WEEK 7 (Vesicular transport) Flashcards
What are the three mechanisms in which proteins are transported into organelles?
- transport through nuclear pores
- transport across membranes
- transport by vesicles
Define Protein targeting/Protein sorting
the biological mechanism by which proteins are transported to their appropriate destinations in the cell or outside it
What does the fate of any protein molecule synthesised in the cytosol depend on?
Its amino acid sequence which contains a sorting signal that directs the protein to the organelle in which it is required
What is the function of transport vesicles?
Transport vesicles bud from one membrane and fuse with another, carrying membrane components and soluble proteins between compartments of the end-membrane system and the plasma membrane
What happens in the outward secretory pathway?
Protein molecules are transported from the ER, through the Golgi apparatus to the plasma membrane or to lysosomes
What happens in the inward endocytotic pathway?
Extracellular molecules are ingested (endocytosed) in vesicles derived from the plasma membrane and are delivered to early endosomes and to lysosomes via late endosomes
Why are vesicles that bud from membranes called coated vesicles?
Since they usually have a distinctive protein coat on their cytosolic surface
What happens to the vesicles after budding from its parent organelle?
The vesicle sheds its coat which allows its membrane to interact directly with the membrane to which it will fuse
What are the functions of the vesicular coat?
- helps shape the membrane into a bud
- captures molecules for onward support
Describe how Clathrin protein coat molecules help shape membranes into vesicles
Clathrin protein coat molecules form basketlike cages that help shape membranes into vesicles
What are Clathrin Triskelions?
The basic subunits of the Cathrin coat and are composed of 3 heavy chains and 3 light chains
What is Dynamin?
A small GTP-binding protein which assembles as a rib around the neck of each deeply invaginated coated pit
What is the function of Dynamin?
Together with other proteins recruited to the neck of the vesicle, the dynamin causes the ring to constrict which pinches off the vesicle from its parent membrane
Describe the stages of cargo molecules budding off into vesicles
1) Cargo receptors with their bound cargo molecules are captured by ADAPTINS which also bind Clathrin molecules to the cytosolic surface of the budding vesicle
2) Dynamin proteins assemble around the neck of budding vesicles
3) With the help of other proteins recruited to the neck of the vesicle, Dynamin causes the vesicle to pinch off
What do the different types of adaptins reflect?
The differences in the cargo molecules to be transported
What is the function of COP-coated vesicles?
COP-coated vesicles are involved in transporting molecules between the ER and the Golgi apparatus and from one part of the Golgi apparatus to another
Each type of transport vesicle carries a unique combination of ______ ____________ which serve as molecular markers for each membrane type
Rab proteins
Describe the stages of Vesicle docking
1) TETHERING - A filamentous tethering protein on a membrane binds to a rab protein on the surface of a vesicle (this interaction allows the vesicle to dock on its particular target membrane)
2) DOCKING - A v-SNARE on the vesicle then binds to a complementary t-SNARE on the target membrane
3) FUSION - The SNARE proteins catalyse the final fusion of the two membranes
What is the difference between the functions of Rab and tethering proteins and complementary SNARE proteins?
Rab and tethering proteins provide the initial recognition between a vesicle and its target membrane
Complementary SNARE proteins ensure that transport vesicles dock at their appropriate target membranes
What must occur for fusion to happen?
The two bilayers must come within 1.5nm of each other so that their lipids can intermix
For this close approach, water must be displaced from the hydrophilic surfaces of the membranes - a process that is energetically highly unfavourable ad thus prevents membranes from fusing randomly
Why must all membrane fusions in cells be catalysed by specialised proteins that assemble to form a fusion complex?
Since the close approach that can only occur when water is displaced from the hydrophilic surfaces of the membranes is highly energetically unfavourable therefore the fusion complex is required to cross the energy barrier
What catalyses the fusion process?
SNARE proteins
What happens once fusion is triggered?
- the v-SNARES and t-SNAREs wrap around each other
- after fusion, the SNAREs are pried apart so that they can be used again
What happens to the majority of proteins that enter the ER lumen or ER membrane?
they are converted to glycoproteins
What are the key concepts of Glycosylation in the endoplasmic reticulum?
- The oligosaccharide is originally attached to a specialised lipid called DOLICHOL in the ER membrane
- Many proteins are glycosylated on ASPARAGINES in the ER
- When an appropriate asparagine enters the ER lumen, it is glycosylated by addition of a branched oligosaccharide side chain
- Each oligosaccharide chain is transferred as an intact unit to the asparagine from a lipid called dolichol, catalysed by the enzyme OLIGOSACCHARYL TRANSFERASE
What do proteins that function in ER have?
ER retention signal
How does the ER control the quality of proteins that it exports to the Golgi apparatus?
Proteins that fold incorrectly or multi-metric proteins that fail to assemble properly are retained in the ER and bind to chaperone proteins
Chaperone proteins hold proteins in ER until proper folding occurs; if it doesn’t occur, proteins are degraded
Where are antibody molecules synthesised?
Endoplasmic Reticulum
Describe the causes and symptoms of Cystic Fibrosis
Cystic fibrosis is a genetic disorder that affects mostly the lungs, pancreas, liver, kidneys and intestine and is caused by a mutation that produces a plasma-membrane transport protein that is slightly misfolded.
SYMPTOMS:
- difficulty breathing
- coughing up mucus as a result of frequent lung infections
- sinus infections
- poor growth
- fatty stool
- clubbing of fingers and toes
- infertility in some males
Where is the mutation that causes Cystic Fibrosis found?
CFTR gene = cystic fibrosis transmembrane conductance regulator gene
CFTR gene provides instructions for making a channel that transports CHLORIDE IONS into and out of cells. The most common mutation is a deletion of three nucleotides that results in a loss of the amino acid PHENYLALANINE. Mutation is the long (q) arm of chromosome 7.
How is Cystic Fibrosis diagnosed?
- diagnosis requires clinical symptoms consistent with CF in at least one organ system
- evidence of CFTR dysfunction based on an abnormal sweat chloride test
- presence of mutations in the CFTR gene
- newborn screening (no clinical symptoms required fro infants)
What is the most commonly used test for diagnosing cystic fibrosis and how does it work?
THE SWEAT CHLORIDE TEST which checks for increased levels of salt in the sweat
1) A chemical is used to make the skin sweat when triggered by a weak electric current
2) Sweat is collected on a pad or paper and then analysed
3) A diagnosis of cystic fibrosis is made if the sweat is saltier than normal
How is cystic fibrosis treated?
There is no cure but there are therapeutic treatments
- Antibiotics
- Mucus-thinning medications
- Bronchodilators
(relax the muscles around the trachea which helps increase air flow. Can be taken through an inhaler or nebuliser)
- Cystic fibrosis transmembrane conductance regulator (CFTR) modulators
(class of drugs that improve the function of the defective CFTR gene)
What does the accumulation of misfolded proteins in the ER lumen trigger?
An unfolded protein response (UPR)
- program prompts the cell to produce more ER, more chaperones and other proteins concerned with quality control
- misfolded proteins are recognised by several types of transmembrane sensor proteins in the ER membrane, each of which activates a different part of the UPR
- some sensors stimulate the production of transcription regulators that activate genes encoding chaperones or other proteins of the ER quality control system; another sensor inhibits protein synthesis, reducing the flow of proteins through the ER
What happens when the expanded ER cannot cope?
UPR directs the cell to self-destruct by undergoing apoptosis
What happens to many of the oligosaccharide chains that are added to proteins in the ER?
They undergo further modifications in the Golgi apparatus
Describe the entering & exiting of proteins in the smooth endoplasmic reticulum
Proteins enter at Cis-face: move through and exit Golgi or return to ER
Proteins exit at Trans-face: transported to plasma membrane or lysosomes
What is the most extensive membrane system in a eukaryotic cell?
Endoplasmic Reticulum
What are key characteristics of the endoplasmic reticulum?
- entry point for proteins destined for other organelles as well as the ER itself
- proteins destined for the Golgi apparatus, lysosomes, endosomes & cell surface all first enter the ER from the cytosol
- once in the ER, proteins do not return to the cytosol but rather travel via vesicles
- proteins enter the endoplasmic reticulum while being synthesised
What happens to ribosomes that are translating proteins with no ER signal sequence?
they remain free in the cytosol
What happens to the ribosomes at the end of each round of protein synthesis?
The ribosomal subunits are released and rejoin the common pool in the cytosol
What are the two protein components that help guide the ER signal sequences to the ER membrane?
- A SIGNAL RECOGNITION PARTICLE (SRP)
(present in the cytosol, binds to both the ribosome and the ER signal sequence when it emerges from the ribosome) - SRP RECEPTOR
(embedded in the ER membrane, recognises the SRP)
Describe the stages of the transfer of soluble proteins into the ER lumen
1) an SRP particle in the cytosol binds to both the ribosome and the ER signal sequence when it emerges from the ribosome
2) an SRP receptor embedded in the ER membrane recognises the SRP
3) The SRP-ribosome complex binds to an SRP receptor in the ER membrane
4) The SRP is released, passing the ribosome from the SRP receptor to a protein translocator in the ER membrane
5) Protein synthesis resumes and the translocator starts to transfer the growing polypeptide across the lipid bilayer
What is the function of the protein translocator?
It binds to the signal sequence and threads the rest of the polypeptide across the lipid bilayer as a loop
Describe what happens to the signal sequence that is released into the ER lumen
1) Protein translocator binds to the signal sequence and threads the rest of the polypeptide across the lipid bilayer as a loop
2) During the translocation process, the signal peptide is cleaved from the growing protein by a signal peptidase
3) Cleaved signal sequence is ejected into the bilayer where it is degraded
4) Once the C-terminus of a soluble protein has passed through the translocation channel, the protein will be released into the ER lumen and the pore of the translocation channel closes
What are the three different types of endocytosis?
- PINOCYTOSIS = involves the ingestion of fluid and molecules via small pinocytotic vesicles
- PHAGOCYTOSIS = involves the ingestion of large particles such as microorganisms and cell debris
- RECEPTOR-MEDIATED ENDOCYTOSIS
What is an example of Receptor-mediated endocytosis?
The ability of animal cells to take up the cholesterol they need to make new membrane
The Low-Density Lipoprotein Receptor (LDL-R) mediates the endocytosis of cholesterol-rich LDL
Whether it’s occupied or not, an _______ receptor typically makes one round trip into the cell and back every 10 mins, making a total of several hundred trips in its 20-hour life-span
LDL
Most extracellular material taken up by pinocytosis is rapidly delivered to ________________
endosomes
The fate of receptor proteins following their endocytosis depends on what?
The type of receptor
Describe the three pathways from the endosomal compartment
- most are returned to the same plasma membrane domain from which they came
- some travel to lysosomes, where they are degraded
- some proceed to a different domain of the plasma membrane, thereby transferring their bound cargo molecules across the cell from one extracellular space to another, a process called transcytosis. Late endosomes contain some lysosomal enzymes, so digestion of cargo proteins and other macromolecules begins in the
endosome and continues as the endosome gradually matures into a lysosome.
Which molecule is displaced when a vesicle and its target membrane fuse?
Water
Why can’t the oligosaccharides on glycosylated proteins be used as a source of energy for the cell?
Since the oligosaccharides aren’t free
