S1: Fate of Newly Synthesised Proteins

Question 1

What are free ribosomes?

Answer 1

• Soluble proteins for release into the cytoplasm are synthesised by free ribosomes
• Intitial codons in mRNA do not code for hydrophobic amino acids

Question 2

What are bound ribosomes?

Answer 2

• Proteins for secretion from the cell or for incorporation into membrane or lysosomes are synthesised by membrane bound ribosomes
• Initial codons in mRNA code for a short sequence of hydrophobic amino acids called a signal sequence

Question 3

What is a signal sequence?

Answer 3

The initial codons (so at the N-terminus, the first part of the protein to be synthesized) in the mRNA code for a short sequence of hydrophobic amino acids, called a “signal sequence”.

Question 4

What determines whether the ribosome that translates the polypeptide will be free or bound?

and How?

Answer 4

Signal sequence

The signal sequence allows a channel in the ER the open up and the signal remains bound to channel as rest of polypeptide enters into the ER lumen. When it has fully entered, the signal sequence is cleaved off and released.

Question 5

What are internal hydrophobic sequences?

Answer 5

They are additional sequences of codons in the mRNA that code for hydrophobic amino acids.

These sequences of AA’s get ‘stuck’ in the membrane as the protein is fed through (the channel into the lumen) so they become membrane proteins.

Question 6

What is the ‘nuclear localisation sequence’?

Answer 6

These are patterns of codons that code for basic amino acids

They will appear on proteins that are destined for the nucleus

Question 7

What are ‘mitochondrial target signals’?

Answer 7

They are alternate patterns of hydrophobic and basic amino acids. This is because while mitochondria do have their own ribosomes and make some of their own proteins, the majority of mitochondrial proteins are synthesised in the cytosol and migrate to the mitochondria.

Question 8

Organelles involved in protein synthesis

Answer 8

The mRNA leaves the nucleus and is translated on a ribosome on the rough ER (for proteins that will be secreted or part of membrane), it then will move to the Golgi apparatus where it’ll be packaged into vesicles, these will then fuse with the plasma membrane and empty the contents into the ECF, this is secretion or it will add new membrane to the surface of the cell, including some the hydrophobic proteins with it.

There will also be messages translated in the cytosol, proteins of which will be used in the cell.

Question 9

What are the two types of ER?

Answer 9

Rough ER

Smooth ER

Question 10

Describe characteristics of RER

Answer 10

• Synthesis of proteins for subsequent packaging and secretion from the cell or for insertion into intracellular structures such as other membranes
• It is involved in the initial steps of glycosylation (joining carbohydrates onto proteins)
• Site of disulphide bond formation (which is important for the folding of the protein into its tertiary structure, chaperones help)

Question 11

Describe characteristics of smooth ER

Answer 11

• Modification of newly synthesised proteins –> addition of carbohydrate, phosphate and lipid group
• In the liver the SER is responsible for detoxification of foreign compounds such as drugs and environment pollutants (making drugs more soluble)

Question 12

Describe the formation of disulphide bonds in the ER

Answer 12

• On the surface of the ER is where there is formation of disulphide bonds between cysteine residues
  (S-H group in side chain)
• The formation of disulphide bonds is catalysed by the enzyme protein disulphide isomerase (PDI) which resides in the ER
• PDI is involved in breaking and reforming the disulphide bonds until the protein is the correct structure

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Question 13

What group is PDI (protein disulphide isomerase) part of?

Answer 13

Folding chaperones

Question 14

What do folding chaperones do?

Answer 14

The polypeptide chains interact with chaperones, which promote the folding of proteins to the correct structure. Folding chaperones also allow re-folding if it has folded into an inappropriate structure.

Question 15

List factors that can cause protein misfolding

Answer 15

• Cells are exposed to elavated temperatures resulting in increased expression of some chaperones (heat shock proteins) so proteins are more likely to misfold
• HSP are also expressed involved more in stressed individuals

Question 16

What happens is the degradative process for misfolded proteins becomes overwhelmed?

Answer 16

Protein aggregates can accumulate causing cellular dysfunction and cell death (apoptosis)

Question 17

Explain the destruction for unwanted proteins

Answer 17

• Unwanted proteins are marked for degradation by tagging lysine residues (on the protein to be destroyed) with a polymer of ubiquitin (polypeptide that acts as an tag)
• Ubiquitin marks the protein so it can be recognised by proteasomes
• The individual amino acids are cleaved off as the protein goes through the hollow core of the proteasomes

Question 18

Why are ubiquitin ligases important?

Answer 18

Ubiquitin ligases are very important in the body, there are about 600 ligases encoded by the human genome (many involved in inflammatory, cardiovascular, metabolic diseases or cancer)

Question 19

What are proteasomes?

Answer 19

Cylindrical complexes of protein with a hollow core in which degradation occurs by an energy dependent process

They destroy proteins that have been synthesised by the cell itself, not external ones

Question 20

Describe the destinations of proteins synthesised within the ER

Answer 20

• Proteins synthesised within the ER are transported to the golgi apparatus
• Portions of the ER are pinched off, forming transport vesicles which carry proteins to the golgi apparatus

Question 21

What is vesicular trafficking regulated by?

Answer 21

Vesicular trafficking is vehicle transport from one site in the cell to another

It is regulated by specific coat proteins on the vesicle surfaces which are regulated by small GTPase.

Question 22

What does membrane fusion between transport vesicle and golgi apparatus depend on?

Answer 22

SNAREs

They allow the protein to migrate to the appropriate site - they site on the cytosolic face of the vesicle

Question 23

What are the two fates on the genes products?

Answer 23

1. Secreted protein
2. Membrane protein

• Secreted protein exons do not contain a membrane spanning region, so they are just secreted
• Membrane protein, exon 3 has a membrane spanning region so it gets locked in the membrane and vesicle end up as part of the membrane

Question 24

Explain how there can be different outcomes from one transcript

Answer 24

e.g. with antibodies it needs to have a membrane bound and secreted form.

Processing of the transcript therefore determines the genes fate - not all the exons would have to be used so it depends on the splicing

Question 25

What is the structure of the golgi apparatus?

Answer 25

• It is a set of flattened bags
• The surface closest to the nucleus is called the ‘cis’ face where the vesicles enter the golgi apparatus
• ‘Trans’ is the part that faces the plasma membrane this is where vesicles leave the golgi apparatus

Question 26

Function of the Golgi Apparatus

Answer 26

1. Further modifications of newly synthesised proteins particularly by addition of carbohydrate residues (glycosylation)
2. Synthesis of glycolipids (glycosylation of lipids)
3. Proteolytic conversion of proforms of proteins into mature forms
4. Packaging of proteins destined for export into secretory vesicles or granules
5. Targeting of proteins (via vesicles) to lysosomes

Question 27

Where does protein glycolysation start and end?

Answer 27

Protein Glycolysation Starts in the ER and is completed in the Golgi Apparatus

Carbohydrate addition is initiated in the ER, concomitant with protein synthesis.

Question 28

What does glycolysation depend on?

Answer 28

Many secreted and membrane proteins are glycosylated.

Glycosylation depends on recognition of specific sequence patterns (sequence of amino acids) in the target protein.

Question 29

What are the two common types of glycoproteins?

Answer 29

N-linked glycoproteins(carbohydrate built on asparagine)

O-linked glycoproteins (carbohydrate built on serine or threonine)

Question 30

Give examples of N-linked glycoproteins

Answer 30

Serum glycoproteins involved in haemostasis (blood clotting) and most membrane proteins

Question 31

Explain the structure of N-linked glycoproteins

Answer 31

Branched carbohydrate chains attached to asparagine residues when they are in the following order : Asn - X - Ser/Thre

Typical sugars involved in these structures are N-acetylglucosamine, mannose, galactose and sialic acid

Carbohydrates are important receptors for a number of ligands e.g. sialic acid is the receptor for the flu virus haemagglutin (a protein that binds to sialic acid residues).

Question 32

Explain the structure of O-linked glycoproteins

Answer 32

They typically have shorter chains (compared to N) attached to serine or threonine residues

Typical sugars are N-acetylgalactosamine, galactose, sialic acid and fucose

carb built on serine or threonine

Question 33

Examples of O- linked glycoproteins

Answer 33

Mucins and blood group antigens (ABO groups are determined by glycosyltransferase genes, the oligosaccharidechain is found on both glycoproteins and glycolipids)

Mucins provide a protective barrier on epithelial surfaces

Question 34

Explain protein maturation in the golgi apparatus

Answer 34

• Most biologically active proteins are synthesised initially as large precursor molecules
• Proteolytic cleavage occurs at few specific sites where specific enzymes in the Golgi cleave precursor proteins to their biologically active form

Question 35

How can you generate multiple hormones from one polypeptide?

Answer 35

POMC cleavage

Question 36

What are the two pathways for protein secretion?

Answer 36

Constitutive pathway: As the vehicle containing protein becomes mature and then continuously moves towards the plasma membrane and continuously fuse releasing their contents e.g. albumin from hepatocytes

Regulated pathway: Protein accumulates in vesicles but will not be released/fuse until controlled factor reaches a certain level. It is controlled by Ca2+ dependent signalling.

Question 37

How are lysosomal enzymes synthesised?

Answer 37

• lysosomal enzymes may be converted from inactive precursors to active enzymes in a process acting in the golgi
• some lysosomal proteins are autocatalytic (it can cleave other molecules and make them into active protease) whereas other enzymes require an additional protease

Activation involves proteolytic removal of part of the polypeptide chain

Question 38

What happens to proteins glycosylated with residues of mannose-6-phosphate?

Answer 38

Proteins glycosylated with residues of mannose-6-phosphate will leave the Golgi in transport vesicles that eventually fuse with lysosomes.

Question 39

List some post translational modifications

Answer 39

1) Glycosylation (addition of carbohydrate groups)
2) Phosphorylation (addition of phosphate groups) – a key way in which proteins are regulated
3) Acetylation (addition of acetyl groups)
4) Methylation (addition of methyl groups)
5) Lipoprotein formation (addition of lipid groups)

Question 40

Give an example of regulating protein activity by phosphorylation

Answer 40

PHOSPHORYLASE (releases glucose-1-phosphate from glycogen)

Phosphorylase is usually inactive in cells (as phosphorylase b). Phosphorylase kinase gets activated after exposure to glucagon and adrenaline and it converts phosphorylase b to phosphorylase a which is active. Phosphorylase a can then perform its function.

To switch off phosphorylase a, protein phosphatase (activated after exposure to insulin) removes the phosphate and converts it back to phosphorylase b (inactive).