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

1. Principles of Membrane
Trafficking
in Polarized Epithelia
K. Erden GOKAY, M.D.,K. Erden GOKAY, M.D.,
Ph.D.Ph.D.
Ay-GRUP SSA Ltd., Turkey
Research Scientist

3. Classic (old) view:Classic (old) view:
 All endosomes eventually get acidified (maturation)
and deliver their cargo to the Lysosome.

4. EGF-R trafficking:EGF-R trafficking:
 Ligand triggers
endocytosis.
 Eventually both the
ligand and the receptor
are degraded.

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).

8. Proper membrane trafficking isProper membrane trafficking is
required for:required for:
 Metabolic regulation:
Trf-R, LDL-R, Glut-4
 Regulation of signaling pathways:
EGF-R, βAR, Furin (TGFβ)
 Antigen presentation:
MHC-II
 Neurotransmitter metabolism:
SSV regeneration (Dynamin)
 Generation and maintenance of cell polarity:
PIg-R (sIgA) {epithelial integrity}

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)?

13. Endotubin:Endotubin:
1195 a.a. single TMD
type I glycoprotein with
a short (36aa)
cytoplasmic domain.

14. Endotubin expression in the native tissue:Endotubin expression in the native tissue:

15. A model polarized simple epithelium:A model polarized simple epithelium:
• Madin-Darby Canine
Kidney (MDCK) cell
line.
{Renal Collecting tubule like}

16. Endotubin expression inEndotubin expression in
polarized MDCK cells:polarized MDCK cells:

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

19. Endotubin-positive endosomes do notEndotubin-positive endosomes do not
label with Rab-11:label with Rab-11:

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)

22. Brefeldin A wash-out:Brefeldin A wash-out:
2-min 10-min

23. Transferrin Pulse-Chase:Transferrin Pulse-Chase:
Transferrin bound to
cell surface receptors
transiently co localizes
with endotubin in non-
polarized MDCK cells.

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.

25. Site directed mutagenesis:Site directed mutagenesis:

26. Targeting of endotubin mutants:Targeting of endotubin mutants:
STOP1180 T1186A E1189A I1178A/L1179A
• Apical
• Perinuclear
• Basal

27. Targeting of endotubin mutants:Targeting of endotubin mutants:
E1189A
F1180A
Apical Ricin uptake for 15 minutes

28. Domain selective Biotinylation:Domain selective Biotinylation:

29. TT11861186 is phosphorylated:is phosphorylated:
1: MDCK
2: wt-endotubin
3: STOP1180
4: T1186A

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:

33. Endotubin - Tac chimeras:Endotubin - Tac chimeras:

34. Tac vs Tac-ET expression:Tac vs Tac-ET expression:
Tac
Wildtype
Tac-ET
C1

35. Transferrin - BfA:Transferrin - BfA:
Tac-ET
C1
Tac
Wildtype

36. Tac-ET is present in apical earlyTac-ET is present in apical early
endosomes:endosomes:
Tac-ET (C1) Apical Ricin 15 min Merge

37. Targeting of Tac-ET chimeras inTargeting of Tac-ET chimeras in
polarized MDCK cells:polarized MDCK cells:
Tac-WT C1 C2 C3
• Apical
• Perinuclear
• Basolateral

38. Domain selective Biotinylation:Domain selective Biotinylation:

39. C4 chimera:C4 chimera:

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

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.

43. Membrane trafficking and cell polarity:

44. Endosomal compartments:Endosomal compartments:
 Model polarized
epithelial cell.

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.

54. A look to the plasma membrane inner
surface:

55. Dynamin dominant negative mutant:
 Dynamin is a
GTPase required
for vesicle budding.

56. SNAREs are not sufficient for
membrane fusion:
 NSF is an ATPase that is requred for
membrane fusion.

57. Endomembrane trafficking pathways
are highly conserved:
MammalianMammalian Yeast homologYeast homolog
 Rab 1a Ypt1p
 Arf Sar1p
 ARNO Sec7p
 NSF Sec18p