Moving Proteins into Membrane

Question 1

Give an overview of protein sorting pathways?

Answer 1

10 000 proteins per cell - very crowded
Must be targeted to organelles for cells to function correctly
RNA polymerases need to be delivered to the nucleus
Transport proteins need to reach the cell surface

Many proteins are synthesised by cytosolic ribosomes and remain in the cytosol
As many as 50% of the proteins produced in a cell are delivered to one of the various membrane bound organelles

Question 2

What are the two mechanisms of protein sorting pathways?

Answer 2

Signal based targeting

Vesicle based targeting

Question 3

Describe signal based targeting?

Answer 3

Targets newly synthesised protein from the cytosol to an organelle
Can occur during translation or following protein synthesis (PTM)

Membrane proteins: inserted into lipid bilayer
Hydrophobic exterior

Soluble proteins: translocation of the entire protein across the membrane into the aqueous interior of an organelle
Hydrophilic amino acids - facing solution

Question 4

Give an overview of the rough endoplasmic reticulum?

Answer 4

This is the major site of protein synthesis
RER membrane is continuous with the membrane of the nucleus
Major site of protein synthesis
Represents the start of the secretory pathway

Some proteins are synthesised at the ER and do not associate with membranes
Can be directed to the ER during translation and ultimately end up in theER lumen
Proteins are then packaged for vesicular release from the cell (secretory pathway)
Dictated by signals in the polypeptide chain

Question 5

What is a key feature of the rough endoplasmic reticulum?

Answer 5

RER is the major branch point for the traffic of proteins

Proteins destined for secretion or incorporation into the ER, Golgi, lysosomes, orplasma membrane are initially targeted to the ER
In mammalian cells, most proteins are transferred into the ER whilst translated on membrane-boundribosomes

Proteins destined to remain in the cytosol or to be incorporated into thenucleus,mitochondria or peroxisomes are synthesized on free ribosomes and released into the cytosol once theirtranslation is complete

Question 6

How are proteins targeted to the ER?

Answer 6

1. Translocated into theRER during their synthesis on membrane boundribosomes(co-translational translocation)
   OR
2. Transported oncetranslation is complete on free ribosomes in the cytosol (post-translational translocation

Mostproteins enter the RER co-translationally

Question 7

What allows proteins to be targeted to the ER?

Answer 7

Proteins destined for the ER contain a signal sequence (N-terminal)
Directs the protein/ribosome complex to the ER membrane
Proteins inserted across membrane as they are translated - co-translational translocation

Question 8

Describe the signal sequences on proteins?

Answer 8

Hydrophobic N-terminal signal sequences
Variable, no sequence homology
16-30 residues in length
Contain one or more positively charged residues adjacent to a continuous stretch of 6-12 hydrophobic amino acids

Question 9

What experiments can be done to find co-translational translocation?

Answer 9

Microsomes: vesicle-like artefacts re-formed from pieces of the ER when eukaryotic cells are homogenised
Cells homogenized: fractures the plasma membrane and shears the rough ER into microsomes
Microsomes with bound ribosomes can be isolated by differential and sucrose density-gradient centrifugation

Pulse-chase experiments
Cells incubated with radiolabelled AAs: newly synthesized proteins are radiolabelled
Microsomes then isolated, purified and treated with a protease and/or detergent
Proteins in ER are protected from digestion in the absence of the detergent which dissolves the ER membrane
Thus radiolabelled proteins are inside microsomes (the lumen of the RER) following their synthesis

Question 10

How do secretory proteins then enter the ER?

Answer 10

Signal recognition particle
Signal sequence recognition - mediated by signal recognition particle (SRP) and its receptor
Binds to – ER signal of nascent protein and large ribosomal subunit

SRP consists of 6 proteins bound to a 300nt RNA (ribonucleoprotein)
p54 subunit of the SRP contains a hydrophobic cleft which binds to the hydrophobic AAs of the signal sequence (large ribosomal subunit)

Arrests further development of the polypeptide chain - prevent full translational before translocation

Question 11

Describe the function of the signal recognition particle?

Answer 11

SRP bound to the signal sequence is recognized by the SRP receptor - an integral protein of the ER membrane
SRPR also bound to GTP
Hydrolysis of GTP leads to the release of SRP from the SRPR
Nascent polypeptide chain is then transferred to a translocon - a protein pore in the ER membrane that is associated with the SRP receptor

Question 12

Describe the translocon?

Answer 12

Binds the signal sequence after its release by the SRP
Forms a channel through which the polypeptide is passed

Features
Sec61 is a heterotrimer and makes up the core of the translocon
TRAM is tightly associated with the translocon and is required for translocation
Other complexes associated with the translocon are signal peptidase and oligosaccharyl transferase

Question 13

What happens once the protein enters the ER lumen?

Answer 13

The signal sequence is cleaved by signal peptidase (ER transmembrane protein)
Polypeptide chain enters ER lumen through the translocon

Question 14

Describe signal peptidase?

Answer 14

Cleaves off signal sequence
Localised to lumen of ER
Not all signal sequences are cleaved - if ER membrane protein
No specific cleavage site
Small, neutral side chains A, C, G T, S at -1 and -3

Question 15

What is the driving force of unidirectional movement across the ER?

Answer 15

Sec63 complex/ BiP (Binding immunoglobulin protein)
HSP (heat-shock protein) molecular chaperone
Located in ER lumen
Contains a peptide binding domain and an ATPase domain
Binds to and stabilises partially or unfolded proteins

Question 16

Describe the mechanism of BiP?

Answer 16

Sec63 hydrolyses BiP.ATP
Conformational change in BiP: binds to incoming polypeptide chain
Polypeptide chain grows - additional BiP.ADP proteins bind
Prevents polypeptide chain from “sliding back

BiP stabilizes the polypeptide as it folds to a mature conformation

Question 17

Give an overview of insertion of membrane proteins at the ER?

Answer 17

Integral membrane proteins remain embedded in a lipid membrane
Can follow the same route as a soluble secretory protein
It retains it’s topology to maintain its position within the membrane

Question 18

Describe the mechanism of inserting Type I membrane proteins?

Answer 18

Signal peptidase cleaves the signal sequence
Hydrophobic TM domains (approx. 20 aa with α-helix secondary structures) enter the translocon
Translocation stops, the translocon opens laterally allowing the TM domain to embed into the ER membrane: Stop-Transfer Anchor sequence (STA)
After embedding, translation continues producing a protein with a C-terminal cytoplasmic domain = component on both sides of the membrane

Question 19

Describe the mechanism of inserting Type II membrane proteins?

Answer 19

No N-terminal signal sequence - instead it is located internally
Also recognised by the SRP, targets the nascent polypeptide chain to the ER where it is inserted into the translocon pore
Signal sequence acts as a transmembrane domain
Released by the translocon – embeds into the ER bilayer: signal-anchor (SA) sequence
Remainder of the protein translated and enters the ER through the translocon

C-terminal is in the ER lumen and the N-terminal is in the cytoplasmic domain (opposite of type I membrane protein)

Question 20

Give an overview of the classes of integral membrane proteins?

Answer 20

Type I: Cleaved N-terminal ER sequence, N-terminal luminal C-terminal cytosolic

Type II: No cleavable N-terminus, C-terminal luminal, N-terminus cytosolic

Type III: Same orientation as Type I but no cleavable signal sequence - differs from Type II by the position of positive charges

Type IV: Contain 2 or more TM domains

GPI linked: Lack membrane spanning segments - instead are linked to an amphipathic phospholipid anchor embedded in the membrane

Question 21

Give a comparison of type II and II membrane proteins?

Answer 21

Type III transmembrane proteins have an internal signal sequence (not cleavable) as for Type II, however:
Type II: positive charges on N-terminal side of the SA sequence
Type III: positive charges on C-terminal side

Positive charges on the C-terminal side cause the polyprotein to be anchored with the C-terminus in the cytosol and the N-terminus in the ER lumen

Question 22

Describe tail anchored single pass proteins?

Answer 22

They have a tail anchor in their hydrophobic tail
Many of these TA proteins are PTM targeted to the ER
It involved an ATPase, Get3, which binds to the C-terminal hydrophobic fragment of the protein
The GEt3-bound protein is recruited by a dimeric receptor Get1/Get2
ATP is hydrolysed
Hydrophobic C-terminus is released and embedded into the membrane

Question 23

Describe type IV membrane proteins?

Answer 23

Alternative mixed and stop-transfer anchor or internal signal anchor sequences
Different orientations depending on cleavable signal sequence, number of stop-transfer anchor sequences and internal signal anchor sequences

Two types - cytosolic or luminal (depends on the number of TM helices)
Each membrane spanning α-helix can direct the protein to the ER, anchor the protein to the ER membrane, or stop-transfer through the ER membrane

Question 24

How can we predict the topology of a protein?

Answer 24

Hydropathy profile - assigns a value to each amino acid; positive values for hydrophobic amino acids, negative values for hydrophilic amino acids
Identifies long segments of sufficient hydrophobicity to be N-terminal signal sequences, stop-transfer sequences or stop anchor sequences
Calculated for each 20 amino acid stretch

Question 25

Describe phospholipid anchors for proteins?

Answer 25

Attached by a covalent amphipathic molecule Glycosylphosphatidylinositol (GPI)
Sequence of amino acids near the N-terminus recognised by a GPI transamidase
Cleaves off original stop anchor sequence and transfers the ER luminal portion to a pre-formed GPI membrane anchor
GPI anchored proteins cluster in lipid rafts

Question 26

What are some post translational modifications in the secretory pathway?

Answer 26

The nascent polypeptide can be modified in a number of ways that alter its structure and function

ER

1. N-linked glycosylation
2. Disulphide bond formation
3. Modifications to aid folding
4. Oligomer formation

Golgi

5. O-linked glycosylation
6. Proteolytic processing

Cell surface
7. Protein shedding

Question 27

Describe the first two stages of N-linked glycosylation?

Answer 27

Step 1 - Attachment of a sugar molecule (glycan) to the nitrogen atom (amide nitrogen) of an asparagine residue
Requires recognition of a consensus sequence: Asn-X-Ser/Thr (aa except proline)

Step 2 - Precursor oligosaccharide is formed, the completed glycan is then transferred to the nascent polypeptide in the lumen of the ER membrane
Reaction is driven by the energy released from the cleavage of the pyrophosphate bond between the dolichol-glycan molecule

Question 28

What is the last stage of N-linked glycosylation?

Answer 28

Step 3 - N-linked glycans undergo extensive processing reactions once transferred to the nascent polypeptide
Three glucose residues and several mannose residues are removed by ER resident glucosidases & mannosidases

Step 4 - ER chaperones bind to the 3 glucose residues on the core N-linked glycan - Calreticulin and Calnexin
Aid folding of the protein attached to the glycan
Following folding, the three glucose residues are removed

If the protein fails to fold, the three glucose residues are reattached, allowing the protein to reassociate with the chaperones

Question 29

Describe di-sulphide bond formation?

Answer 29

Occurs in the oxidising environment of the ER lumen
Oxidative linkage of sulfhydryl groups (known as thiol groups)
Occurs between two cysteine residues
Typically absent in cytosolic proteins

Mechanism
Formed in the presence of PDI - protein disulphide isomerase
Acts as a reducing agent, oxidising the thiol group
Requires ER oxidoreduction (Ero1)
Ero1 oxidises PDI, allowing it to exchange disulphides on the protein

Question 30

What is the rearrangement of disulphide bonds?

Answer 30

S-S form between residues that occur sequentially
Can yield bonds between the wrong residues
Pro-insulin: bonds must form at positions 1-4, 2-6, 3-5
Accelerated by PDI - it wants these bonds to be in their most favourable form

Braking non-native disulphide bonds so the protein can finish folding properly - to form the native disulphide bond

Question 31

Describe oligomer formation?

Answer 31

Hemagglutinin protein of influenza virus (IAV)
Trimeric spikes protrude from the IAV particle
HA trimers are formed within the ER of an infected host cell
Co-translationally inserted into the ER as a precursor HA0
Transported to the cell surface via the Golgi - this is a post-translational event

Misfolded proteins that fail to fold, exit the ER and are degraded - ERAD

Question 32

Describe O-linked glycosylation?

Answer 32

Specific glycosyl transferases add activated sugars to the O-atom of serine or threonine residues
Enzyme only resident in the cis-Golgi

Question 33

Describe proteolytic processing?

Answer 33

Conversion of an inactive or protein to its functional form
Pairs of basic amino acids (RR,KK,RK,KR) recognised by specific endoproteases in the Golgi
They cleave for the pro (inactive) form into the active form

Question 34

Describe proteolytic shedding?

Answer 34

Generates soluble forms of membrane proteins
Typically occurs at the cell surface

Example: ADAM (a disintegrin and metalloprotease) family
Cleaves and removes the ectodomains of many membrane proteins