Fate of Newly Synthesised Proteins: The ER and Golgi

Question 1

Describe the structure of the ER

Answer 1

A network of membrane tubules continuous with the nuclear envelope.

Question 2

What are the 6 functions of the ER?

Answer 2

• Protein Synthesis
• Glycosylation: Attachment of sugars to protein (proteins in lysosome and membrane are heavily glycosylated).
• Folding and assembly of multi-protein complexes
• Lipid Synthesis (cholesterol and phospholipids):
• Ca2+ sequestration: intracellular calcium ion storage
• Detoxification by cytochrome P450 enzymes: Destroy or modify toxic molecules

Question 3

How are proteins transported into the ER lumen?

Answer 3

1) The N terminus of the polypeptide translated is the first part to exit the ribosome. It contains a signal sequence (SS) if the protein is destined for the ER (a sequence of at least 8 hydrophobic amino acids).
2) The SS causes translation to stall and interacts with a Signal Recognition Particle (SRP), a large protein complex that guides the RNA-ribosome-protein complex to the surface of the ER.
3) GTP allows binding of SRP and SRP receptor, whilst the (above) complex interacts with the Peptide Translocation Complex (PTC) (aka ribosome receptor).
4) When the ribosome attaches to the PTC, the SRP is removed, by hydrolysis of GTP, and translations resumes on the ER membrane.
5) THE PTC forms a channel through which the polypeptide is threaded through into the ER lumen.
6) The signal peptide remains (attached to the membrane?), to be cleaved off by a signal peptidase.
7) When protein synthesis is complete, The protein is released into the ER and the ribosome comes off the ER membrane and disscociates.

Question 4

What type of cell is insulin formed in?

Answer 4

Pancreatic beta cells

Question 5

How does mature insulin form?

Answer 5

Mature Insulin is formed from its large precursor Preproinsulin by proteolytic processing.
Removal of a 24 amino acid segment (signal sequence) at the amino acid terminus of Preproinsulin and the formation of 3 disulphide bonds produces Proinsulin.
Further proteolytic cuts remove the C peptide from Proinsulin to produce Mature Insulin, composed of A and B chains.

Question 6

What are the protein modifications that occur in the ER?

Answer 6

1) Proteolysis (signal peptide): When the signal peptide reaches the lumen of the ER it is cleaved off
2) Disulphide bond formation
3) Glycosylation (N-glycosylation?)
4) Deglycosylation
5) Protein folding and assembly into complex (tertiary and quaternary)

Question 7

What happens if proteins fail to do any of the modification steps properly?

Answer 7

They will not be incorporated into transport vesicles and are instead transported back to the TLC and degraded.
(An example of a failed modification step is a signal peptide not being properly cleaved)

Question 8

How do vesicles form?

Answer 8

Part of the ER membrane buds off, surrounded by proteins that form the COPII coat. When the vesicle forms, the coat proteins come off and dissolve in the cytoplasm to be used to form a new vesicle later.

Question 9

Describe the structure of the Golgi Apparatus

Answer 9

• The cis face is where the new vesicles fuse whilst the trans face is where they leave.
• The middle stacks are known as the medial cisternae
• The cisternae are often not connected
• There is usually more than one Golgi stack in the cell which form a ribbon organisation.

Question 10

What are the roles of the Golgi?

Answer 10

• Protein Modification: Glycosidases (take off sugar units) , glycosyltransferases, O-linked glycosylation (sugars attach to serine residues, O stands for oxygen in hydroxyl group of serine), Sulfatases, Proteases
• Lipid synthesis: Sphingomyelin (type of lipid found in animal cell memebrane), glucosylceramide (precursor of glycosphingo lipids).
• Protein and Lipid sorting: Vesicles transport to secretory granules (store enzymes or secreted hormones temporarily e.g. insulin), plasma membrane proteins (e.g. receptors),the basolateral (outer surface) vs apical (inner lumen side) membrane in epithelial cells, endosomes and lysosomes.

Question 11

Describe how the COPII coat is made for ER transport.

Answer 11

Membrane-bound, active Sar1-GTP recruits COPII adaptor proteins to the membrane. They select certain transmembrane proteins and cause the membrane to deform. The adaptor proteins then recruit the outer coat proteins which help form a bud. A subsequent membrane fusion event pinches off the coated vesicle (other coated vesicles are thought to form in a similar way).