Metabolism 8- Membrane Trafficking

Question 1

How does the lysosome appear on an EM

Answer 1

Opaque

Question 2

What is exocytosis

Answer 2

In exocytosis vesicles from inside the cell fuse with the plasma membrane to release their contents into the external medium.

Question 3

What is endocytosis

Answer 3

Material transported by endocytosis is captured from the external medium.

Question 4

Describe, simply, how proteins are sorted

Answer 4

The fate of the protein molecule depends on whether it has a sorting signal sequence attached. which directs the protein to the organelle where it is required. If this sorting signal is not present then the protein will remain in the cytosol. Different sorting signals are required to direct the proteins to different organelles.

Question 5

How do we know the importance of these signal molecules

Answer 5

Genetic engineering allows us to add signal molecules to proteins that would normally reside in the cytosol, experiments show that with the signal sequence attached these proteins move to a specific organelle

Question 6

What are the 3 types of intracellular transport

Answer 6

1. Gated transport (e.g. nuclear import)
2. Transport across membranes (e.g. import of newly synthesized proteins into ER, import of proteins into mitochondria)
3. Vesicular transport (e.g. inter-organellar transport)

Question 7

Describe how proteins enter the nucleus.

Answer 7

Nuclear envelope is perforated by nuclear pores that form gates through which molecules enter or leave. Some pore components form mesh-like structures that prevent passage of large molecules but allow small, water soluble molecules to pass freely.
Nuclear proteins are imported by so-called import receptors that recognise nuclear localisation signals as the ‘address label’- address label removed.

Cytosolic fibrils bring the protein to the receptor.
The signal sequence is called a nuclear localisation sequence, typically contains positively charged lysines or arginines.
Nuclear import receptors disrupt the mesh of proteins in the pore, by grabbing repeat sequenced within the mesh.
Bumps from one repeated sequence to the next until it enters the nucleus. the cargo is delivered, the receptor returns to the cytosol.

Question 8

Why does nuclear import require energy

Answer 8

Order is created, GTP hydrolysis provides the energy, catalysed by GTPase Ran.

Question 9

Where are the two populations of ribosomes within the cell found

Answer 9

There are two populations of ribosomes in cells.

Free in cytosol

OR

Bound to ER membrane

Both types of ribosomes are structurally and functionally identical. They originate from a common pool of ribosome subunits and differ only in the type of protein they are making at any given time.

Question 10

What is the difference between the proteins in which cytosolic and ER ribosomes synthesise

Answer 10

Ribosomes bound to the ER membrane make all the proteins that are being translocated into the ER.
Free ribosomes are unattached to any membrane and make all of the other proteins encoded in nuclear DNA.

Question 11

What happens to the ribosome once it has finished translating a protein on the ER membrane

Answer 11

At the end of each round of protein synthesis, the ribosomal subunits are released and rejoin the common pool in the cytosol

Question 12

How do the proteins enter the ER

Answer 12

Ribosome is passed to a protein translocator on the ER membrane, where translation recommences. The signal sequence if found at the N-terminus ( the end synthesised first), where it opens the translocator, and the polypeptide is threaded through as it is synthesised. It is cleaved by a transmembrane signal peptidase, the signal sequence is then degraded.

Question 13

What is the destination for proteins in the ER

Answer 13

Stay in ER

Golgi– vesicles

Question 14

Describe post-translation modifications in the ER

Answer 14

Modifications:
Folding
Formation of disulphide bonds
- Initial glycosylation (addition of sugars)
- Specific proteolytic cleavages
- Assembly of multimeric proteins
Unassembled or misfolded proteins are retained in the ER and exported back into the cytosol where they are degraded
Many diseases result from blocked ER exit due to misfolding, e.g. Cystic Fibrosis

Question 15

Why don’t disulphide bonds form in the cytosol

Answer 15

The environment there is reducing

Question 16

What is the purpose of post-translational modification in the ER

Answer 16

The proteins will encounter changes in pH and degradative enzymes in the cytosol- need to be stabilised
They can hold the protein in the ER until it is correctly folded
They can guide it to the appropriate organelle.

Question 17

Describe the role of misfolded proteins in Cystic Firbrosis

Answer 17

ABC transporter-class chloride channel in epithelial cell plasma membranes
Mutations of the CFTR gene affect functioning of the chloride channels in the membrane, leading to CF
The most common mutation (ΔF508) results from deletion (Δ) of three nucleotides which causes loss of the phenylalanine (F or Phe) at the 508th position on the protein
As a result, the CFTR does not fold normally and is degraded

Question 18

What is SMED-SL

Answer 18

example of disease due to misfolding of protein.
Spondylo-meta-epiphyseal dysplasia with short limbs and abnormal calcifications
DDR2 is a cell surface protein kinase receptor for collagens

Question 19

Describe inward exocytosis pathways

Answer 19

In the inward endocytic pathway (green arrows) extracellular molecules are ingested (endocytosed) in vesicles derived from the plasma membrane and are delivered to endosomes and lysosomes.

Question 20

Describe exocytosis pathways

Answer 20

Vesicular transport between membrane-bound organelles is highly organised. In the outward secretory pathway (red arrows) proteins are transported from the ER through the Golgi to the plasma membrane, or via endosomes to lysosomes.

Question 21

Describe transport from the ER to Golgi to vesicles

Answer 21

Transport from the ER to the Golgi and from the Golgi to other compartments is carried out by budding and fusion of transport vesicles. This transport system extends outwards from the ER to the plasma membrane, and inward from the plasma membrane to lysosomes.

Question 22

What is the function of the protein coat surrounding vesicles.

Answer 22

It helps to shape the membrane into a bud

Captures molecules for onward transport.

Question 23

What is an example of a protein coat

Answer 23

Clathrin. Clathrin-coated vesicles bud from the Golgi body in the outward secretory pathway and from the plasma membrane in the inward endocytic pathway.

Question 24

How is the clathrin coat formed

Answer 24

The vesicle starts as a clathrin coated pit during invagination.
Clathrin assemles into basket-like network, starting to shape membrane into vesicle
A small GTP binding protein called dynamin froms a ring around the invaginated pit, causing the vesicle to pinch from the plasma membrane.
The clathrin coat is then degraded, allowing the vesicle to interact with its target organelle.

Question 25

How do vesicles carry the correct cargo proteins

Answer 25

Molecules for outward transport are recognised by cargo receptors, adaptins capture specific cargo by trapping the cargo receptors that select them. Different adptins for the transport of different cargo

Question 26

Describe the steps in vesicular transport

Answer 26

Cargo Sorting and Vesicle Formation- donor compartment membrane
Vesicle Movement- along actin and microtubules
Vesicle Tethering/ Docking
Vesicle Fusion- Acceptor compartment membrane.

Question 27

Describe the two requirements for membrane fusion

Answer 27

Membranes need to be 1.5nm apart to fuse

Vesicle Tethering/Docking proteins bring vesicle and target membrane together

Question 28

Describe the process of membrane docking

Answer 28

The surface of vesicles have a family of GTPases called rab proteins. These are recognised by thethering molecules on the cytosolic side of the target membrane. Rab proteins are specific to each organelle. Thethering proteins are specific to each membrane.
Transmembrane proteins called SNAREs bind to v-SNAREs, firmly docking the vesicle in place.

Question 29

Why are SNAREs important

Answer 29

For the bilayers to fuse, water must be displaced from the hydrophilic surfaces of the membranes, an energetically unfavourable process. The SNAREs catalyse this process by wrapping around each other, acting like a winch that pulls the two lipid bilayers into close proximity.

Question 30

What are the three types of endocytosis

Answer 30

Receptor-mediated endocytosis

Pinocytosis (fluid phase)

Phagocytosis (fluid / particles e.g. microbes)

Question 31

Describe the transport of proteins in the Golgi body

Answer 31

The Golgi stack has two distinct faces, an entry face (or cis face) and an exit, or trans face. The cis side is next to the ER, while the trans face points towards the plasma membrane. Soluble proteins and membrane enter the cis Golgi network via transport vesicles derived from the ER.

Proteins are transported through the Golgi from cis to trans, and are sorted further at the trans Golgi network.

Question 32

Describe the further modifications that take place in the Golgi body

Answer 32

Many of the sugar chains that are added in the ER undergo further modifications in the Golgi. On some proteins, more complex oligosaccharide side chains are added and removed by a series of enzymes that reside in sequence as the protein passes through the Golgi stack.
Cis cisterna- Mannosidase I
Medial Cisterna- Mannosidase II, GlcNAc transferase I

Trans cisterna- GAL transferase, NANA transferase
Golgi body determines whether the proteins are destined for lysosomes, plasma membrane

Question 33

What is the difference between constitutive secretion and regulated secretion

Answer 33

Constitutive- all cells- Steady stream of vesicles buds from the trans-Golgi network and fuses with the plasma membrane
Regulated- specialised cells- The products are concentrated and stored in secretory vesicles until an extracellular signal stimulates their secretion

Question 34

Describe the molecules in endocytosis

Answer 34

Early endosome
Late endosome
Lysosome OR cargo unpackaged and used in cell.