29 - Protein Targeting and Modification

Question 1

Proteins must be ____ to cross the membrane

Answer 1

linear

Chaperone proteins are acquired to assist the protein in attaining its final and correct conformation

Similar chaperone proteins exist within organelles

Question 2

Targetting sequences are usually N terminal and removed, what are the two exceptions to this?

Answer 2

Peroxisome signalling sequences are usually C terminal and the signal is not removed.

Nucleus signalling sequence is usually internal and not removed as well.

Question 3

Give the steps of signal peptide targeting the secretory pathway

Answer 3

1. Polypeptide synthesis begins
2. Polypeptide synthesis is inhibited by binding of SRP
3. SRP docks to the SRP receptor to form the ribosome-translocon complex
4. SRP and SRP receptor dissociate and polypeptide synthesis resumes
5. signal sequence is excised
6. Protien extrudes, folds, and is anchored to membrane
7. Ribosome dissociates

Question 4

Give three properties of signal peptides

Answer 4

• Amino terminal has 2-40 amino acids that are positively charged
• The central part of the peptide has 7-20 hydrophobic amino acids
• The C-terminal has a beta strand/turn

Signal sequences are transposable (experimental evidence)

Question 5

Give four post-translational modifications of proteins

Answer 5

• Signal peptide cleavage (co-translational)
• Disulfide bond formation (shuffling)
• Fatty acylation (S-linked, N-linked, O-linked)
• Glycosylation (N-linked, O-linked)

Question 6

Describe N-linked glycosylation of proteins

Answer 6

Carbohydrates covalently linked to asparagine residues in polypeptide sequence.

Glycosylation mechanism recognizes Asn-X-Ser/Thr where X can be any residue except proline.

Addition of carbohydrate is initiated in the ER, but the final structure is attained by modification of the sugar as it is tansported through the golgi apparatus.

Question 7

How is N-linked glycosylation initiated?

Answer 7

The first step in the synthesis of the glycoprotein is the
addition of a preformed oligosaccharide chain to the polypeptide as the polypeptide is being synthesized (co-translational)

The oligosaccharide chain is assembled on an isoprenoid containing lipid called dolichol phosphate

This metabolite is anchored in the ER membrane via its long hydrophobic isoprenoid tail

Question 8

How is the dolichol linked oligosaccharid in N-linked glycosylation assembled?

Answer 8

Involves a series of glycosyltransferase reactions resulting in Glc3Man9GlcNAc2-dolichol phosphate

Amino sugar residues are added as UDP (for GlcNAc, glucose) or GDP (for mannose) activated forms

Cytosolic synthesis of dolichol-mannose, dolichol-glucose and translocation for addition of 4 mannose, 3 glucose residues to make Man9Glc3 form

Question 9

What happens after the dolichol linked oligosaccharide of an N-inked glycosylation is assembled?

What properties must a acceptor sequence have to by glycosylated?

Answer 9

The oligosaccharide chain is added en bloc to the polypeptide with the release of dolichol pyrophosphate

The reaction is catalysed by oligosaccharyltransferase

Not all Asn-X-Ser/Thr sequences are acceptors of the oligosaccharide, the sequence must also be accessible to the enzyme in a turn or bend in the protein structure

Question 10

What happens as a glycosylated protein is transported through the ER and the golgi apparatus?

Answer 10

The carbohydrate moiety is modified by a series of enzymatic steps

Terminal glucose residues are removed in the ER

Mannose trimming and addition of other sugar monomers occurs in the golgi apparatus.

Question 11

How does glycosylation affect SDS-PAGE analysis?

Answer 11

Causes slower mobility.

We use glycosidases (bacterial enzymes that hydrolyze sugar linkages) to get rid of this. Eg.

• N-glycosidase (hydrolyzes GlcNAc bond to Asn)
• Endoglycosidase H (hydrolyzes Man9GlcNAc bond to Asn)
• Neuraminidase (removes terminal sialic acid residues)

Question 12

In what order are carbohydrates in on glycosylated protein oligosaccharides?

Answer 12

Asn

• N-acetyglucosamine + L-Fucose
• N-acetylglucosamine
• Mannose
• Mannose x2
• N-acetylglucosamine x3
• Galactose x3
• Sialic acid x3

If it gets to the sialic acid

Question 13

What is N-linked glycosylation?

Answer 13

N-linked glycosylation, is the attachment of the sugar molecule oligosaccharide known as glycan to a nitrogen atom (amide nitrogen of asparagine (Asn) residue of a protein), in a process called N-glycosylation

Provides structural components to the cell wall and extracellular matrix.

Modify protein properties such as stability and solubility.[7] (More stable to high temperature, pH, etc.)