L14 Flashcards
what are basolateral sorting signals similar to
endocytosis signals
what are some basolateral sorting signals
tyrosine-based motifs (Yxxϕ)
or
dileucine (LL) motifs
what are some possible apical sorting signals
GPI anchors, O- or N-linked sugar chains,
amino acid motifs in transmembrane
domains or cytoplasmic part of protein
however there is no consensus apical sorting signalling
there is no consensus apical sorting signal, but trafficking to apical membrane is saturable. what does this indicate
This indicates that binding proteins are required to specifically traffic apical proteins.
what are the apical sorting signals for CFTR and ENaC
we dont know
what do sorting signals in epithelial cells depend on
the cell type and the specific protein being sorted
is it possible that sorting signals act in hierarchy
example
yes
for some transmembrane
proteins basolateral signals are dominant over apical sorting
signals
describe the direct route for exocytosis of proteins in epithelial cells
Proteins sorted in trans golgi network (TGN) into apical or
basolateral bound vesicles.
Apical and basolateral bound proteins go straight to their appropriate membrane
describe the indirect route for exocytosis of proteins in epithelial cells
All proteins exocytosed to ONE membrane (either apical or basolateral NOT BOTH)
Proteins retrieved from membrane through endocytosis and trafficked to endosome
from the endosome…
Apical proteins trafficked to apical membrane, and Basolateral proteins trafficked to basolateral membrane
what is the endosome
This is like a post office which will redirect the proteins to the membrane that they are supposed to go to
describe the random sorting route for exocytosis of proteins in epithelial cells
Proteins trafficked to either membrane randomly
Proteins retrieved from membrane through endocytosis and trafficked to endosome
Apical proteins redirected to apical membrane
Basolateral proteins redirected to basolateral membrane
what is the difference between direct, indirect and random route
direct = no endosome
indirect = all into one membrane and then sorted in the endosome
random = proteins to random membrane and the sorted in the endosome
what does most experimental evidence support (in terms of the routs taken)
the direct route
how can yo experimentally test to see what route is taking place within a cell
they grew epithelial cells in culture, expressing basolateral and apical proteins tagged with
different coloured fluorescent tags (green for apical and blue for basolateral).
Experiment: cool to 20 degrees to accumulate
proteins at the trans-Golgi network, warm
up cells to 37 degrees and use live cell imaging to ‘watch’ the proteins.
VSV-G-blue (basolateral marker protein tagged with a blue fluorophore) and greenGPI (apical marker protein tagged with a green fluorophore) were sorted into distinct vesicular carriers at the TGN.
this shows evidence for the direct pathway but some people argue that the cell is not fully polarised
describe the exocyst model
this model is only for basolateral bound proteins
Complex of eight proteins important for plasma membrane
targeting and tethering of vesicles.
Required for polarised exocytosis in yeast.
In polarised epithelial cells localises with ZO-1 at top of lateral membrane domain.
how could you experimentally test for the exocyst model
Experiment: Antibody binding to one exocyst protein prevented
correct trafficking of a basolateral protein, but not of an apical
protein
what protein trafficking model is only for basolateral proteins
the exocyst model
Which statement about protein sorting and targeting in epithelia is
CORRECT?
A. The exocyst is the site for delivery of proteins to the basolateral
membrane.
B. Experimental evidence shows that the trans-Golgi network can sort apical and basolateral proteins into separate vesicle populations.
C. In the indirect sorting pathway all proteins are first delivered to the
same membrane domain.
D. All of A, B and C are correct.
D
what is the process of vesicle formation (from membrane to endosome)
- Receptors recruit adaptin and clathrin to the plasma membrane
2a. Receptor binds ligand “weighing down” the membrane, causing an indentation/budding (receptor-mediated endocytosis)
2b. Unspecific uptake of extracellular fluid, causing an indentation/budding (fluidphase endocytosis) - Vesicle continues to bud off from the membrane
- Vesicle buds off from the membrane (contains endocytosed material)
- Clathrin-coated vesicle carriers endocytosed material away from the membrane
- Clathrin and adaptin dissociate from the vesicle
- Clathrin-coated vesicle approaches endosome. v-SNARE on vesicle connects with
t-SNARE on the target membrane (endosomal membrane) - Clathrin-coated vesicle fuses with endosome, “emptying” its contents into the endosome
describe caveolae mediated endocytosis
Small lipid-raft invaginations of the
membrane
Calveolin associates with the lipid rafts to
form calveolae
Ligands bind to receptors and calveolae bud off from the membrane
Calveolae fuse with the caveosome (a type
of early endosome)
Caveosome can then fuse with other
endosomal compartments, lysosome or plasma membrane
what is caveolae mediated endocytosis important for
Important in the the trafficking of cholesterol which helps maintain the fluidity of the plasma membrane
what are known as endocytosis motifs found in plasma membrane proteins
Tyrosine based amino acid motifs e.g. YXXϕ, NPXY; (ϕ=a hydrophobic amino acid).
Hydrophobic amino acid motifs e.g. LL;
Monoubiquitin are known endocytosis motifs found in plasma membrane proteins
what motifs do ENaC subunits have
YXXϕ (tyrosine) motifs and are monoubiquitinated
what motifs do CFTR subunits have
YXXϕ (tyrosine) and LL
motifs (hydrophobic amino acids)
what motifs do Na/K ATPase subunits have
YXXϕ (tyrosine) and LL
motifs (hydrophobic amino acids)
what are endocytosis motifs bound by
clathrin binding addaptins
what is the specific adaptin in
plasma membrane clathrin-coated pits
AP2
what is AP2 made up of
it has 4 subunits
α and β adaptins (bind to clathrin to form the clathrin coated vesicle)
μ2 adaptin (binds YXXϕ (tyrosine motifs in proteins)
and σ2 subunits.
NOTE σ = sigma
therefore has 4 subunits
a, b, u, and o
Endocytosis of transmembrane proteins occurs through interaction with the AP2 complex
BECAUSE
the transmembrane proteins contain amino acid sequences such as YXXϕ.
both statements are true and causal
explain epithelia endocytosis
1a. Apical proteins are endocytosed to
an apical sorting endosome
1b. Basolateral proteins are endocytosed to a basolateral sorting
endosome
- Endocytosed material (apical and basolateral) goes to the common endosome
3a. Basolateral proteins can be
recycled back to the basolateral membrane
3b. Apical proteins are, believed, to recycle back to the apical membrane via an apical recycling endosome
what is the difference between basolateral and apical endocytosis and recycling
apical go from the common endosome to an apical recycling endosome before they get inserted back into the apical membrane
basolateral go from the common endosome straight back into the basolateral membrane
describe endocytosis and recycling/degradation of apical ENaC
ENaC is ubiquitinated and
endocytosed
Trafficked through apical sorting endosome to the
common endosome
if ubiquitin is removed ENaC is recycled back to apical membrane
if ubiquitin remains
ENaC is trafficked to late endosome and onto the lysosome for degradation
explain endocytosis and recycling or degradation of the basolateral transferrin receptor (TfR)
- Once ligand-bound transferrin is endocytosed to basolateral sorting membrane
- Trafficked on to common endosome
- TfR is either recycled back to basolateral membrane or trafficked through the late endosome to the lysosome for degradation
what is transcytosis
Endocytosed from the basolateral membrane and the transytosed to the apical membrane
or vice versa
explain how the immunoglobulin A receptor - IgAR is transcytosed
IgAR endocytosed from basolateral membrane and, trafficked to basolateral sorting endosome
it is then trafficked on to apical recycling endosome through common endosome
IgAR exocytosed to apical membrane
what are the 3 types of endocytosis
- Receptor mediated endocytosis
- Fluid phase endocytosis
- Caveolae mediated endocytosis
