Endomembrane Systems

Question 1

Give an example of a cytochemical methods to identify enzyme activity on the ER.

Answer 1

Immunoflouresence methods, i.e. Using antibodies against enzymes present.

Question 2

How can we stain RER to observe under the light microscope?

Answer 2

Via basophilic staining (blue/purple)

Question 3

What are features of the RER under a TEM?

Answer 3

• Membranes organised in stacks.

- Ribosomes

Question 4

What are features of the SER under a TEM?

Answer 4

• Tubule structure

- No ribosomes

Question 5

What are features of the ER under a TEM?

Answer 5

• Formed by an intricate network of tubules and cisternae.
• Lipoprotein membrane with the same structure as the PM.
• The joining part between RER and SER is visible.

Question 6

What kinds of cells is the RER abundant in?

Answer 6

The RER is very abundant in cells specialised for synthesis and secretion of proteins. E.g. pancreatic cell.

Question 7

What kinds of cells is the SER abundant in?

Answer 7

The SER is abundant in cells specialized in synthesis and secretion of steroid hormones. E.g. Adrenal cell.

Question 8

What is the organisation of the SER in skeletal muscles and the heart?

Answer 8

In skeletal muscles (myocytes) and heart (cardiomyocytes) the SER is highly developed and placed around the contractile myofibrils: SER form the terminal.

The ER is specialised for their function. Particularly the SER has an organisation with terminal cisternae + tubules that connect these terminal cisternae.

Question 9

What are microsomes and how are they obtaines?

Answer 9

Homogenizing the cells, ER is fragmented into vesicles into vesicles called miocrosomes that can be isolated by fractional centrifugation.

Microsomes are small fragments of the ER that are more easy to study.

Question 10

What are the 3 functions of the SER?

Answer 10

1. Detoxifying compounds such as pesticides and carcinogens (hepatocytes - cells of liver).
2. Synthesis of phospholipids and steroids (adrenocortical cells and leydig cells).
3. Ca2+ transport and accumulation.

Question 11

Where and how does detoxication take place?

Answer 11

The SER is highly developed in the liver cells and has the function of detoxification.

Any substance carried in the circulation can be rapidly acted on by the liver where the metabolism or ‘elimination’ of lipophilic substances such as drugs, anesthetics, pesticides, toxins, and hormones takes place.

Question 12

Where and how does glycerophospholipid take place?

Answer 12

In particular, this process starts on the cytosolic side.

Newly synthesised phospholipids transferred to bilayer on the lumen of the ER through flippase.

Question 13

Why is Ca2+ released in some situation?

Answer 13

Ca2+ release in the SER causes muscle cells contraction.

Ca2+ release from SER causes initiation of embryonic development:

Spermatozoa binds to PM and promotes the second messenger IP3, which binds to the receptor on lumen and releases Ca2+.

Question 14

What are the 4 functions of RER?

Answer 14

1. Biosynthesis of peptide chains intended to secretory vesicles, lysosomes and PM to allow sorting.
2. Correct conformation of proteins.
3. Assembly of multimeric proteins.
4. Initial phase of protein glycosylation.

Question 15

Describe the overall procedures of protein synthesis.

Answer 15

Starts with a mRNA that is translated to free ribosomes in the cytoplasm.

Signal sequence is formed and recognized by signal recognition particles.

On the surface of the ER, receptor for srp and budding from srp and receptor.

Ribosomes and mRNA are translocated at the level of RER.

Protein synthesis continues inside lumen.

Signal cleaved by specific peptide

Once translation completed, ribosome dissociate and move to cytoplasm.

Protein located inside the lumen of er and will go through other modifications.

Question 16

How are proteins conformed to their final structures?

Answer 16

3D shapes as bonding occurs between R groups. E.g. Hydrophobic interactions, ionic bonding, disulfide bridges, hydrogen bonding.

Question 17

What are chaperones? What is their function?

Answer 17

Chaperones are protein or protein complexes that allow the proteins to reach the correct secondary and tertiary structure necessary for their function:

If chaperone fails in function, the incorrectly folded proteins are destined to degradation in a structure called the proteasome, after being marked by binding to a small protein called ubiquitin, this marking mechanism is called ubiquitination.

Question 18

How does the proteasome function?

Answer 18

Complex of proteins found inside cytoplasm.

Misfolded protein attacked by several ubiquitin molecules.

Promote translocation of protein towards proteasome,
Once inside, it will be degraded in several aminoacids.

Released into the cytoplasm to be reused.

Possible with ATP.

Question 19

How does the initial phase of glycolysation proceed?

Answer 19

An oligosaccharide chain of 14 sugars (olio N-linked) is linked onto the protein synthesized in the RER.

The process will end in the golgi apparatus.

Question 20

Where and how does N-linked glysolysation take place?

Answer 20

The 1st phase takes place in RER after synthesis of the initial oligosaccharide chain on a lipid molecule, the dolichol phosphate, followed by the transfer of the chain on the asparagine of the new protein.

Question 21

Describe the structure of the Golgi Apparatus.

Answer 21

Made up of flattened membranous sacs called cisternae.

The side facing the nucleus is the cis facing side, the middle is the intermediate region and the side facing towards the cytoplasm is the trans side.

Vesicles arriving are called the cis golgi network, whilst vesicles coming out from trans side are called the trans golgi network.

Question 22

What is the function of the Golgi Apparatus?

Answer 22

• Receiving vesicles from the ER.
• Modifying the N-linked oligosaccharides of proteins.
• O-linked oligosaccharide synthesis and polysaccharides.
• Synthesis of sphingomyelin and glycolipids.
• String proteins to different destinations.

Question 23

Describe the vesicular shuttle model.

Answer 23

This implies that the proteins during modification from 1 cisternae to another, are transported through shuttling of this protein through vesicles.

The protein reaches the cis side and then will move towards the cisternae with continious formation of vesicle from one cisternae to the other.

Then reaches the trans golgi network and moves towards the membrane where it fuses with the pm and releases its content.

Question 24

Describe the cisternal maturation model.

Answer 24

The whole cisternae moves from cis to intermediate and then trans golgi network. Lastly it will disaggregate to form several vesicles which will move to its destination.

Question 25

How does the ER and GA sort proteins to their correct areas?

Answer 25

Transport vesicles are used to transport material from the ER to the GA.

The whole process is highly regulated by the cell, though there may be some errors, this can be recovered.

After the transport vesicles are used to transport material from the ER to the GA, they are then sent to their final destinations.

Some transmembrane proteins of these vesicles serve as receptors for the molecules to be concentrated and sorted in a specific final component.

Question 26

How do transport proteins bud?

Answer 26

A vesicle that buds from a donor membrane will have v group of proteins in its membrane. The vesicle buds are transported towards the target membrane. Here there is the t-SNARE that recognizes v-SNARE and will fuse these together and therefore the membranes will also fuse.

Question 27

Give an example of GA sorting and recovery.

Answer 27

Protein contains KDEL signal, which binds to KDEL receptors containing the KDXX signal.

If a ER-resident protein is missorted, this is identified by the KDEL sequence. The KDEL peptide interacts with the KDEL receptor and a second vesicle is formed to transport the bound protein and receptor back to the ER.

Due to a lower pH, the receptor unbounds with the peptide and is transported back to the ER.

Question 28

Describe exocytosis.

Answer 28

In almost all cells there is a constitutive exocytosis or secretion (no concentration material secreted).

In the regulated exocytosis, an extracellular signal leads the vesicles to fuse (accumulation of the material secreted).

In some cells (neurons, glandular epithelial cells) exocytosis can be polarized.

The microtubule cytoskeleton seems to direct the vesicles to the membrane.

Question 29

What is a constitutive pathway?

Answer 29

Continuous production of a small number of proteins that move from the GA towards the membrane.

Question 30

What is a regulated secretory pathway?

Answer 30

Accumulation of vesicles parked with proteins, once signal arrives (Ca2+), this determines fusion of vesicles with the PM.

Question 31

Describe the types of endocytosis.

Answer 31

Pinocytosis - endocytosis in the liquid phase, clathrin-independent.

Phagocytosis - receptor mediated endocytosis, clathrin independent.

Receptor- mediated endocytosis, clathrin independent

Question 32

Describe phagocytosis.

Answer 32

Formation of pseudopodia formed by filaments of actin.

Phagocytic vacuole or phagosome.

Question 33

Describe the process of clathrin dependent phagocytosis.

Answer 33

Clathrin works with adaptation.

Clathrin molecules coat vesicles to form hexagons and pentagons.

Structure of a triskelion and its assembly to form planar or curved gratings.

Then various types of adapter proteins allow the attachment of clathrin to the membranes, thus regulating the function.

Structure of a curved reticulum formed by triskelion of clathrin and complexes of adapter proteins.