Lecture 5.2: Targeting Proteins and Protein Modification

Question 1

Protein Sorting: Cytosolic Ribosomes

Answer 1

Protein destined for cytosol
Post-translational import into organelles
Proteins are assembled in cytosol, and either remains in cytosol or is imported into an organelle such as the nucleus, mitochondria…etc

Question 2

Protein Sorting: ER Ribosomes

Answer 2

Protein destined for membrane or secretory pathway via co-translational insertion
The proteins either remain in the ER or are transported to the Golgi where they are modified, packaged and sorted
These are exported via secretory vesicles, lysosomes or vesicles to the plasma membrane

Question 3

What is required for protein sorting?

Answer 3

• A signal (address), intrinsic to the protein
• A receptor that recognises signal and directs it to the correct membrane
• A translocation machinery
• Energy to transfer the protein to its new place

Question 4

Protein import into mitochondrial matrix

Answer 4

• Protein with signal kept unfolded by chaperones
• Signal binds to receptor
• Protein fed through pore in outer membrane of mitochondria
• Protein moves through channel in adjacent inner membrane
• Targeting signal cleaved leaving only the functional protein

Question 5

Pyruvate Dehydrogenase Deficiency

Answer 5

Mutation at codon 10 in N-MTS of PDH E1a subunit results in Arg to Pro substitution
• Reduced uptake into mitochondria
• X-linked dominant disorder

Question 6

What is a Nuclear Localisation Sequence (NLS)?

Answer 6

It is an amino acid sequence that ‘tags’ a protein for import into the cell nucleus by nuclear transport.

Question 7

Swyer syndrome

Answer 7

• Loss or mutation of NLS in sex determining region Y (SRY) protein
• NLS is nuclear localisation signals
• XY genotype
• But outwardly female
• SRY required for testis differentiation

Question 8

Leri-Weill Dyschondrosteosis & Langer Mesomelic Dysplasia

Answer 8

• R173C mutation in NLS of SHOX
• SHOX = transcription factor
• Required for skeletal development
• Short stature

Question 9

Rhizomelic Chondrodysplasia Punctata

Answer 9

• Mutation in Pex7
• Receptor for a subset of peroxisomal enzymes

Normally:
• Receptor binds peroxisomal protein’s translocation sequence
• Receptor + translocation machinery → import

Question 10

Targeting proteins to the ER/secretory pathway (4)

Answer 10

1) Protein Synthesis on bound ribosomes on ER
2) Budding and fusion of ER-to-Golgi vesicles on cis Golgi face
3) Cisternal progression: cis to medial to trans Golgi
(There is also retrograde progression of vesicles Golgi-to-ER)
4) At trans-Golgi network proteins are packaged into secretory vesicles, vesicles or transport vesicles which combine with late endosome’s and form lysosomes

Question 11

Types of Secretion from Cells: Constitutive Secretion

Answer 11

Proteins are secreted from a cell continuously, regardless of external factors or signals

Question 12

Types of Secretion from Cells: Regulated Secretion Cells (3) + what is it

Answer 12

Regulated secretion terminates in secretory vesicles that store secreted material until a signal triggers fusion with the plasma membrane

• Endocrine Cells – secreting hormones
• Exocrine Cells – secreting digestive juices
• Neurocrine Cells – secreting neurotransmitters

Question 13

Functions of the Endoplasmic Reticulum

Answer 13

• Insertion of proteins into membranes
• Specific proteolytic cleavage
• Glycosylation
• Formation of S-S bonds
• Proper folding of proteins
• Assembly of multisubunit proteins
• Hydroxylation of selected Lys and Pro residues

Question 14

Why is glycosylation of proteins important?

Answer 14

• Correct protein folding
• Protein stability
• Facilitates interactions with other molecules
• Deficiencies in N-linked glycosylation lead to severe inherited human
disease: Congenital disorders of glycosylation (CDG)

Question 15

N-linked glycosylation

Answer 15

• Sugars are added on an asparagine side chain (involves an amino group
- hence N-linked)
• Oligosaccharide preassembled on lipid (dolichol) carrier
• Occurs in ER
• Further sugar modification takes place in ER & Golgi

Question 16

Modifications in the Golgi Apparatus: cis to trans face

Answer 16

Cis Face Network: Sorting, phosphorylation of ogliosaccharides on lysosomal proteins
Cis Cisterna: Removal of Man
Medial Cisterna: Removal of Man, addition of GlcNAc
Trans Cisterna: Addition of Gla, addition of NANA
Trans Face Network: sulphation of tyrosines and carbohydrates + sorting

Question 17

Glycosylation

Answer 17

Glycosylation is the reaction in which a carbohydrate, i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule in order to form a glycoconjugate

Question 18

Delivery of lysosomal enzymes to the lysosome: what signal is needed?

Answer 18

Requires a mannose-6-phosphate signal
As a lysosomal enzyme passes through the Golgi apparatus, a phosphate group is added to the hydroxyl group of carbon 6 of a mannose sugar

Question 19

Delivery of lysosomal enzymes to the lysosome: what enzymes needed (2)?

Answer 19

Requires the activity of two enzymes:
• Nacetyl glucosamine phosphotransferase
• Phosphodiesterase

Question 20

Inclusion-Cell Disease

Answer 20

• A fatal autosomal recessive disorder
• Deficiency in N-acetyl glucosamine phosphotransferase
• Lysosomes become bloated with un-degraded material

Question 21

O-linked Glycosylation

Answer 21

• Occurs in Golgi
• Attachment of sugar to hydroxyl group of serine, threonine
• Important in proteoglycans
• Component of extracellular matrix and mucus secretions

Question 22

Disulphide Bond Formation

Answer 22

• Occurs in ER
• Between 2 cysteine amino acids
• Redox reaction with a protein disulphide isomerase (PDI)
• PDIs are ER proteins
• C-terminal KDEL motif – signal to return them to ER
• KDEL = a retrieval pathway

Question 23

What happens if there are folding problems in proteins?

Answer 23

• Many proteins spontaneously fold into correct conformation,
• Some require chaperone proteins to help them

BUT:
• Protein may be trapped in misfolded conformation
• Protein contains mutation resulting in mis-folding
• Protein may be incorrectly associated with other sub-units

Question 24

ER Chaperone Proteins: role

Answer 24

Act as sensors to “monitor” extent of protein mis-folding:
• Mediate increased transcription of chaperones
• Mediate reduction in translation

• Retain unfolded proteins in the ER

Question 25

ER Chaperone Proteins: BiP

Answer 25

“Binding Immunoglobulin Protein”

Binds to exposed amino acid sequences that would normally be buried in the
interior of a folded protein

Question 26

ER Chaperone Proteins: Calnexin and Calreticulin

Answer 26

Binds to oligosaccharides on incompletely folded proteins

Question 27

What happens if protein mis-folding cannot be corrected?

Answer 27

• Protein may be returned to cytosol for degradation (proteasome)
• Protein may accumulate to toxic levels in the ER resulting in disease

Question 28

What proteins are secreted via constitutive secretion?

Answer 28

Collagen
Fibronectin
Laminin
Immunoglobulins
Albumin
Prohormones
Receptors

Question 29

What proteins are secreted via regulated secretion?

Answer 29

Insulin
Glucagon
β-endorphin
ACTH
α-MSH
β-lipotropin
Trypsin

Question 30

Collagen Fibres Structure

Answer 30

Basic unit is tropocollagen
3 polypeptides in a helical structure
H-bonds between a chains stabilise structure
Non-extensible
Non-compressible
High tensile strength

Question 31

Ehlers-Danlos Syndrome (EDS)

Answer 31

Mutation in collagen type I, III or V or lysyl oxidase deficiency

Stretchy skin

Question 32

Processing of insulin in pancreatic β-islet cells: ER lumen

Answer 32

Disulphide bonds form in the ER lumen (required for the biological activity of insulin)

Specific folding stabilised by DS bonds

Connecting peptide removed, leaving complete 2 chain insulin molecule

Reduction irreversibly separates the 2 chains

Question 33

Proteolytic processing of pro-insulin requires the action of which three enzymes?

Answer 33

PC3 Endoprotease
PC2 Endoprotease
Carboxypeptidase

Question 34

When does proteolytic processing of pro-insulin occur?

Answer 34

After budding from the trans-Golgi network

Question 35

Effect of Insulin Release

Answer 35

• Blood glucose ↑
• Glucose enters β-islet cells through GLUT2 transporter
• So intracellular [glucose] ↑, in turn causing Ca2+ in flux and release from ER
• [Ca2+ ] ↑ releases insulin containing vesicles

Question 36

Why can proteolytic processing can be very complex and yield different products?

Answer 36

Different amounts of processing enzymes in different cell locations

Question 37

Why is proteolytic processing so common in the secretory pathway?

Answer 37

Can give rise to very small products (e.g. enkephalins – 5aa) that would
be too short to enter ER via co-translational mechanism

Some secreted proteins e.g. hydrolytic enzymes would be destructive if
activated inside the cell

Multiple bioactive products can be produced from the same polypeptide