Microbiology 12: (Prigent) Modification in the secretory pathway

Question 1

Functions of the endoplasmic reticulum?

Answer 1

Insertion of proteins into membranes
Formation of S-S bonds
Proper folding of proteins
Glycosylation
Specific proteolytic cleavage
Assembly of multi subunit proteins

Question 2

Describe disulphide bond formation in the ER - protein involved?

Answer 2

Catalysed by protein disulphide isomerase (PDI)
- an ER resident protein

Contains its own disulphide bond in catalytic site

Electrons transferred from thiol groups (-SH) to the PDI S-S

-> PDI becomes reduced, S-S bonds formed in protein (oxidised)

Can also correct incorrectly formed disulphide bonds

Question 3

Enzymes responsible for acceleration of interconvertion of cis and trans isomers? Mechanism?

Answer 3

Peptidyl-prolyl isomerases
- catalyses isomerisation of prolyl groups

important (in industry) as this isomerisation is often rate limiting step in protein folding

Question 4

Describe N-linked glycosylation - where does it occur?

Answer 4

Sugars are added onto an asparagine side chain ( a reaction involving an amino group hence ‘N’ linked)

Occurs in ER

Question 5

Describe assembly of precursor oligosaccharide and addition to protein forming glycoprotein

Answer 5

Oligosaccharide is assembled onto dolichol phosphate, a lipid carrier molecule in the membrane

• sugars added 1 step at a time in form of nucleotide sugars
• N-acetylglucosamine delivered in UDP form
• UDP -> UMP as phosphate is added to dolichol as well as attachment of acetylglucosamine
• Delivery of GDP mannose sugars
• membrane ‘flipping’
• further addition of mannose and glucose
• oligosaccharide attached to asparagine residue on protein
• finish with trimming of excess sugars

left with 2 N-acetylglucosamine, 8 mannose

Question 6

Main chaperone proteins in protein folding process in ER? Function?

Answer 6

Chaperone proteins - assist protein translocation, mediate the unfolded protein response, retain unfolded proteins in the ER

Binding immunoglobulin protein (BiP) - binds to exposed amino acid sequences that would normally be buried in the interior of a folded protein - binds to proteins to prevent them leaving ER before complete assembly

Calnexin (membrane) and Calreticulin (soluble) - bind to incompletely folded oligosaccharides on incompletely folded proteins

Question 7

What happens if their are folding problems in the ER?

Answer 7

Calnexin and BiP bind irreversibly to misfolded proteins

• > triggers unfolded protein response pathway
• > Increases transcription of chaperones and folding catalysts

Question 8

Examples of proteins in unfolded protein response?

Answer 8

Ire1 membrane protein -> BiP bound to it to keep it in inactive state

When BiP dissociates, Ire1 proteins dimerise (similar to TRK) -> enable endonuclease activity

results in increased transcription of chaperone proteins

ATF-6, also kept inactive by BiP

• > BiP released with high unfolding, results in cleavage and release of suluble ATF-6
• > acts as TF for chaperones etc.

Question 9

Describe function and mechanism of ubiquitin?

Answer 9

Ubiquitin binds to lysine chains on misfolded proteins
-> polyubiquitination

Targeted for degradation via proteosomes

Question 10

Describe golgi network addition of phosphate to mannose sugars - result?

Answer 10

Provide the signal by which lysosomal enzymes are delivered to the lysosome -> ‘labelled’ in golgi network

Newly synthesised lysosomal enzymes contain signal patch -> recognised by NAG-phosphotransferase

-> addition of phosphate to mannose sugar to form mannose-6-phosphate, signals enzyme is destined for lysosome

Question 11

Product of ER -> Product of golgi?

Answer 11

ER -> 2 N-acetylglucosamine, 8 Mannose

Golgi -> high-mannose oligosaccharide (5 mannose)

• or -
• > complex oligosaccharide (addition of many sugars)

Question 12

What is the purpose of glycosylation?

Answer 12

Makes protein intermediates more soluble + prevents aggregation

Modifications to no. of N-linked sugars in each step mark progression of folding + mediate chaperone binding

Sugars with limited flexibility limit approach of other proteins like proteases -> reduce proteolysis

Extensive glycosylation e.g. in mucus coat protects against infection

Recognition of sugars in extracellular space by lectins important in developmental processes + cell-cell recognition

Question 13

Describe post-golgi processing via proteolytic cleavage - example?

Answer 13

Endoproteases like Furin endoproteases recognise aa’s e.g. arginines and cleave within the protein

Proinsulin - insulin processed this way