WEEK 5
LECTURE 1: PROTEIN PROCESSING AND TRAFFICKING IN THE RER
ROUGH ER + SMOOTH ER FUNCTIONS
• RER
o Many ribosomes
o Co-translational transport
o Protein modification
o Formation of vesicles to transport proteins from ER to Golgi
• SER
o No ribosomes
o Fatty acid + phospholipid synthesis
o Carb metabolism
o Calcium sequestering to regulate Ca+ concentration in cytosol
POST TRANSLATIONAL MODIFICATIONS (PTMS) IN THE ER
1. Glycosylation
2. protein folding
3. disulphide bond formation
4. proteolytic cleavage
• modifications occur in luminal part of membrane, not on transmembrane domain or
cytosolic part
1. PROTEIN GLYCOSYLATION: N-LINKED
• covalent addition of polysaccharides
• on proteins. Secreted from cell + embedded in
cell membrane
• needed for proteins that mediate cell interactions with extracellular matrix +
receptor-ligand recognition
• N-linked glycosylation:
o Add sugar to NH2 of R-group of asparagine
• Further modifications to the sugar can occur in ER and Golgi
• Required for proper transport of proteins, folding + stability, function (influences
substrates)
, 2. PROTEIN FOLDING IN THE ER
• Recognize amino acids carrying N-linked sugars
• Lectins recognize modified proteins + help fold (similar to chaperones)
o Calnexin + calreticulin
§ Calnexin in ER membrane
BIP
• ER resident HSP70 chaperone
• Function depends on ability to recognize + bind to
unfolded proteins
• Co-chaperones Hsp40 + nucleotide exchange factor
• Transfer proteins from ER through translocon by
binding to proteins as soon as they appear on luminal
side of membrane during co-translational transport
• Initiates unfolded protein response in ER
3. DISULPHIDE BOND FORMATION IN ER
• Disulphide bond: covalent linkage between sulfhydryl groups of 2 cysteine residues
• In proteins secreted from cell + on outer leaflet of membrane
o Helps maintain folds of protein
• Can occur within a single protein (intramolecular) or 2 different proteins
(intermolecular)
• In eukaryotic cells: oxidative rxn to form disulphide bond only in ER
• Lumen of ER: oxidizing environment (favours reverse rxn)
• Cytoplasm: reducing environment
DISULPHIDE BONDS IN RNASE A
• Pancreatic ribonuclease A contains 4 disulphide bridges
• Secreted into intestine
• Digestion of RNA by cleaving it into small pieces
• Acidic condition of small intestine normal cause proteins to denature
• Prevented by disulphide bonds
PROTEINS DISULPHIDE ISOMERASE (PDI)
• Resident ER protein that promotes oxidation
• Forms intermediate with 2 cysteine residues to
accelerate rate of rxn
• Oxidized PDI contains a disulphide bridge
• PDI intermediate with 1 cysteine residue in a
protein to form intramolecular cysteine bond
• PDI spontaneously converted back to oxidized
form
• Can correct inappropriate disulphide bridges
LECTURE 1: PROTEIN PROCESSING AND TRAFFICKING IN THE RER
ROUGH ER + SMOOTH ER FUNCTIONS
• RER
o Many ribosomes
o Co-translational transport
o Protein modification
o Formation of vesicles to transport proteins from ER to Golgi
• SER
o No ribosomes
o Fatty acid + phospholipid synthesis
o Carb metabolism
o Calcium sequestering to regulate Ca+ concentration in cytosol
POST TRANSLATIONAL MODIFICATIONS (PTMS) IN THE ER
1. Glycosylation
2. protein folding
3. disulphide bond formation
4. proteolytic cleavage
• modifications occur in luminal part of membrane, not on transmembrane domain or
cytosolic part
1. PROTEIN GLYCOSYLATION: N-LINKED
• covalent addition of polysaccharides
• on proteins. Secreted from cell + embedded in
cell membrane
• needed for proteins that mediate cell interactions with extracellular matrix +
receptor-ligand recognition
• N-linked glycosylation:
o Add sugar to NH2 of R-group of asparagine
• Further modifications to the sugar can occur in ER and Golgi
• Required for proper transport of proteins, folding + stability, function (influences
substrates)
, 2. PROTEIN FOLDING IN THE ER
• Recognize amino acids carrying N-linked sugars
• Lectins recognize modified proteins + help fold (similar to chaperones)
o Calnexin + calreticulin
§ Calnexin in ER membrane
BIP
• ER resident HSP70 chaperone
• Function depends on ability to recognize + bind to
unfolded proteins
• Co-chaperones Hsp40 + nucleotide exchange factor
• Transfer proteins from ER through translocon by
binding to proteins as soon as they appear on luminal
side of membrane during co-translational transport
• Initiates unfolded protein response in ER
3. DISULPHIDE BOND FORMATION IN ER
• Disulphide bond: covalent linkage between sulfhydryl groups of 2 cysteine residues
• In proteins secreted from cell + on outer leaflet of membrane
o Helps maintain folds of protein
• Can occur within a single protein (intramolecular) or 2 different proteins
(intermolecular)
• In eukaryotic cells: oxidative rxn to form disulphide bond only in ER
• Lumen of ER: oxidizing environment (favours reverse rxn)
• Cytoplasm: reducing environment
DISULPHIDE BONDS IN RNASE A
• Pancreatic ribonuclease A contains 4 disulphide bridges
• Secreted into intestine
• Digestion of RNA by cleaving it into small pieces
• Acidic condition of small intestine normal cause proteins to denature
• Prevented by disulphide bonds
PROTEINS DISULPHIDE ISOMERASE (PDI)
• Resident ER protein that promotes oxidation
• Forms intermediate with 2 cysteine residues to
accelerate rate of rxn
• Oxidized PDI contains a disulphide bridge
• PDI intermediate with 1 cysteine residue in a
protein to form intramolecular cysteine bond
• PDI spontaneously converted back to oxidized
form
• Can correct inappropriate disulphide bridges
