Epithelia Polarity and Protein Trafficking Flashcards
What is polarity?
A difference in structure, composition or function between the two poles of a cell, such as apical/basolateral in an epithelial cell, axon/dendrites in a neuron.
In epithelial cells this also means location of a protein in a specific location (e.g., apical or basolateral) in the cellular membrane.
Are all cells polar?
Yes
What are the two distinct domains that epithelial cells develop?
Apical domain
Basolaterial domain
What are the steps taken to establish polarised epithelium?
1) Interactions between neighbouring cells and between cells and basement membrane
2) Adherens junctions form
3) Activation of small GTP proteins to form polarity complexes.
4) Formation of tight junctions
5) Positioning of the three protein polarity complexes (PAR / CRB / SCRIB)
What connects cells to basal lamina?
Hemidesmosomes
What is cell-cell interaction between?
Tight Junctions, Adherens Junction and Desmosomes
What occurs in order for the epithelial cells to form epithelium (e.g., during wound healing)?
Stem cells divide - new cells form contract with basal lamina though hemidesmosomes - then new cells form intercellular connections with neighbouring epithelial cells, helping to form an enlarged epithelium/fill gap.
What does the formation of adheren junctions initiate?
Epithelia formation
What makes the initial cell to cell contact in the formation of adherens junctions?
Nectin proteins
What does two e-cadherins on neighbouring cells form?
homodimer
What does the formation of a homodimer require?
Ca2+
What does the cytoplasmic tail of E-cadherin bind to?
Alpha Catenin
What links E-cadherin to actin cytoskeleton?
Alpha catenin
What does alpha catenin link to and what does the link form?
Alpha catenin links E-cadherin to actin cytoskeleton and also links nectin and cadherin complexes.
What are the small GTP binding proteins?
cdc42 and RAC1
what does aPKC do?
initiate enzyme activating and can phosphorylate target proteins
What are the three polarity complexes?
PAR (Partitioning defective)
CRB (Crumbs proteins)
SCRIB (Scribble proteins)
What are the three functions of a tight junction?
Barrier
Gate
Fence
What do tight junction components recruit?
Polarity protein complexes (PAR, CRB, SCRIB)
What is the position of PAR complex?
Apical near TJ
What is the positioning of the CRB complex?
Apical near TJ
What is the positioning of the SCRIB complex?
basolateral
What does the positioning of the protein polarity complexes create?
Polar epithelium
What has to be pulled apart for cell division?
Polarity complexes (then they are reformed so they have to be able to change rapidly)
If you inhibit the PAR complex what happens?
TJ falls apart
What does transcription of Snail, Zeb and Twist cause?
Reduction of Claudius, occludins and JAMS
What is Epithelial Mesenchymal Transition (EMT)?
The transition from high differentiated cells (epithelium) to undifferentiated cells (mesenchymals).
The epithelial–mesenchymal transition is a process by which epithelial cells lose their cell polarity and cell–cell adhesion, and gain migratory and invasive properties to become mesenchymal stem cells; these are multipotent stromal cells that can differentiate into a variety of cell types. EMT is the process that causes cancer metastasis.
Do polarity complexes play a role in EMT?
Yes
What does TGF-beta bind to?
TGF receptor
What two things happen when TGF-beta binds to the TGF receptor?
Activation of SMAD proteins (transcription factor)
and
Downregulation of PAR complex
What does the activation of SMAD proteins cause?
Down-regulation of E-cadherin, occludins, ZOs and claudins
What two things cause EMT?
Down-regualtion of PAR complex and down-regualtion of E-cadherin, occludins, ZOs and claudins
What four things does a mutation in polarity complex proteins cause?
1) Decreased formation or lack of tight junctions so barrier, gate and fence function compromised
2) changes in cell-cell adhesion and cell movement
3) changes in location of apical and basolateral proteins
4) cancer
What happens when epithelia are fully polarised with AJ and TJ ?
inhibit cell division pathway and promotes differentiation (epithelia takes on specialised function)
Trafficking requires proteins that are…
In the right place and right form/orientation
What is meant by the topology of plasma membrane proteins?
The orientation with respect to the membrane
What alters protein trafficking pathways by increasing or reducing cell surface population disrupting ion transport and physiology?
Genetic polymorphisms or mutations
In Liddle’s syndrome ENaC endocytosis is inhibited causing what?
Severe hypertension
When is caused by very little deltaF508-CFTR reaching the apical membrane?
CF (cystic fibrosis)
What occurs in the ER?
production/protein synthesis
What occurs in the Golgi apparatus?
“post office” - proteins received from the ER are further processed and sorted for transport to their eventual destinations.
What is a hydrophobic signal sequence?
A particualr sequence (amino acid address label) that determines where a protein goes.
What do proteins in the secretory pathway contain to ensure they are taken to the correct location?
A hydrophobic signal sequence located at the N-terminus or further into the protein
What is the address label of proteins localised in the ER?
“KDEL”. Standing for lysine/aspartic acid/glutamic acid/leucine
Where can address labels send proteins?
Nucleus
ER
Mitochondria
Lysosomes
Peroxisome
What directs proteins to the ER?
Hydrophobic signal sequence
How does a hydrophobic signal sequence direct a protein to the endoplasmic reticulum (ER) for secretion out of the cell?
1) Protein Synthesis Begins: Translation starts at the ribosome, which produces the protein. The first part of this protein contains a signal sequence.
2) Signal Sequence Recognition: The signal sequence, typically a hydrophobic segment, is recognized by the signal recognition particle (SRP). The SRP binds to the signal sequence and temporarily halts further translation.
3) Targeting to the ER: The SRP then guides the ribosome-protein complex to the ER membrane, where it binds to the SRP receptor.
4) Translocation into the ER: Once docked, the ribosome is transferred to a protein channel called a translocon in the ER membrane. The SRP is released, and protein synthesis resumes, with the growing polypeptide being threaded through the translocon into the ER lumen.
5) Signal Sequence Cleavage: As the protein enters the ER, the signal sequence is cleaved off by a signal peptidase.
6) Protein Maturation and Completion: The protein continues to be synthesized and enters the ER lumen, where it may undergo additional folding, modification, and maturation. Once synthesis is complete, the ribosome dissociates.
7) Final Protein Processing: The cleaved signal sequence is degraded, and the mature protein is prepared for eventual secretion out of the cell.
Where is the N-terminus?
Extracellular (ER)
Where is the C-terminus?
Intracellular (cytosol)
How many transmembrane domains does the Na+/K+-ATPase alpha subunit have?
10
How many transmembrane domains does the CFTR have?
12
What type of environment is provided by a tranlocon?
Hydrophobic
What is the difference between the hydrophobic regions of a protein with 1 and 2 transmembrane domains?
In a protein with 2 transmembrane domains the start-transfer sequence is not cleaved off
What is the common feature between ENaC, CFTR and Na+K+-ATPase?
All glycosylated (N-linked) - means the addition of a sugar
What is the benefit of N-linked glycoslylation/addition of sugars?
Aids protein folding and stability
Allows ENaC to interact with extracellular matrix
What are the abnormalities in glycosylation linked to?
Cancer and Rheumatoid arthritis
What are the post translational modification in the ER?
N-linked Glycosylation
Addition of GPI anchors
Folding and assembly
What can glycosylphosphatidylinositol (GPI) replace?
They may replace transmembrane domains
Where are GPI’s?
On apical membrane
What do GPI do?
Hold proteins close to the membrane
Where are disulphide bridges/bonds formed?
a, beta and yENaC subunits
What does the chaperone protein do?
Determine if a protein is incorrectly folded or misassembled - if so the chaperone protein sends it for ERAD degradation
What subunits does Na+K+-ATPase assemble into?
alpha plus Beta complexes
What is ERAD?
ER assisted degradation = ER Quality Control System
How does the ERAD work?
ERAD = Quality Control = identifies and removes misfolded or unassembled proteins in the endoplasmic reticulum (ER).
ER recognises whether proteins are ready to move onto the Golgi or if they need to stay in the ER longer or whether they need to be destroyed.
How does ERAD work (5 steps)?
Substrate recognition by chaperone protein > retrotranslocation > Ubiquitination > removal of sugars > degradation in proteasome
What is the most common mutation causing CF?
Fdel508-CFTR (which prevents CFTR from reaching the apical membrane)
Why is delta-Fdel508-CFTR unable to progress to the Golgi?
Because cannot be folded into the correct structure therefore it gets stuck in ER and is tagged by ubiquitin pathway for destruction in ERAD pathway.
How are proteins that pass the quality control in the ER transported to the Golgi?
Via vesicles = COPII
What is the difference in function between COP1 and COP2?
COP1 = takes protein back to ER
COP2 = takes protein to cis Golgi
How can the Golgi be further defined?
cis, medial and trans
What are examples of post translational processing in the Golgi?
Modification of sugars/N-linked glycoslyation
Addition of sugars to serine/threonines of proteins (O-linked glycosylation)
Sulfation of sugars and some tyrosines
Proteins packages into clathrin vesicles
Where in the Golgi does protein sorting occur?
trans-golgi (TGN)
What are the two important proteins involved in clathrin-coated vesicles?
t-SNARE
v-SNARE
What is the difference between the constitutive pathway and regulated pathway?
Constitutive = always happening to provide constant supply of some proteins to membrane
Regulated = proteins stored in vesicles and only tarnsported to membrane when signalled.
ENaC, CFTR and Na+K+-ATPase complexes move to plasma membrane via what pathway?
Constitutive pathway
(but also evidence that ENaC use the regulated pathway which is where proteins are stored in vesicles until signal induces exocytosis).
Where do clathrin coated vesicles move proteins from and to?
From the Golgi to the plasma membrane
v-SNARE protein on vesicles bind to what?
to the t-SNARE protein on the target membrane
What do SNARE proteins couple with in order to force vesicle fusion with cell membrane?
Rab
What is an endosome?
A membrane bound organelle within the cell that transports and sorts proteins and other substances within the cell.
What is a lysosome?
A membrane bound organelle that contains enzymes that break down waste materials, damaged cell parts, and foreign substances like bacteria.
For transmembrane proteins are basolateral or apical signals dominant?
Basolateral signals are dominant over apical sorting signals.
What apical sorting signals are unknown?
Those for CFTR and ENaC
What is required to specifically traffic apical proteins?
Binding proteins
What are sorting signals?
Sorting signals are molecular tags that help direct proteins to the correct surface of polarized cells in order to perform their functions properly - either the apical or the basolateral surface.
What are examples of basolateral sorting signals?
Tyrosine-based motifs (Yxx)
Dileucine (LL) motifs
Vesicular stomatitis virus glycoprotein
What are examples of apical sorting signals?
GPI anchors
O or N linked sugar chains
Amino acid motifs
Where in the Golgi apparatus does sorting occur?
TGN = trans-golgi network
What are the three pathways for protein exocytosis in epithelial cells?
1) direct route
2) indirect route
3) random sorting
What is the direct route for protein exocytosis in epithelial cells?
Proteins are sorted in TGN into apical or basolateral bound vesicles.
Apical and basolateral bound proteins go straight to their appropriate membrane.
What is the indirect route for protein exocytosis in epithelial cells?
All proteins exocytosed to one membrane
Protein are then retrieved from membrane (ENDOcytosis) and trafficked to endosome where protein trafficked to their respective membrane.
What is the difference between exocytosis and endocytosis?
Exocytosis is the process by which cells expel materials out of the cell. Whereas, Endocytosis is the process by which cells take in materials from the outside environment.
What is the random sorting route for protein exocytosis in epithelial cells?
Proteins are trafficked to either membrane randomly
Then protein are retrieved from membrane through endocytosis and trafficked to endosome where they are sent to their respective membrane.
Explain the experimental evidence for the direct sorting route of protein exocytosis in epithelial cells:
That basolateral and apical proteins go directly to their respective membrane by being sorted into distinct vesicle carriers at the TGN.
This was discovered by watching epithelial cell proteins which were tagged with different colour fluorescent tags (green for apical and blue for basolaterial).
What is endocytosis?
Taking up of particles or fluid from extracellular space
What are the two types of endocytosis?
Cathrin-mediated endocytosis
(either receptor mediated or fluid mediated)
Caveoloe-mediated endocytosis
What recognises the receptor in clathrin-mediated endocytosis?
Adaptin
Is receptor-mediated endocytosis or fluid-phase endocytosis more regulated?
Receptor-mediated endocytosis is highly regulated as it is ligand specific. Fluid-phase endocytosis is less regulated.
What does clathrin bind to?
Adaptin
During clathrin-coated endocytosis what is caused by the receptor binding to the ligand?
Indentation/budding in the membrane
What is the difference between receptor-mediate endocytosis and fluid-phase endocytosis?
Receptor mediated is ligand specific - therefore during fluid-phase endocytosis there is an unspecific uptake of extracellular fluid that causes indentation/budding rather then it being caused by a receptor binding to a ligand.
What does the v-SNARE on vesicle connect with on the target membrane?
t-SNARE
What is caveolae-mediated endocytosis?
Endocytosis involving small lipid-raft invagination of the membrane.
Caveolin associates with lipid rafts to form caveolae.
When ligands bind to receptors the caveolae bud off from the membrane.
Caveolae fuse with the caveosome which can then fuse with other endosomal compartments, lysosomes or plasma membrane
What is a caveosome?
A type of early endosome
What is Ubiquitination?
The cellular process of attaching ubiquitin marker proteins for endocytosis
What is the ROMK1 channel regulated by?
Ubiquitination
What does an apical recycling endosome do?
Take protein back to the membrane
What is TfR?
Basolateral transferrin receptor (TfR)
What is IgAR?
Immunoglobulin A receptor
Where is IgAR exocytosed to?
Apical membrane
What is meant by protein “half-life”?
Protein half-life refers to an experimental measurement = the time that half of the original population of protein remains or the time that half of the original population of protein has been degraded.
It’s a measure of protein stability, indicating how long a protein persists before being broken down by cellular processes like proteolysis (the breakdown of proteins into amino acids).
When are hydrolytic enzymes active?
At acidic pH
How do lysosomes degrade cellular proteins?
By breaking down peptide bonds
Why are proteins degraded?
Regulate
Replacement
Inactivate
Recycle
Where are proteins degraded?
In the lysosome or the proteasome
What does the lifetime of a protein depend on?
The function of a protein and the cellular pathway it is involved in
What are the three main systems for protein degradation?
Lysosomal degradation
Autophagy
Ubiquitin-proteasome degradation
What is autophagy?
Self eating
When do lysosome enzymes work best?
Acidic pH
What do lysosome enzymes break down?
proteins, sugars and nucleic acids
How is the low pH (acidic environment) of lysosomes maintained?
Two transport pumps that pump H+ and Cl- into the lysosome.
What are the five steps of the autophagy process?
1) uptake of random area of cytoplasm or defective organelles = phagophore
2) Phagophore forms a complete vesicle = autophagosome
3) autophagosmoe begins to fuse with lysosome
4) autolysosome formed and degradation begins
5) organelles and any uptake substances degraded and recycled
What is a phagophore?
Cup-shaped membrane structure involved in the early stages of autophagy. It is the precursor to the autophagosome, which is the double-membraned vesicle that eventually engulfs cellular components for degradation.
What is a autophagosome?
Complete vesicle
What is the name of the highly regulated process for degrading unwanted proteins, termination or activating signalling pathways, regulating the cell cycle and providing an importance source of amino acids for de novo protein synthesis?
Ubiquitin-proteasome system for protein degradation
What are proteins in the Ubiquitin-proteasome system tagged with?
Covalently tagged with a small 8 kDa protein called ubiquitin (Ub)
What two things is ubiquitin recognised to be a signal for?
1) degradation
2) endocytosis (monoubiquitin)
What is the difference between mono-ubiquitinated and poly-ubiquitinated?
Mono-ubiquitination: A single ubiquitin molecule is attached to one site on a protein.
Poly-ubiquitination: Multiple ubiquitin molecules are linked together in a chain and attached to a protein.
What enzymes are involved in ubiqutinating a protein?
E1 (activating enzyme)
E2 (conjugating enzyme)
E3 (ligase)
What is deubiquitinating and when does it occur?
It is the process of removing ubiquitin from proteins and it occurs when it is decided that the protein shouldn’t be degraded
What does DUBs prevent?
Degradation - it is the deubiquinating process.
What is the proteasome?
The proteasome is a protein complex in cells that breaks down and recycles damaged, misfolded, or unneeded proteins.
Proteins only - no other substates.
Does proteasome have mono or poly Ub regulation?
Poly
What is released and what is recycled from proteasome?
Peptides are released and amino acids are recycled
How does endocytosis of ion channels occur?
1) ENaC and ROMk1 (cell membrane ion channels) are tagged with monoubiquitination tag that promote endocytosis of the channel and to the apical sorting endosome
2) The ubiquinated protein is then moved from apical sorting endosome to the common common endosome where fate of the channel is determined
3) It is either receyled back to the cell membrane by deubiquitination (removal of the ubiquitin tag) or it is moved on to lysosome for degradation
What is the genetic form of hypertension syndrome?
Liddle’s syndrome
What is Liddle’s syndrome caused by?
When ENaC cannot be tagged with ubiquitin due to the loss of the binding site for an E3 ubiquitin ligase therefore less ENaC endocytosis and more ENaC at the plasma membrane and more Na+ reabsorption leading to high ECF volume and elevated blood pressure.
What enzymes are involved in lysosome mediated degradation?
Acid hydrolases
What do lysosomes degrade?
Proteins, sugars, DNA and RNA
What enzymes are involved in proteasome mediated degradation?
Peptidases
What are the co-factors of proteasome mediated degradation?
series of enzymes: E1, E2, and E3
What are the general targets of proteasome mediated degradation?
Cell surface, cytosolic, nuclear, ER proteins (ERAD)
What enzymes are involved in autophagy mediated degradation?
Acid hydrolyses
What is degraded during autophagy mediated degradation?
Damaged organelles, microbes, cytosolic components
What is the structure of autophagy mediated degradation?
Autophagosomes
What is the key difference between a lysosome and a proteasome?
Lysosome can degrade proteins and other cellualr components whereas a proteosome only degrades proteins.
What is the process of tagging a protein for ubiquination?
ATP binds to Ub protein
Ub to E1, E2 and E3
Ub then marks protein
