This is a summary of a presentation our CEO has given at Northwestern University, Chicago - U.S.A. in 2003. Many aspects of protein targeting and membrane trafficking in eukaryotic cells are discussed.
Tags: Gokay-BIOTECH, K.Erden Gokay, Cell Biology, protein sorting, endotubin
5. LDL-R trafficking:LDL-R trafficking:
Ligand dissociates
from the receptor upon
delivery to a relatively
acidic compartment.
Ligand and receptor
are sorted into distinct
compartments where
ligand is degraded but
the receptor is
recycled.
6. Trf-R trafficking:Trf-R trafficking:
Receptor is
constitutively
internalized.
The ligand is exposed
to a relatively acidic
environment.
Both the ligand and the
receptor are recycled.
7. PIg-R trafficking:PIg-R trafficking:
Receptor is targeted
basolaterally for ligand
binding.
Ligand induces
internalization.
Ligand bound receptor
is delivered apically for
apical secretion of the
ligand (s-IgA).
9. Endosomes are a heterogeneous group ofEndosomes are a heterogeneous group of
compartments:compartments:
10. Principles of Membrane Trafficking:Principles of Membrane Trafficking:
Coat assembly
and budding
Fission and
transport
Un-coating and
priming
Docking and
fusion
11. A common sorting site?A common sorting site?
•Transcytotic cargo
as well as
basolaterally
recycling cargo has
been shown to meet
in an apical
compartment (ARE)
prior to sorting,
12. Question:Question:
•How does apicalHow does apical
endomembraneendomembrane
trafficking differ fromtrafficking differ from
basolateral trafficking?basolateral trafficking?
•What is the nature ofWhat is the nature of
apical endosomalapical endosomal
targeting cue(s)?targeting cue(s)?
17. Endotubin-positive structures are apicalEndotubin-positive structures are apical
derived early endosomes:derived early endosomes:
• 20 min Apical
Ricin uptake
• 30 min
Basolateral
Ricin uptake
• Anti-LAMP-1
18. Endotubin-positive endosomes do notEndotubin-positive endosomes do not
contain Transferrin:contain Transferrin:
• Apical
• Perinuclear
• Basolateral
20. Endotubin positive apical endosomalEndotubin positive apical endosomal
compartment is distinct from the ARE:compartment is distinct from the ARE:
Red = Transferrin
Green = Endotubin
21. Coat assembly is a regulated process:Coat assembly is a regulated process:
• Arf family of small
GTPases regulate
Coatomer (Cop-1,
Cop-2) assembly
onto the donor
compartment
membrane.
• Arf-GEF = ARNO
(Cytohesin)
24. Biogenesis of endotubin-positive apicalBiogenesis of endotubin-positive apical
endosomes:endosomes:
Endotubin targets
into an apical
derived early
endosomal
compartment distinct
from the common
(apical) recycling
endosome.
30. Conclusions:Conclusions:
In polarized epithelia endotubin targets into
an apical early endosomal compartment
distinct from the ARE.
This targeting process relies on cytoplasmic
signals present on intracellular domain of the
molecule.
Apical targeting and endosomal targeting
(internalization) signals are independent from
each other.
31. Other possible targeting signals:Other possible targeting signals:
Association with glycolipid rich rafts.
Differential glycosylation (N-linked and O-
linked) of the ectoplasmic domain.
Other protein-protein interactions (lectin like
sorting protein) ?
32. Endotubin does not partition intoEndotubin does not partition into
raft domains:raft domains:
40. Acknowledgements:Acknowledgements:
* Jean M. WILSON, Ph.D.
Carol GREGORIO, Ph.D.
Paul St. JOHN, Ph.D.
Mani RAMASWAMI, Ph.D.
Tamara Lee COLTON, M.S.
Jennifer SALATA
• The University of Arizona, Dept. of Cell Biology and Anatomy.
Tucson - ARIZONA
41.
42. Arf is required for maintenance ofArf is required for maintenance of
apical early endosomes:apical early endosomes:
Brefeldin-A treatment
causes fusion and
tubulation of
Endotubin positive
endosomes with
basolateral early
endosomes.
When the drug is
washed out, proper
polarized sorting
resumes.
46. Glucose transporters:
In the human genome so far there are 11
GLUT isoforms identified (facilitative hexose
transporter gene family) but there is only one
insulin-responsive transporter is known.
In theory there are 3 ways by which insulin
may modulate Glut-4 function:
- Altering its transport activity
- Upregulating its expression
- Altering its endomembrane trafficking
47. Glut-4 trafficking:
At steady state the
transporter targets to
endosomes.
Upon initiation of
insulin signaling
activation of PI-3
kinase, PKCζ and
PKB (Akt) cascades
result in exocytosis of
the transporter.
48. Furin trafficking:
Furin is an
endopeptidase required
for cleavage (secretion)
of TGF-β from the
transmembrane
precursor.
Phosphorylation of
Furin cytoplasmic
domain via CK-II alters
its trafficking pathway.
49. Synaptic vesicle regeneration:
Following
neurotransmitter
release, rapid
endoctosis is required
to regenerate SSVs.
Shibire (Dynamin-ts)
mutation in the fly is
characterized with a
temperature sensitive
paralysis.
50. Basic concepts in membrane trafficking:
Despite high degree of regulation and
specificity, isolated trafficking events are not
error-proof (reliably unreliable).
However, sorting efficiency is boosted up via
iterative sorting.
There is high degree of redundancy in
membrane trafficking regulators.
Membrane trafficking events are quite plastic
and alternate pathways do exist.
51. Budding and fusion cycle:
Aggregation
and coat
assembly.
Budding and
transport.
Priming (un-
coating).
Docking and
fusion.
52. Role players:
Adaptors and coat regulators:
Adaptins, Arf, ARNO
Coat proteins:
Coatomer, Clathrin, Caveolin
Pinching-off and Priming:
Dynamin, Rab, Rabaptin, Rabphylin, Arf-GAF
Docking molecules:
t-SNAREs, v-SNAREs
Fusion proteins:
NSF, SNAP
53. Clathrin coated pits:
Clathrin pre-assembles
into triskelions
composed of 3 heavy
and 3 light chains.
Upon cargo binding,
adaptins (AP-1, AP-2
and AP-3) mediate
clathrin triskelions to
polymerize into clathrin
cages.