The Endomembrane System and the Golgi Apparatus

Question 1

What is the endomembrane system?

Answer 1

Where proteins are further processed and transported to their final location
- The endomembrane system is intricately involved in the processes of transporting cargo out of or into the cell, termed the exocytic pathway and endocytic pathway

Question 2

What is the importancce of endomembrane system?

Answer 2

The endomembane system is critical for allowing transport of various cargo throughout, into, or out of the cell. The cell has produced many intracellular membranes or barriers to compartmentalize the activities of different organelles, as well as developed a difficult to permeate membrane, separating itself from the extracellular space.

These barriers are important to proper cellular functioning, but they can pose a problem for transport of cargo throughout the cell. While small molecules can more easily move around and in/out of the cell, the endomemvrane system provides a means by which the cell can transpot critical larger to various locations within the cell, remove unwanted cargo from within the cell, and attain necessary cargo from outside of the cell.

Question 3

What organelles are involved in the endomembrane system?

Answer 3

1. Endoplasmic Reticulum
2. Golgi Apparatus
3. Vesicles that are formed for transport between organelles
4. Endosomes, lysosomes, proteasomes and peroxisomes

Question 4

What are the sections of the Golgi apparatus?

Answer 4

• Cis Golgi
• Medial Golgi
• Trans Golgi

Question 5

Outline vesicle-mediated transport.

Answer 5

1. Small vesicles bud off from lipid membranes of organelles or the cell as a whole, with the purpose of transporting cargo, such as soluble proteins or membrane bound proteins
2. These vesicles can transport cargo into or out of the cell, or just to a different location within the cell
3. Once reaching their final destination, they fuse with other lipid membranes to deposit their cargo

Question 6

What are endosomes?

Answer 6

Endosomes hold cargo coming into the cell from the extracellular space through a process called endocytosis

Question 7

What are lysosomes?

Answer 7

Lysosomes break down protein, lipids, and sugars into their molecular building blocks.

Question 8

What are peroxisomes?

Answer 8

Peroxisomes break down molecules, specifically those that generate hydro peroxide as a by-product. This way, cells can neutralize hydrogen peroxide as it is produced to prevent cell damage.

Question 9

What are proteasomes?

Answer 9

A group of enzymes required for initiation of protein breakdown. This is an essential protective process in the cell - proteins that are no longer working properly need to be degraded so as to not cause damage.

Question 10

Outline the signal sequences for proteins destined for: endomembrane system, cytosol and secreted from the cell.

Answer 10

Protein in the Endomembrane System
- Proteins targeted to the ER, Golgi complex, endosomes, lysosomes, the plasma membrane or other organelles contain target signal sequences or are tagged within the endomembrane system. These proteins may eventually end up in various other areas of the cell, as transmembrane proteins, or in the extracellular space, depending on how they are tagged within the endomembrane system.

Proteins Destined for the Cytosol
Proteins destined for the cytosol lack signal peptides, since the free ribosomes will translate them in the cytosol.

Proteins that Need to be Secreted from the Cell
Proteins that are to be secreted out of the cell also have distinct signal sequences. These proteins can be produced by cells and are essential for body function, such as hormones.

Question 11

Outline protein transport into the ER.

Answer 11

1. Transport of proteins into the ER begins during translation. This is accomplished by the presence of a signal that is translated as part of the protein, termed the ER signal sequence.
2. This sequence interacts wth a receptor, termed the signal recognition particle (SRP), which binds to the ribosome that is translating the protein. This pauses translation. The binding of GTP simply indicated this is an energy consuming process.
3. During this pause in translation, the SRP docks onto the ER membrane by interaacting with a complex called th translocon, that allows and facilitates the translocation of the growing protein into the ER.
4. Translation restarts, and once the protein has translocated into the ER, the signal peptide sequence is cleaved off the protein, translation finishes, and the protein folds inside the ER lumen. The finished protein is destined to be soluble, and not attached to a membrane.

Question 12

How do proteins become embedded into the plasma membrane?

Answer 12

• The translocon for transmembrane proteins recognizes a signal that is similar to the ER signal sequence, termed the transmembrane signal anchor sequences
• This sequence not only acts as a signal to direct these proteins to the ER but will also be the transmembrane domain of the protein once it is a functional mature protein within a membrane

Question 13

What are the modifications of protein done by the ER?

Answer 13

Once in the lumen of the ER and folded, proteins are often modified in a process termed post-translational mofication. Post-translational modification is the covalent modification of the protein after translation.

These modifications may include:

• The addition of proteins, sugars, lipids and new functional groups ike phosphatess and methyl groups, which can change the final target location, structure or function of the protein
• The cleaving of the peptide bonds in the protein
• Most proteins have the signal sequence, which is typically located at the N-terminus removed

Question 14

What enzymes are involved in protein folding in the ER?

Answer 14

PDI - Protein Disulphide Isomerase
Folding of a protein is often facilitated by the formation of a disulfide bond between cysteine amino acids. Enzymes called protein disulfide isomerases help form disulfide bonds between cysteines in the protein to help it fold properly.

BiP - Binding Protein
Another protein, termed chaperonins, help fold the polypeptide by binding hydrophobic patches in recently translated proteins.

Question 15

Why do you think the cell does not allow cargo to free float between organelles?

Answer 15

Without vesicles, these organelles are isolated organelles without the ability to shuttle cargo between them. It would also make the cell unable to secrete cargo out of the cell.

Question 16

How does cargo get to its intended destination?

Answer 16

1a. Cargo Selection
Vesicles need to be packaged with the correct cargo. Signal sequences and receptors on the membrane help gather the required cargo to the area of the membrane that will be turned into a besicle. This process is important since the ability to accurately transpot cargo to the correct location allows the cell to be more efficient.

1b. Coat Proteins
Coat proteins play an important role in cargo selection. Coat proteins, orginally present in the cytosol, bind to the area on the membrane which will become the outside of the vesicle. These coat proteins cause receptors and other proteins with signal sequences to cluster on the surface of the membrane and then allow the vesicle to bud off from this area of the membrane.

2. Budding
   Next, the coat proteins interact with proteins termed cytosolic adaptor proteins. Together they form a mesh-like network that acts like a basket to pull the membrane into a bulging shape, which will become the vesicle.
3. Scission
   Scission requires another set of proteins to pinch the newly formed vesicle off the membrane
4. Uncoating
   Now that the coat and adaptor proteins have successfully created a vesicle, they are disassembled, uncoating the vesicle. This allows the cell to reuse the coat and adaptor proteins in the formation of another vesicle.
5. Transport
   Vesicles are typically attached to a type of protein, termed motor proteins, in the cytosol, which allow for the movement of vesicles throughout the cell.
6. Tethering
   Once the vesicle has reached its destination, the vesicle uses a tether protein to attach to a receptor on the accept membrane. This allows the vesicle to be close enough to try to find the correct acceptor target proteins.
7. Docking
   If the target proteins are found, the vesicle then docks on the surface, allowing for fusion of the vesicle with the membrane.
8. Fusion
   Dusion entails the lipid memvbrane becoming continous with that of the acceptor membrane. This allows the cargo contained within the vesicle to be exuded into the acceptor organelle.
9. Disassembly
   Finally, the remaining tether proteins and other receptors are disassembled and recycled back to the intial organelle.

Question 17

What are the three common coat proteins?

Answer 17

• The ER uses the protein COPII to shuttle to the Golgi
• Proteins that need to be returned to ER use COPI to coat vesicles that are recycled back to the ER
• Clathrin is used by the Golgi to form vesicles for exocytosis, endocytosis and transport to the endosomes

Question 18

What is the cis-Golgi network?

Answer 18

The cis-Golgi Network (CGN) receives proteins from the ER that have entered the enomembrane pathways.

• Within the CGN, sugar groups called oligosaccharides can be added to proteins
• Existing oligosaccharides can also be modified

Question 19

What is the trans-Golgi network?

Answer 19

The trans-Golgi Network (TGN) performs the final packaging to send materials to different organelles, and sorts cargo to specific destinations.

Question 20

How does the Golgi help vesicles get to their destination?

Answer 20

Vesicles are targeted to each of the following areas:
1. Other organelles: depending on the cell type, the different organelles in a cell will need various proteins. The TGN is where the cell packages these products to go to their final location. So, each cell will have very different requirements for their TGN.

2. Back to the ER: some proteins that have arrived from the ER contain an ER Retention signal. Once being processed by the GA, these proteins are packed up with COPI coating proteins and then recycled back to the ER. This is a common mechanism found in the cell, similar to how proteins in the nucleus return after exporting mRNA to the cytosol.
3. The Lysosomes: The mannose-6-phosphate ((M6P) receptors in the TGN recognize proteins with M6P tags, and send then to the lysosoome where the cell degrades proteins, DNA, lipids and sugars.
4. Outside of the Cell:
   This is accomplished by a process known as exocytosis.

Question 21

How does the cell regulate exocytosis?

Answer 21

1. Constitutive secretion is uncontrolled and always happening.
   Example: extracellular matrix proteins such as collagen, which are produced and excreted continuously.
2. Regulated secretion in cells required a signal to stimulate the cell to secrete it.
   - Cells often have specalized storage granules that are released in respose to specific signals
   Example: neurotransmitters in neurons, or insulin from the pancreas

Question 22

What do you think the role of exocytosis is in unicellular organisms? How is this different from the role in multicellular organisms?

Answer 22

In unicellular organisms, exocytosis can be used primarily to get rid of waste or to release protective agents, such as toxins. In multicellular organisms connected by blood vessels, exocytosis is necessary for cells to communicate via hormones, release nutrients for other cells, and cell-to-cell signalling.

Question 23

Outline the Endocytic Pathway.

Answer 23

1. Formation of the Endosome
   - Like in exocytosis, cargo that is being exported from outside the cell is transported into the cell via vesicles
   - Clathrin is a protein involved in vesicle formation during endocytosis, it initiates the invagination of the plasma membrane that will form vesicles that will take cargo to the early endosomes
   - The clathrin coat is disassembled by cytosolic proteins, whcih then cuases the vesicle to transport and fuse with the early endosome
2. The Early Endosome
   - Made up of vesicles from the plasma membrane and vesicles from the TGN
   - Once these vesicle fuse together, the cargo and receptors dissociate in the slightly acific early endosome
   - This acidic environment is formed by proton pumps that add protons to the interior of the endosome, lowering the pH, intitating dissociation of proteins
   - Cargo is retained in the endosome, while dissociated recepors are typically recycled back to the plasma membrane; some cargo and receptors may be transported elsewhere in the cell, depending on their signal sequence
3. The Late Endosome
   - THe early endosome matures to become the late endosome
   - Proton pumps that are present in vesicles arriving from the TGN contribute to the acidity of the interior of the endosome, lowing the pH of the endosome more
   - Continues recycling of receptors and cargo is performed
   - -> Cargo will be degraded as the pH continues to drop, this is similar to how the stomach uses acid to digest food, unfolding the proteins
   - -> Degredation of cargo in the endosome provides essential building blocks such as sugars and amino acids for repurposing
4. The Lysosome
   - When the pH becomes more acidic, enzymes including proteases are activated, leading to conversion to the lysosome
   - A protective layer of proteins forms to prevent the degregation of the organelle

Question 24

What happens with damaged and dangerous cargo?

Answer 24

Cells create waste products that need to be disposed of carefully.

Lysosomes and Proteasomes: Misfolded and damaged proteins undergo breakdown in a few carefully regulated systems

Peroxisomes: Dangerous free radicals, or reactive oxygen specias, are also problematic to the cell, and the cell needs a safe place to use these chemcials

Question 25

How do Lysosomes recive cargo?

Answer 25

Proteins that are misfolded or non-functional are tagged for delivery to the endosome.

Cargo is targeted to the lysosome by a specific mannose-6-phosphate (M6P) sugar tag.

Interestingly, the enzymes that degrade these proteins, or proteases, are also directed to the lysosome with the same M6P tag.

Question 26

Describe digestion in the lysosome.

Answer 26

The lysosome contain high concentrations of proteases, which cleave noth membrane proteins and proteins contained in the lysosome. The lysosome also contains enzymes that cleave and digest fats and sugars, and can even engulf other organelles, like damaged mitochondria. Once large molecules have been broken down into their basic parts, they are transported to the cytosol so the cell can reuse them. Lysosomes are mainly responsible for the breakdown of proteins that are not endogenous to the cell.

Question 27

Describe protein degredation by the proteasome.

Answer 27

Intracellular proteins from the cytosol and ER are degraded in the proteasome. Cytosolic proteins that have been misfolded or damaged are tagged with polyubiquitin chain. Ubiquitin is a small protein that is attached to another protein that has been selected for destruction. Multiple ubiquitin are required for the protein to be degraded. This process of ubiquination targets the protein for destruction in the proteasome. Ubiquination is another form of post-translational modification.

Proteasomes are also located in the nucleus so the cell can degrade unwanted nuclear proteins without have oto export them to the cytosol.

Question 28

Describe the function of Peroxisomes.

Answer 28

Peroxisomes are small, membrane-enclosed organelles that contain enzymes that catalyze a variety of metabolic reasons. Essential peroxisome proteins called peroxins, synthesized in the cytosol, are targeted to the peroxisome by specific peroxisomal targeting signals (PTS).

Oxidizing agents like peroxie are very hazardous to the cell. Peroxisomes serve as a place to keep and use these reactive oxygen species safely. These nclude ions free radicals and peroxides. Although they are hazardous, they carry out important ecomposing functions for some cargo such as uric acid, amino acids and long chain fatty acids.