Homotypic fusion between early endosomes requires the phosphatidylinositol 3-phosphate (PI3P)-binding protein, Early Endosomal Autoantigen 1 (EEA1). We have investigated the role of other proteins that interact with EEA1 in the fusion of early endosomes derived from Baby Hamster Kidney (BHK) cells. We confirm a requirement for syntaxin 13, but additionally show that the assay is equally sensitive to reagents specifically targeted against syntaxin 6. Binding of EEA1 to immobilised GST-syntaxin 6 and 13 was directly compared; only syntaxin 6 formed a stable complex with EEA1. Early endosome fusion requires the release of intravesicular calcium, and calmodulin plays a vital role in the fusion pathway, as judged by sensitivity to antagonists. We demonstrate that both EEA1 and syntaxin 13 interact with calmodulin. In the case of EEA1, binding to calmodulin requires an IQ domain, which is adjacent to a C-terminal FYVE domain that specifically binds to PI3P. We have assessed the influence of protein binding partners on EEA1 interaction with PI3P and find that both calmodulin and rab5-GTP are antagonistic to PI3P binding, whilst syntaxins 6 and 13 have no effect. These studies reveal a complex network of interactions between the proteins required for endosome fusion.

REFERENCES

Bock
J. B.
,
Klumpermann
J.
,
Davanger
S.
,
Scheller
R. H.
(
1997
).
Syntaxin 6 functions in trans-Golgi network vesicle trafficking.
Mol. Biol. Cell
8
,
1261
1271
Bock
J. B.
,
Lin
R. C.
,
Scheller
R. H.
(
1996
).
A new syntaxin family member implicated in targeting of intracellular transport vesicles.
J. Biol. Chem
271
,
17961
17965
Burd
C. G.
,
Emr
S. D.
(
1998
).
Phosphatidylinositol(3)-phosphate signaling mediated by specific binding to RING FYVE domains.
Mol. Cell
2
,
157
162
Callaghan
J.
,
Simonsen
A.
,
Gaullier
J.-M.
,
Toh
B.-H.
,
Stenmark
H.
(
1999
).
The endosome fusion regulator early-endosomal autoantigen 1 (EEA1) is a dimer.
Biochem. J
338
,
539
543
Christoforidis
S.
,
McBride
H. M.
,
Burgoyne
R. D.
,
Zerial
M.
(
1999
).
The rab5 effector EEA1 is a core component of endosome docking.
Nature
397
,
621
625
Clague
M. J.
(
1999
).
Membrane transport: Take your fusion partners.
Current Biol
9
,
258
260
Colombo
M. I.
,
Beron
W.
,
Stahl
P. D.
(
1997
).
Calmodulin regulates endosome fusion.
J. Biol. Chem
272
,
7707
7712
Diaz
R.
,
Mayorga
L.
,
Weidman
P. J.
,
Rothman
J. E.
,
Stahl
P. D.
(
1989
).
Vesicle fusion following receptor-mediated endocytosis requires a protein active in Golgi transport.
Nature
339
,
398
400
Gaullier
J.-M.
,
Simonsen
A.
,
D'Arrigo
A.
,
Bremnes
B.
,
Stenmark
H.
(
1998
).
FYVE fingers bind PtdIns(3)P.
Nature
394
,
432
433
Gorvel
J. P.
,
Chavrier
P.
,
Zerial
M.
,
Gruenberg
J.
(
1991
).
Rab 5 controls early endosome fusion in vitro.
Cell
64
,
915
925
Holroyd
C.
,
Kistner
U.
,
Annaert
W.
,
Jahn
R.
(
1999
).
Fusion of endosomes involved in synaptic vesicle recycling.
Mol. Biol. Cell
10
,
3035
3044
Jones
A. T.
,
Clague
M. J.
(
1995
).
Phosphatidylinositol 3-kinase activity is required for early endosome fusion.
Biochem. J
311
,
31
34
Jones
A. T.
,
Mills
I. G.
,
Scheidig
A.
,
Alexandrov
K.
,
Clague
M. J.
(
1998
).
Inhibition of endosome fusion by wortmannin persists in the presence of activated rab5.
Mol. Biol. Cell
9
,
323
332
Li
G. P.
,
Dsouzaschorey
C.
,
Barbieri
M. A.
,
Roberts
R. L.
,
Klippel
A.
,
Williams
L. T.
,
Stahl
P. D.
(
1995
).
Evidence for phosphatidylinositol 3-kinase as a regulator of endocytosis via activation of rab5.
Proc. Natl. Acad. Sci.USA
92
,
10207
10211
Lowe
S. L.
,
Peter
F.
,
Subramaniam
V. N.
,
Wong
S. H.
,
Hong
W.
(
1997
).
A SNARE involved in protein transport through the Golgi apparatus.
Nature
389
,
881
884
McBride
H. M.
,
Rybin
V.
,
Murphy
C.
,
Giner
A.
,
Teasdale
R.
,
Zerial
M.
(
1999
).
Oligomeric complexes link rab5 effectors with NSF and drivemembrane fusion via interactions between EEA1 and syntaxin 13.
Cell
98
,
377
386
Mills
I. G.
,
Jones
A. T.
,
Clague
M. J.
(
1998
).
Involvement of the endosomal autoantigen EEA1 in homotypic fusion of early endosomes.
Curr. Biol
8
,
881
884
Mu
F.-T.
,
Callaghan
J. M.
,
Steele-Mortimer
O.
,
Stenmark
H.
,
Parton
R. G.
,
Campbell
P. L.
,
McCluskey
J.
,
Yeo
J.-P.
,
Tock
E. P. C.
,
Toh
B.-H.
(
1995
).
EEA1, an early endosome-associated protein.
J. Biol. Chem
270
,
13503
13511
Nielsen
E.
,
Christoforidis
S.
,
Uttenweiler-Joseph
S.
,
Miaczynska
M.
,
Dewitte
F.
,
Wilm
M.
,
Hoflack
B.
,
Zerial
M.
(
2000
).
rabenosyn-5, a novel rab5 effector, is complexed with hVPS45 and recruited to endosomes through a FYVE finger domain.
J. Cell Biol
151
,
601
612
Patki
V.
,
Lawe
D. C.
,
Corvera
S.
,
Virbasius
J. V.
,
Chawla
A.
(
1998
).
A functional PtdIns(3)P-binding motif.
Nature
394
,
433
434
Peters
C.
,
Andrews
P. D.
,
Stark
M. J.
,
Cesaro-Tadic
S.
,
Glatz
A.
,
Podtelejnikov
A.
,
Mann
M.
,
Mayer
A.
(
1999
).
Control of the terminal step of intracellular membrane fusion by protein phosphatase 1.
Science
285
,
1084
1087
Peters
C.
,
Mayer
A.
(
1998
).
Ca2+/calmodulin signals the completion of docking and triggers a late step of vacuole fusion.
Nature
396
,
575
580
Peterson
M. R.
,
Burd
C. G.
,
Emr
S. D.
(
1999
).
Vac1p coordinates Vps21 Rab GTPase and Vps34 PtdIns 3-kinase signaling, essential for Pep12-and Vps45p-dependent vesicle docking/fusion at the endosome.
Curr. Biol
9
,
159
162
Poirer
M. A.
,
Xiao
W.
,
Macosko
J. C.
,
Chan
C.
,
Shin
Y. K.
,
Bennett
M. K.
(
1998
).
the synaptic SNARE complex is a parallel four-stranded helical bundle.
Nat. Struct. Biol
5
,
765
769
Pryor
P. R.
,
Mullock
B. M.
,
Bright
N. A.
,
Gray
S. R.
,
Luzio
J. P.
(
2000
).
The role of intraorganellar Ca2+in late endosome-lysosome heterotypic fusion and in the reformation of lysosomes from hybrid organelles.
J. Cell Biol
149
,
1053
1062
Quetglas
S.
,
Leveque
C.
,
Miquelis
R.
,
Sato
K.
,
Seagar
M.
(
2000
).
Ca2+-dependent regulation of synaptic SNARE complex assembly via a calmodulin and phospholipid-binding domain of synaptobrevin.
Proc. Natl. Acad. Sci. USA
97
,
9695
9700
Schiavo
G.
,
Gu
Q. M.
,
Prestwich
G. D.
,
Sollner
T. H.
,
Rothman
J. E.
(
1997
).
Calcium-dependent switching of the specificity of phosphoinositide binding to synaptotagmin.
Proc. Natl. Acad. Sci. USA
93
,
13327
13332
Simonsen
A.
,
Gaullier
J. M.
,
D'Arrigo
A.
,
Stenmark
H.
(
1999
).
The rab5 effector EEA1 interacts directly with syntaxin-6.
J. Biol. Chem
274
,
28857
28860
Simonsen
A.
,
Lippe
R.
,
Christoforidis
S.
,
Gaullier
J.-M.
,
Brech
A.
,
Callaghan
J.
,
Toh
B.-H.
,
Murphy
C.
,
Zerial
M.
,
Stenmark
H.
(
1998
).
EEA1 links PI(3)K function to rab5 regulation of endosome fusion.
Nature
394
,
494
498
Spiro
D. J.
,
Boll
W.
,
Kirchhausen
T.
,
Wessling-Resnick
M.
(
1996
).
Wortmannin alters the transferrin receptor endocytic pathway in vivo and in vitro.
Mol. Biol. Cell
7
,
355
367
Sutton
R. B.
,
Fasshauer
D.
,
Jahn
R.
,
Brunger
A. T.
(
1998
).
Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4Å resolution.
Nature
395
,
347
353
This content is only available via PDF.