Protein Processing And Targetting In Cells

Question 1

Proteins for the mitochondrial matrix

Answer 1

• protein with signal kept unfolded by chaperones
• signal binds to receptor
• protein fed through pore into outer membrane
• moves through channel in adjacent inner membrane
• targeting signal then cleaved

Question 2

Pyruvate dehydrogenase deficiency

Answer 2

Arg –> Pro substitution
Receptors can’t recognise targeting sequences as well so there is a reduced uptake into the mitochondria
Results in build up of lactic acid and neurological problems

Question 3

Secretion from cells

Answer 3

• constitutive secretion
• regulated secretion
  Endocrine cells - secrete hormones
  Exocrine cells - secrete digestive juices
  Neurocrine cells - secrete neurotransmitters

Question 4

Insertion into the ER membrane

Answer 4

Required for delivery of membrane proteins destined for the plasma membrane or internal membrane of secretory pathway

Question 5

Stop Transfer Anchor Sequence

Answer 5

A hydrophobic sequences exists the protein which anchors the protein in the membrane and prevents further transfer into the ER lumen

Question 6

Functions of endoplasmic reticulum

Answer 6

• insertion of proteins into membranes
• specific proteolytic cleavage
• glycosylation
• formation of disulphide bonds
• ensures proper folding of proteins
• assembly of multi subunit proteins
• hydroxyl atom of selected Lys and Pro residues

Question 7

Glycosylation

Answer 7

• ensures correct protein folding
• increases protein stability
• facilitates interactions with other molecules
• deficiencies in N-linked glycosylation lead to severe inherited human disease: Congenital Disorders of Glycosylation (CDG)

Question 8

Protein Disulphide Isomerase

PDI

Answer 8

• protein passes through the ER which catalyses disulphide bond formation

Question 9

KDEL

Answer 9

KDEL carrying protein has a higher affinity

Allows it to bind and then be released back into the ER

Question 10

Folding problems

Answer 10

ER chaperone proteins attempt to correct misfolding
BiP - binds to exposed amino acid sequences that would normally be buried in the interior of a folded protein
Calnexin and Calreticulin - binds to oliosaccharides on incompletely folded proteins
IF IT CANNOT BE CORRECTED
protein returned to cytosol for degradation
Or may accumulate to toxic levels in the ER and result in disease
(e.g. Alzheimer’s, CF, diabetes mellitus)

Question 11

Collagen

Answer 11

most abundant protein in the body (25-35%)
connective tissue - tendons, ligaments, etc.
Secreted protein produced by fibroblast cells in the connective tissue
Assembly of collagen fibres has to occur outside of the cell otherwise it would be catastrophic

Question 12

Collagen fibres

Answer 12

300nm rod-shaped protein
3 polypeptides (alpha helixes)
Glycine in every third position (Gly-X-Y)n - allows chains to come together - stabilised by H-bonds (prolyl hydroxylase - allows increased H-bonding)
Triple helix - RIGHT HANDED - non-extensible, non-compressible and has a high tensile strength

Question 13

Distribution of collagen types

Answer 13

Type I - skin, bone (90% of all body collagen)
Type II - cartilage, intervertebral discs
Type III - foetal skin, cardiovascular system
Type IV - basement membranes
Type V - placenta, (skin)

Question 14

Formation of a collagen fibre

Answer 14

The conversion of procollagen to tropocollagen is done extracellularly
Removal of the N and C terminal properties outside of cell
Lateral association of collagen molecules followed by covalent cross-linking

Electron light and electron dense bands alternate
Then the aggregation of fibrils

Question 15

Insulin

Answer 15

Proinsulin is one polypeptide chain has three disulphide bonds (in the ER) which is cleaved to form two polypeptide chains which has three disulphide bonds to form mature insulin (occurs post Golgi)
Requires three enzymes

Question 16

Proteolytic processing

Answer 16

One polypeptide chain can yield several different enzymes depending on the way it is processed - cleaved and rejoined in different sequences
Common in the secretory pathway as hydrolytic enzymes would be destructive if they were active in the cell

Question 17

To ER

Answer 17

Signal sequence at N-terminus 
Unfolded during transfer 
Signal recognition protein/receptor
Signal cleaved by signal peptidase 
Requires energy - hydrolysis of GTP by SRP

Question 18

To nucleus

Answer 18

NLS - nuclear localising signal BASIC
on surface of folded protein
Folded during transfer 
Importin recognises NLS 
Signal is retained 
Requires energy - hydrolysis of GTP

Question 19

To mitochondria

Answer 19

Amphipathic signal for initial targeting to matrix located on the N-terminus
Held partially unfolded by chaperones during transfer
Mitochondrial-import simulating factor and Tom&Tim channel complexes
Signal is cleaved
Requires energy - ATP hydrolysis

Question 20

To Lysosomes

Answer 20

Post translational addition of mannose-6-phosphate
Signal patch to distinguish lysosomal proteins from other mannose-labelled proteins
Folded during transfer (delivered via vesicle)
M-6-P receptor in trans-Golgi
Phosphate removed by phosphatase
Requires energy for phosphotransferase (not directly)

Question 21

For retention in ER

Answer 21

KDEL signal (Lys-Asp-Glu-Leu) on the C-terminus
Folded during transfer (delivered via vesicle)
KDEL receptor in cis-Golgi
Signal is retained
Doesn’t require energy - binding and release is dependent on pH

Question 22

What is required for protein sorting?

Answer 22

a signal (address), intrinsic to the protein shown as a pre___ protein
a receptor that recognises the signal and directs the protein to the correct membrane
translocation machinery
energy to transfer the protein to its new place