What must be used in addition to secretory and endocytosis pathways?
Epithelia must sort proteins into apical and basolateral populations and direct these to the correct membrane domain
What are basolateral sorting signals?
They are amino acid sequences similar to endocytosis signals
What are examples of basolateral sorting signals?
tyrosine-based motifs (Yxxø) or dileucine motifs (LL), however, apical proteins also contain these
What basolateral motifs does ENaC have?
YXXL
What basolateral motif does CFTR have?
YXXø and LL
What are apical sorting signals?
GPI anchors, O or N linked sugar chains, amino acid motifs in the transmembrane domains or cytoplasmic part of the protein
What is the apical sorting signal?
It is unknown but trafficking to the apical membrane is saturable which indicates that binding proteins are required to specifically traffic apical proteins
What are the three models for protein exocytosis in epithelial cells?
Direct, indirect, and random sorting routes
What is the direct route of protein exocytosis?
Proteins are sorted in TGN into apical or basolateral bound vesicles, apical and basolateral bound proteins then go to their appropriate membrane
What is the indirect route of protein exocytosis?
All proteins are exocytosed to one membrane, proteins are retrieved from the membrane and trafficked to the endosome, apical proteins are trafficked to the apical membrane and basolateral proteins to the basolateral membrane
What is the random sorting route of protein exocytosis?
Proteins are trafficked to either membrane randomly, proteins are retrieved from the membrane and trafficked to the endosome, apical proteins are redirected to the apical membrane and basolateral proteins to the basolateral membrane
How are epithelial cells recognised for experimental evidence of the direct route?
Epithelial cells are grown in culture expressing apical and basolateral proteins which are tagged with fluorescent tags
What is the process for experimental evidence for the direct sorting route?
Cells are cooled to 20 degrees to accumulate proteins at the trans-golgi network, warmed to 37 degrees and live cell imaging is used to 'watch' the proteins
What is seen in the experimental evidence for the direct sorting route?
The basolateral marker protein and apical marker protein were sorted into distinct vesicular carriers at the TGN
What is the problem with the experimental evidence for the direct sorting route?
The cells were grown in culture so weren't polarised and don't have epithelial cell characteristics
What is used for basolateral protein trafficking?
Exocyst
What is an exocyst?
A complex of eight proteins important for plasma membrane targeting and tethering of vesicles which is required for polarised exocytosis
What occurs to polarised epithelial cells with exocyst?
They localise the exocyst complex with ZO-1 at the top of the lateral membrane domain
What is an experiment used with the exocyst complex?
Antibody binding to one exocyst protein prevented correct trafficking of a basolateral protein, but not an apical protein
What is the process of endocytosis for clathrin coated vesicles? (1)
Receptors recruit adaptin and clathrin to the plasma membrane
What is the process of endocytosis for clathrin coated vesicles? (2)
Receptor binds ligand 'weighing down' the membrane, causing an indentation/budding (receptor mediated endocytosis) or unspecific uptake of extracellular fluid, causing an indentation/budding (fluid-phase endocytosis)
What is the process of endocytosis for clathrin coated vesicles? (3)
Vesicle continues to bud off from the membrane
What is the process of endocytosis for clathrin coated vesicles? (4)
Vesicle buds off from the membrane (contains endocytosed material)
What is the process of endocytosis for clathrin coated vesicles? (5)
Clathrin-coated vesicle carriers endocytosed material away from the membrane
What is the process of endocytosis for clathrin coated vesicles? (6)
Clathrin and adaptin dissociate from the vesicle
What is the process of endocytosis for clathrin coated vesicles? (7)
Clathrin-coated vesicle approaches the endosome, v-SNARE on vesicle connects with t-SNARE on the target membrane (endosomal membrane)
What is the process of endocytosis for clathrin coated vesicles? (8)
Clathrin-coated vesicle fuses with the endosome, 'emptying' its contents into the endosome
What is calveolae-mediated endocytosis?
Endocytosis for membrane proteins
What is the process of calveolae-mediated endocytosis? (1)
Small lipid-raft invaginations of the membrane
What is the process of calveolae-mediated endocytosis? (2)
Calveolin associates with the lipid rafts to form calveolae
What is the process of calveolae-mediated endocytosis? (3)
Ligands bind to receptors and calveolae bud off from the membrane
What is the process of calveolae-mediated endocytosis? (4)
Calveolae fuse with the caveosome (type of early endosome)
What is the process of calveolae-mediated endocytosis? (5)
Caveosome can then fuse with other endosomal compartments, lysosome or plasma membrane
What is calveolae-mediated endocytosis important for?
Trafficking of cholesterol
What are the endocytosis signals for receptor-mediated endocytosis?
Tyrosine based amino acid motifs, hydrophobic amino acid motifs, and monoubiquitin
What is monoubiquitin?
All known endocytosis motifs found in plasma membrane proteins
What are tyrosine based amino acid motifs?
YXXø, NPXY
What are hydrophobic amino acid motifs?
LL
What endocytosis signal does ENaC subunits contain?
YXXø motifs and are monoubiquinated
What endocytosis signals do CFTR subunits contain?
YXXø and LL
What endocytosis signals do Na+/K+ ATPase subunits contain?
YXXø and LL
What are endocytosis motifs bound by?
Clathrin-binding adaptins
What is AP2?
The specific adaptin in the plasma membrane clathrin-coated pits
What is AP2 made up of?
alpha and beta adaptins, µ2 adaptin and ∂2 subunits
What do alpha and beta adaptins bind to?
Clathrin
What does µ2 bind to?
YXXø
Why is endocytosis in epithelia different to other cells?
Because they are polarised
What is the process for endocytosis in epithelia? (1)
Apical proteins are endocytosed the an apical sorting endosome and basolateral proteins to a basolateral sorting endosome
What is the process for endocytosis in epithelia? (2)
Endocytosed material (apical and basolateral) goes to the common endosome
What is the process for endocytosis in epithelia? (3)
Basolateral proteins can be recycled back to the basolateral membrane, apical proteins are recycled back to the apical membrane by an apical recycling endosome
What is the endocytosis and recycling or degradation pathway of apical ENaC?
ENaC is ubiquitinated and endocytosed and trafficked through apical sorting endosome to the common endosome
What occurs if ubiquitin is removed from ENaC?
It is recycled back to the apical membrane
What occurs if ubiquitin remains on ENaC?
It is trafficked to the late endosome and onto the lysosome for degradation
What is the pathway endocytosis and recycling or degradation of the basolateral transferrin receptor? (1)
Ligand-bound transferrin is endocytosed to basolateral sorting endosome
What is the pathway endocytosis and recycling or degradation of the basolateral transferrin receptor? (2)
Ligand-bound transferrin is trafficked onto the common endosome
What is the pathway endocytosis and recycling or degradation of the basolateral transferrin receptor? (3)
Basolateral transferrin receptor is either recycled back to the basolateral membrane or trafficked through the late endosome to the lysosome for degradation
What is the transcytosis of the immunoglobulin A receptor? (1)
IgAR is endocytosed from the basolateral membrane and trafficked to the basolateral sorting endosome
What is the transcytosis of the immunoglobulin A receptor? (2)
It is trafficked on to the apical recycling endosome through the common endosome
What is the transcytosis of the immunoglobulin A receptor? (3)
IgAR is exocytosed to the apical membrane