Lecture 6: endosomal sorting

Question 1

‘Endosomes are sorting stations in the cell’
What’s meant by this?

Answer 1

When (early) endosomes enter a cell with cargo, this endosome system is going to sort out what part needs to be degraded and what part can be recycled.

Question 2

Shortly describe how endosome sorting works.

Answer 2

Endosomes take up cargo from outside the cells in vesicles. The cargo is transported into these vesicles with the help of escort proteins. These vesicles are important for the sorting process. All content that is not sorted away, is degraded in lysosomes.

Question 3

Endosomes contain two different domains. What are these and what are their function?

Answer 3

1. Tubular sub-domains that take up cargo (lipids and proteins) that is suitable for recycling.
2. Vacuolar domain, where inward budding events take up cargo that needs to be degraded.

Question 4

How are early endosomes recognized compared to late endosomes?

Answer 4

The amount of particles taken up/the amount of intraluminal vesicles. Loads of intraluminal vesicles belong to late endosomes.

Question 5

So the amount of intraluminal vesicles can already tell you something about the maturation of endosomes. What elses is an indicator of maturation?

Answer 5

pH, a more matured endosome has a lower pH (5) than an early endosomes (pH 7)

Question 6

So the amount of intraluminal vesicles or the pH can already tell you something about the maturation of endosomes. What elses is an indicator of maturation?

Answer 6

The presence of certain Rab GTPases located in the cytosol. Early endosomes are typically labeled by Rab5, while late endosomes are typically labeled by Rab7.

Question 7

So the amount of intraluminal vesicles, the pH or Rab GTPases can already tell you something about the maturation of endosomes. What elses is an indicator of maturation?

Answer 7

Phosphorylated lipids. Early endosomes have PI(4,5)P2 (phosphorylated on C-ring 4 and 5) and late endosomes have PI(3,5)P2 (phosphorylated on C-ring 3 and 5).

Question 8

These phosphorylated lipids recruit effector proteins that help with endosome maturation. What are PI(3)P binding proteins or so called Sorting Nexins (SNXs)?

Answer 8

SNXs have a specific domain (PX) that bind to PI(3)P lipids. There are 3 groupes of these proteins, namely:

• SNX-BAR
• SNX-PX
• SNX-other

Question 9

What are the SNX-BAR proteins?

Answer 9

These proteins contain two PX-domains and 2 BAR-domains. The BAR domains dimerize, it forms an arc/bridge structure. These proteins seem to be important in shaping the tubular domains on endosomes for recycling.

Question 10

Induction of a curvature can be achieved through different mechanisms, like lipid composition, membrane proteins, cytoskeleton, scaffolding and helix insertion. How is curvature induced with the help of lipid composition?

Answer 10

The lipid bilayer can be composed differently. Some Lipid bilayers have lipid heads an tails with an equal size, so that the membrane is straight. Some lipid bilayers contain e.g. lipid heads (or tails) that are bigger than other lipid heads (or tails). This kind of composition can be changed in a curvature.

Question 11

Induction of a curvature can be achieved through different mechanisms, like lipid composition, membrane proteins, cytoskeleton, scaffolding and helix insertion. How is curvature induced with the help of transmembrane proteins?

Answer 11

Through oligomeriztion of membrane proteins which induces a curvature in the lipid bilayer.

Question 12

Induction of a curvature can be achieved through different mechanisms, like lipid composition, membrane proteins, cytoskeleton, scaffolding and helix insertion. How is curvature induced with the help of the cytoskeleton?

Answer 12

By applying pressure to the membrane with the help of actin filaments or microtubule motors.

Question 13

Induction of a curvature can be achieved through different mechanisms, like lipid composition, membrane proteins, cytoskeleton, scaffolding and helix insertion. How is curvature induced with the help of scaffolding?

Answer 13

Associating proteins that already have a certain curvature shape. This can be done through:

• indirect scaffolding
• direct scaffolding (negative)
• direct scaffolding (positive)

Question 14

What research was performed (liposome membrane remodelling assay)?

Answer 14

Purification of SNX-BAR proteins and synthesis of lipid vesicles. The goal was to see if tubular structures could be formed.

Question 15

What was seen under the microscope after the liposome membrane remodelling assay? Could all SNX-BAR proteins induce curvature?

Answer 15

No, not all. E.g. SNX1 and 2 could induce curvature, but its binding partners SNX5 and 6 could not. This was also the case for some other SNX-BAR proteins.

Question 16

What other characteristic of these SNX-BAR proteins was discovered?

Answer 16

That different SNX-BAR proteins induced tubules with different diameters and so the proteins are very specific and have their own ‘identity’ (they stay true to their own size in diameter).

Question 17

In the same liposome membrane remodelling assay they researched whether SNX-BAR proteins could homo- or heterodimerize and whether this homo- or heterodimers could induce membrane remodelling. What was found (also think of the SNX-BAR proteins and name them)?

Answer 17

Most SNX-BAR proteins that were able to homodimerize, were able to induce membrane remodelling. For example SNX8, 9, 18 and 33. Most heterodimers could not induce membrane remodelling, except SNX1, 2 and 4. But most importantly, it’s now clear that there’s a certain specificity in dimerization.

Question 18

Next, they investigated what the cause could be of this SNX-BAR specificity. They used SNX5, that normally isn’t able to dimerize with another SNX5. What was the cause of this?

Answer 18

The BAR domain is hydrophobic and within this hydrophobic region there are 4 glutamine residues that are charged and therefore repel each other. This prevents homodimerization. When these glutamine residues were replaced, homodimerization was possible.

Question 19

Fill in:

Homodimerization is required, but not … to form tubulus.

Answer 19

Homodimerization is required, but not sufficient to form tubulus.

Question 20

So if homodimerization is needed, but not sufficient for tubule formation: there needs to be something else that induces tubule formation. Certain SNX-BAR proteins that could induce tubule formation had one thing in common, besides being able to homodimerize. What was this and what SNX-BAR proteins had this in common?

Answer 20

Amphiphatic helices, SNX1, 2, 4, 9, 18 and 33. It was also found that in heterodimerization, the presence of only one amphiphatic helix was sufficient for tubule formation.

Question 21

To complete tubule formation (because homodimerization of the BAR domains is not enough) and to form a 3D structured tubule, other BAR proteins are needed. What do other BAR proteins do?

Answer 21

They form tubules by tip-to-tip interactions

Question 22

What are the three critical steps in tubule formation?

Answer 22

1. Dimerization
2. Amphiphatic helix
3. Oligomerization of SNX-BAR proteins by tip-to-tip interaction.

Question 23

What is a retromer?

Answer 23

Retromer is a protein complex containing SNX proteins and 3 vacuolar protein sorting (VPS) that has been shown to be important in recycling transmembrane receptors from endosomes to the trans-Golgi network (TGN). They are important in membrane deformation (through SNX proteins) and cargo recognition (through VPS proteins).

Question 24

Don’t know if this is really important, but:

Name the functions of SNX9, SNX4, SNX8 and of the following SNX proteins together: SNX1, SNX2, SNX5 and SNX6

Answer 24

• SNX9, endocytosis
• SNX4, recycling from endosome to plasma membrane
• SNX8, retromer-independent sorting-to-TGN
  
  • SNX1, SNX2, SNX5 and SNX6: retromer-independent sorting endosome-to-TGN

Question 25

What is the importance of the three vacuolar protein sorting (VPS)?

Answer 25

They form the core of the retromer and recognize what type of proteins need to be recycled. They can then associate to the SNX proteins that are important in tubule formation for recycling.

Question 26

How do the SNX and VPS protein associate so that they can recognize cargo that needs to be recycled?

Answer 26

The SNX proteins form helical structures and the VPS sit on top of these SNX proteins and form barbwire structures and these structures can associate with motives that recognize certain cargo that needs to be recycled.

Question 27

What is retromer function linked to?

Answer 27

Brain function, through GWAS it was found that retromer mutations are linked to neurodegenerative disorders (Alzheimer’s, Parkinson’s, Down syndrome and lysosomal storage diseases)

Question 28

Why is it so easy to study retromers?

Answer 28

Because they are evolutionairy conserved, from very simple organisms to very complex organisms.

Question 29

Why are classic null mutation methods generally not viable in retromer studies?

Answer 29

Because retromers are a central complex in cell function, so null mutations typically result in a not viable organism.

Question 30

What was shown in a retromer heterozygous flies and mice (one allel knocked out, one allel still functioning)?

Answer 30

That these flies accumulate Aβ plaques and show neurodegeneration. And heterozygous mice show impaired hippocampal memory.

Question 31

What was shown in postmortum AD brain slices?

Answer 31

Reduction of retromer proteins

Question 32

How is amyloid beta produced in Alzheimer’s?

Answer 32

Amyloid beta is formed out of the APP protein. APP is cleaved by two enzymes, called BACE1 and Presenilin 1 or 2.

Question 33

APP is continuously trafficking from the plasma membrane through endosomes, towards the recycling endosome and back to the plasma membrane. Explain how retromer function is important in this.

Answer 33

SORL1 is a trafficking receptor for APP. Retromers can bind to SORL1 and when the retromer encounters SORL1, it will associate to SORL1 and SORL1 then associates with APP. The retromer will drag APP out of the endosome towards the recycling endosome, where it goes back to the plasma membrane.

Question 34

How is Aβ produced in regard to the endosomes and retromers?

Answer 34

In a normal situation, APP is not cleaved. This, because it doesn’t encounter active BACE1. BACE1 is mainly active in late endosomes because of the increased acidity in late endosomes. If the retromer is not present, than SORL1 can not associate with the retromer. Thus, the retromer cannot drag APP bound to SORL1 to the recycling endosome. So APP bound to SORL1 is then moved to the late endosome where BACE1 is active. APP is then cleaved and turned into Aβ.