Membrane trafficking

Question 1

Outline different types of intrecellular transport; types of ribosomes and their characteristics

Answer 1

Types of intracellular transport:

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)

2 types of ribosome in cell:

Free: Makes proteins for use within cell e.g. nuclear protein

Bound to ER membrane: Makes protein for use outside the cell (i.e. secretion) or within plasma membrane or lysosome

Ribosomes bound to the ER membrane make all the proteins that are being translocated into the ER.

Proteins destined for other organelles are first imported into the ER

• Free ribosomes, and ribosomes bound to membrane are the same
• They start in the cytosol in a ‘common pool’.
• Then they get some mRNA, and start translating it
• The protein being made has a signal peptide/signal sequence/localisation signal which directs the protein to the correct area in the cell

Essentially, when the ribosome starts translating any mRNA strand it is in the cytoplasm.

Signal sequences behave like address labels and direct the protein to the correct compartment

1. Signal peptide directs ribosome to ER
   * The ribosome only becomes bound to the rER if the protein it is translating has a signal peptide specific to the rER. In this case, translation halts, and the ribosome moves to the ER membrane, and then begins translation again, with the protein being produced then being directed into the ER lumen, as translation occurs. These proteins are then modified, packaged and secreted, or used in the plasma membrane or the lysosomes.
2. No signal peptide
   * If there is no signal peptide on the protein being produced then the ribosome will remain in the cytoplasm
3. Specific signal peptide NOT for ER, but for different cell compartment (relocated)
   * As well as signal peptides for the rER, there are compartment specific signal peptides. For example, the ribosome, whilst still in the cytoplasm, may translate a protein with a signal peptide which shows the protein is for the nucleus. The ribosome stays in the cytoplasm, but when the protein has been produced, this protein then moves to the nucleus, and enters it. There are also signal peptides for mitochondria and some other compartments.

If you just had one ribosome translating each mRNA, it would be far too slow

• Instead, multiple ribosomes bind to the mRNA, so lots of translation happens at the same time
• Called a polyribosome

Proteins are modified in the ER: Post-Translational Modifications and Quality Control

Proteins which are incorrectly folded are blocked from ER exit, or exported in cytoplasm and degraded

Cystic fibrosis (CF): example of disease due to misfolding of protein

• 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 2

Concepts: explain the terms “exocytosis” and “endocytosis”, define “constitutive” and “regulated” secretion

Answer 2

The type of ribosome that translates proteins destined for secretion or plasma membrane is Ribosomes bound to rER

Eukaryotic cells continually import materials by endocytosis and secrete intracellular materials by exocytosis.

Material transported by endocytosis is captured from the external medium.

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

Exocytosis: How proteins get from the rER to the membrane outside the cell

2 types of secretion

CONSTITUTIVE:

• ALL cells
• Steady stream of vesicles from TGN, fusing with plasma membrane
• Constitutive secretion is mostly to replenish membrane

REGULATED:

• Excitable cells
• Products concentrated and stored in secretory vesicles until a signal stimulates secretion

Endocytosis:

Vesicles containing proteins which have been modified in the ER bud from ER membrane, into a vesicle.

This vesicle fuses with the golgi apparatus

Proteins, after further modification in Golgi, bud from the Golgi membrane and then fuse with the plasma membrane for secretion. Alternatively, the protein might stay in the membrane, or be sent to lysosome instead of secretion.

Question 3

Exocytosis: summarise the pathway and cellular locations for synthesis, post-translational modification and exocytosis of a secreted protein

Answer 3

Outward secretory pathway

1. proteins are transported from the ER through the Golgi to the plasma membrane
2. proteins transported via endosomes to lysosomes

• The Golgi stack has two distinct faces;
  
  1. entry face (or cis face) next to the ER (Soluble proteins and membrane enter the cis Golgi network via transport vesicles derived from the ER)
  2. an exit, or trans face points towards the plasma membrane
• Proteins are transported through the Golgi from cis to trans, and are sorted further at the trans Golgi network.

The secretory or EXOCYTIC pathway:

1. ER
2. Golgi
3. Trans-Golgi network (TGN)
4. Plasma membrane

Steps:

1. Cargo Sorting and Vesicle Formation
2. Vesicle Movement
3. Vesicle Tethering/ Docking
4. Vesicle Fusion

Vesicles that bud off from membranes have a distinctive protein coat on their cytosolic surface and are therefore called coated vesicles. After budding the coat is shed, allowing the vesicle to interact with the membrane to which it will fuse.

Clathrin-coated vesicles have an outer coat made up of the protein clathrin. They bud from the trans-Golgi network on the outward secretory pathway and from the plasma membrane on the inward secretory pathway.

Question 4

Endocytosis: summarise the process of receptor-mediated endocytosis and the roles played by endocytic vesicles, early endosomes, late endosomes and lysosomes

Answer 4

Endocytosis:

• Vesicles containing proteins which have been modified in the ER bud from ER membrane, into a vesicle.
• 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.
• This vesicle uses with the golgi apparatus
• Proteins, after further modification in Golgi, bud from the Golgi membrane and then fuse with the plasma membrane for secretion. Alternatively, the protein might stay in the membrane, or be sent to lysosome instead of secretion.

Material is brought into the cell: 3 types

1. Receptor mediated endocytosis (e.g. the LDL receptor!)
2. Pinocytosis (fluid intake)
3. Phagocytosis (fluid or particles like microbes)

Mechanism (image):

• During endocytosis, membrane invaginates, and pinches off, forming a vesicle containing the endocytosed substance
• Also surrounded by basket like protein called clathrin. This is removed after vesicle formation
• Dynamin helps to pinch off vesicle from membrane

example of Receptor-mediated endocytosis: Low Density Lipoprotein Receptor (liver) (image)

Question 5

Molecular mechanisms: summarise the molecular mechanisms of vesicular transport within cells, and the mechanisms underlying diseases that affect membrane trafficking

Answer 5

Gated transport
Free ribosomes are destined for the nucleus translated in

Import receptors around the nucleus recognise nuclear localisation signals (=signal peptides) on nuclear proteins

Proteins and other substances move in and out of the nucleus via nuclear pores, which act as gates, with mesh-like structures

Vesicular transport

• Proteins move between organelles in vesicles carried on microtubules
• Vesicle buds from one organelle, travels on microtubule, then fuses with another organelle
• Membranes need to be 1.5nm apart to fuse
• Vesicle Tethering/Docking proteins bring vesicle and target membrane together

Diseases involved in membrane transport

1. Disease of endocytosis: Familial Hypercholesterolaemia
   * LDL receptor not produced or not functional, cannot removed LDL (bad cholesterol) from blood
2. Disease of exocytosis: Cystic fibrosis

• Faulty CFTR protein produced due to ∆F508 mutation, so is degraded
• Chloride channels affected, leading to CF

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