifications
    ○ Folding proteins may become N-glycosylated (covalent attachment of 3s of sugars onto asparagine residues or target proteins)
    ○ Disulfide-bonded depending on whether they hv the right AA sequence motifs for these events
    § Disulfide bonds do not form in cytosolic proteins
    § Disulfide bonds can only form b/w 2 adjacent cystines in oxidizing cellular environments (eg. ER lumen, periplasm in gram negative bacteria)
  • IF Protein doesn’t fold properly -> will fail quality control check carried out by ER chaperones
  • Misfolded proteins are not usually allowed to proceed thru the secretory pathway
  • Misfolded proteins are ejected from the ER in a process called retro-translocation (dislocation) then degraded in cytosol

Question 10

What is the N-glycosylation of proteins?

Answer 10

• Covalent addition of an oligosaccharide tree of sugars (core N-glycan) from a lipid carrier to the target protein as the target protein is being extruded into the ER lumen
  
  • Oligosaccharyltransferase (OST): protein that does this
    ○ Allows contact w the ribosome n translecon
  • As the protein is being extruded into the ER, there is a glycan tucked away into the OST
    ○ Transferred as a 3 n covalently attached to specific sites in the native protein
  • As the protein moves thru the secretory pathway, the core N-glycans are modified to make complex N-glycans

Question 11

What is the core N-glycan made up of?

Answer 11

• 2 N-acetyl glucosamine
  
  • 9 mannose residues
    3 glucose residues
• N: Asp, asparaginyl (residue of asparagine)
  
  • X: any amino acid apart from proline
  • Residue followed by hydroxyl group
    ○ S/T: seryl/thereonyl (residue of either a serine or threonine)
  • If the NXS/T is seen by OST, It recognizes N as a place to attach its tree of sugars

Question 12

What are the functions of the N-glycosylation?

Answer 12

• N-glycans act as flags for folding n ER quality control
  ○ Removal of 2 Glc residues from the core N-oligosaccharide in the ER -> allows interactions w ER chaperones required for efficient folding of N-glycosylated proteins
  ○ As proteins proceed thru folding steps, final Glc residue n Man residue are removed
  ○ Removing protein from folding environment n signaling that protein is now ready to be passed to Golgi
  ○ Further sugar additions to N-glycans occur in Golgi -> complex N-glycans -> allows cell to track progress of protein thru secretory pathway
  
  • Increases protein solubility [N-glycan is larger than asparagine residue to which it is attached n made of hydrophilic sugars] -> reduce aggregation problems during folding in the ER
  • Influence folding rates n final protein conformation
    ○ N-glycans are bulky -> constrain α-carbon backbone of the polypeptide
  • N-glycans may influence the activity of a protein (e.g. enzymes) or its interaction with other molecules (e.g. the interaction of an antibody with effector molecules) [final conformation of the protein may be constrained]

Question 13

What conditions are required for disulfide bonds to form?

Answer 13

• Oxidizing conditions (e.g. ER in eukaryotes or bacterial periplasm)
  
  • Form where 2 cystine residues are brought cloes together during protein folding
  • Biological catalysts: protein disulfide isomerase (PDI)
    ○ PDIs can make, break n shuffle disulfide bonds
  • Covalent S-S bonds contribute to the stability of protein tertiary structures of SECRETED proteins

Question 14

What are the 3 roles of PDI?

Answer 14

• Isomerize disulphide bonds
  
  • Make/break disulfide bonds
  • Join / separate 2 proteins

Question 15

How does PDI isomerize (shuffle disulfide bonds)?

Answer 15

• PDI has 2 self hydrol groups when the PDI is in reduced conditions
  
  • Reduced PDI can bind to client proteins that has inappropriate disulfides -> binds as a chaperone (covalently bound complex -> forms mixed disulfide bonds
  • Isomerization of disulfide bonds continues until the client protein reaches stable conformation when it is released from PDI

Question 16

How does PDI make/break disulfide bonds?

Answer 16

• Oxidized PDI-reduced client complex w mixed disulfide
  
  • PDI changes the shape of the disulfide
  • RESULT: oxidized client, reduced PDI
  • This process can be done in reverse to break disulfide bonds

Question 17

How do we know how PDI can join and separate 2 proteins?

Answer 17

• PDI can take 2 separate proteins n join them together
  
  • If reduced PDI is added to ricin, it reduces the ricin to reduced RTA n RTB
  • Only if the PDI is in reduced conditions
  • Reversible process
    ○ Reduced RTA n RTB won’t interact w/o oxidized PDI

Question 18

Describe the process of peptide loading onto MHC Class I molecules in the endoplasmic reticulum

Answer 18

• Heavy chain inserted into membrane n has binding site for peptides in ER lumen
  
  • Held there in inactive but soluble conditions bc bound by BiP
  • BiP is displaced by β2 microglobulin
  • N-glycosylation allows entry into folding environment
  • Interacts w calreticulin makes contact w ERp57 (PDI family)
  • Erp57 is bound by disulfide to tapasin
  • Cage of chaperones form around the complex -> provides folding environment
  • Folding environment leads to formation of groove to which peptide can be inserted
  • Peptide can be displayed in the MHC Class I molecule, where it can act as a flag for other immune cells
  • Typically derived from the cytosol
  • Fed in from the protein channel (TAP transporter), which is recruited by tapasin
    The peptides are derived from proteasomal degradation

Question 19

What happens to an ER protein that fails quality control? Eg. a MHC Class I HC that fails to find ß2

Answer 19

• Much longer residence time w BiP than expected
  
  • Hsc70 molecule can direct proteins down a productive/destroying pathway
  • Long residence time flag that targets for destruction
  • No partner -> can’t target calreticulin
  • Mannosidase removes 1 particular mannose -> removes this protein from the folding environment
  • Protein is unfolded n fed thru multi-subunit dislocon
  • The protein is ubiquitylated during dislocation, which targets it to the proteasome, where it is deubiquitylated
  • ERAD (ER-associated protein degradation)
    ○ Misfolded ER proteins are destroyed in the cytosol

Question 20

Name 3 other examples of secretory system modifications

Answer 20

• Attachment of sugars to oxygen atoms of amino acids (O-glycosylation)
  
  • Proteolytic cleavage
    ○ To active protein such as albumin
    § Albumin is activated in the secretory pathway by furin (endosomal/lysosomal enzyme)
  • Proteolytic activation occurs for
    ○ Active digestive enzyme (e.g. trypsin)
    ○ Hormones (e.g. glucagon, insulin)
    ○ Neurotransmitters (e.g. enkaphalin)
    ○ Proteins that would otherwise aggregate (e.g. mature collagen)
  • Addition of lipids to permit/maintain membrane targeting
    ○ GPCRs are polytopic membrane proteins that hv a myristic acid n lipid anchor
    ○ GPI-linked proteins hv glycophosphatidylinositol membrane anchor linked via sugars to the protein

Question 21

Explain how Alzheimer’s occurs in regards to proteolysis and aggregation

Answer 21

• Amyloid precursor protein (APP) expressed in the brain
  
  • Cleaved by 2 cleavage events: CUT 1 (BACE) n CUT 2 (α-secretase)
    ○ Which one gets there first depends on whether you get toxic aggregate buildup
  • β secretase cuts APP (top part) then ϒ secretase wll cleave the part that is bound to the membrane
  • RESULT: Aβ fragment that aggregates to form precursor amyloid plaques in the brain
  • α secretase cuts first then it cleaves within Aβ -> prevents release of toxic fragment
  • Possible solution is to design BACE inhibitors