Lecture 14 - Membrane Asymmetry

Question 1

Why is uncatalysed transverse diffusion very slow?

Answer 1

Re-orientation of the polar head group through the non-polar core of the membrane is energetically unfavourable

Question 2

Why is transverse asymmetry observed?

Answer 2

The orientation of lipids and proteins is controlled during the assembly and maintenance of membranes

Question 3

What do phospholipid translators do? and where?

Answer 3

Facilitate lipid movement between leaflets at the smooth ER and elsewhere

Question 4

What are lipid rafts?

Lateral asymmetry is shown

Answer 4

Specific membrane proteins and lipids cluster together in a temporary and dynamic fashion (transient)

Membranes show lateral asymmetry with the formation of transient microdomains that are rich in cholesterol and glycosphingolipids - lipid rafts.

Question 5

Possible functions of lipid rafts?

Answer 5

Organising and concentrating membrane proteins for transport in membrane vesicles or working together in protein assemblies

Question 6

Diameter and half life of lipid rafts?

Answer 6

Diameter of 10-200nm

Half life of only 100 ns

Question 7

What lipoproteins partition preferentially into lipid rafts?

Answer 7

GPI anchored proteins and proteins modified by myristoylation and palmitoylation

Question 8

What proteins involved in endocytosis appear to partition into lipid rafts? and in cell signalling?

Answer 8

Calveolin-1 - endocytosis

Heterotrimeric G-proteins - cell signalling

Question 9

What are the 4 principles that underpin protein targeting?

Answer 9

1. Default pathways are followed in the absence of specific instructions
2. Signals can be encoded in the primary sequence of a protein or in tertiary structure (signal patches)
3. Further signals from glycosylation during post-translational processing
4. Vectorial transport - non random transport of proteins to specific locations

Question 10

What is the role of the SRP

i.e. what three things does it do when it binds to the signal sequence

Answer 10

Binding of the SRP (RNA molecule and 6 proteins) to the signal sequence leads to:

Stops translation
Prevents premature protein folding
And “directs” the ribosome to the ER

Question 11

What is the role of GTP in the function of the SRP?

Answer 11

Both the SRP and SRP receptor bind GTP - and the hydrolysis of this GTP releases the SRP and allows protein synthesis to resume on the bound ribosome

Hydrolysis of GTP allows release of the SRP thus allowing protein synthesis to resume on the bound ribosome.

Question 12

The GTP-GDP cycle is:

This equation shows how the equation is driven forward

Answer 12

Free ribosome + nGTP -> Bound ribosome + nGDP

Question 13

Outline protein translocation Into the ER

Answer 13

Protein synthesis resumes after the release of SRP and the growing polypeptide chain is threaded through the membrane into the ER lumen (ATP consuming process).

Question 14

Name the enzyme that removes the signal sequence

Answer 14

A signal peptidase

Question 15

If not threaded through the membrane into the ER lumen, where can the nascent polypeptide go?

Answer 15

A stop transfer sequence can instruct the polypeptide to stay in the membrane

Question 16

What 2 types of polypeptides have a signal sequence?

Answer 16

Those that are going to be secreted or embedded into the plasma membrane

Question 17

What is N-linked glycosylation?

Occurs while the protein is located in the ER membrane

Answer 17

A precursor oligosaccharide is transferred en bloc to proteins.

It is transferred by to the side chain NH2 group of an asparagine in the protein

Question 18

Where is the activated oligosaccharide moiety transferred?

Answer 18

It is transferred to asparagine residues on the lumenal side of the membrane

Question 19

Why are cytosolic proteins not glycosylated in this way?

Answer 19

Because the enzyme oligosaccharyl transferase has its active site exposed on the lumenal side of the membrane

Question 20

Describe the function of dolichol

And name the bond that links dolichol to the oligosaccharide

Answer 20

Dolichol anchors the precursor oligosaccaride in the ER membrane. Dolichol is linked to the O by a high energy pyrophosphate bond that provides the activation energy required to drive the reaction

Question 21

Where does the new glycoprotein go after the ER?

Answer 21

After further modification, it is exported to the Golgi in a vesicle that buds off from the ER

Question 22

Name the two broad classes of N-linked oligosaccharides

Answer 22

The complex O

High mannose O

Question 23

How are complex O generated?

Answer 23

When the original N-linked O added in the ER is trimmed and further sugars are added

High mannose O are trimmed with no new sugars added in the Golgi

Question 24

What are signal patches

Answer 24

Signal patches on some proteins are recognised by an enzyme that phosphorylates mannose residues - mannose 6-phosphate targets the protein to lysosomes

Question 25

Where do unmarked proteins go?

Answer 25

They are transported to the plasma membrane in either lumen or membrane of secretory vesicles

Question 26

Why are most mitochondrial proteins imported?

Answer 26

Most proteins are encoded in nucleus and imported from cytosol Synthesised using cytosolic ribosomes

Question 27

What do chaperone proteins do?

Answer 27

Prevent folding before translocation - allow the N terminal signal sequence to dock with a receptor on the outer mitochondrial membrane

Question 28

What happens to the chaperone proteins after docking?

Answer 28

The proteins are removed by an ATP dependent process

Question 29

How is insertion into the inner mitochondrial membrane driven?

Answer 29

Is driven by membrane potential and the translocation is driven by ATP hydrolysis

Question 30

What is ATP used for during protein import into the chloroplasts?

Answer 30

ATP is the energy source for protein import into the stroma

Question 31

What is the pH dependent TAT pathway?

Answer 31

...

Question 32

How is membrane potential used in insertion? | during import of proteins into the mitochondria

Answer 32

Pumping protons from the matrix space into the intermembrane space (driven by electron transport processes in the inner membrane) maintains the electrochemical gradient. Energy from the electrochemical H+ gradient drives the translocation of positively charged signal sequences by electrophoresis

Question 33

Explain how multiple signals are present in protein import into chloroplasts

Answer 33

Hydrolysis of the N terminal signal peptide in the stroma may reveal a second signal directing the protein into the thylakoid membrane

Question 34

What are three example signal sequences

Answer 34

1. retain in the ER lumen 2. Import into mitochondria 3. import into ER

Question 35

Describe protein import into the mitochondria

Answer 35

Most mitochondrial proteins are encoded by the nucleus and made using cytosolic ribosomes. Chaperone proteins prevent folding before translocation, allowing the N terminal signal sequence to dock with a receptor on the outer mitochondrial membrane After docking, the chaperone proteins are removed by an ATP-dependent process. Insertion into the mitochondrial membrane is driven by membrane potential, and translocation is driven by ATP. On arrival in the matrix, cleavage of the first signal peptide may reveal a second signal that directs the protein into the inner mitochondrial membrane.

Question 36

Protein import into chloroplasts

Answer 36

Operates in a similar way to mitochondria for many proteins. Chaperone proteins prevent premature folding, allowing unfolded polypeptides to be threaded through translocators. Multiple signals may be present - hydrolysis of the N terminal signal peptide in the stroma may reveal a second signal directing the protein into the thylakoid membrane. Energy is required - ATP is the energy source for protein import into the stroma. There is also evidence that some proteins are imported into the the thylakoid lumen in a fully folded state via the pH dependent TAT pathway that is also used for the export of certain proteins into the bacterial periplasm.