The Journal of Cell Biology, Volume 151, Number 3, October 30, 2000 601–612
The Rockefeller University Press, 0021-9525/2000/10/601/12 $5.00
Rabenosyn-5, a Novel Rab5 Effector, Is Complexed with hVPS45
and Recruited to Endosomes through a FYVE Finger Domain
Erik Nielsen,* Savvas Christoforidis,* Sandrine Uttenweiler-Joseph,
*Max-Planck-Institute for Molecular Cell Biology and Genetics, 01307 Dresden, Germany;
Laboratory, 69117 Heidelberg, Germany; and
EP 525/Institut Pasteur de Lille 1, 59021 Lille, France
and Marino Zerial*
European Molecular Biology
Institut de Biologie de Lille, Centre National de la Recherche Scientifique
by specifically recruiting cytosolic effector proteins to
their site of action on early endosomal membranes. We
have characterized a new Rab5 effector complex in-
volved in endosomal fusion events. This complex in-
cludes a novel protein, Rabenosyn-5, which, like the
previously characterized Rab5 effector early endosome
antigen 1 (EEA1), contains an FYVE finger domain
and is recruited in a phosphatidylinositol-3-kinase–
dependent fashion to early endosomes. Rabenosyn-5 is
complexed to the Sec1-like protein hVPS45. hVPS45
does not interact directly with Rab5, therefore Rabeno-
syn-5 serves as a molecular link between hVPS45 and
the Rab5 GTPase. This property suggests that Rabeno-
syn-5 is a closer mammalian functional homologue of
Rab5 regulates endocytic membrane traffic yeast Vac1p than EEA1. Furthermore, although both
EEA1 and Rabenosyn-5 are required for early endoso-
mal fusion, only overexpression of Rabenosyn-5 inhib-
its cathepsin D processing, suggesting that the two pro-
teins play distinct roles in endosomal trafficking. We
propose that Rab5-dependent formation of membrane
domains enriched in phosphatidylinositol-3-phosphate
has evolved as a mechanism for the recruitment of mul-
tiple effector proteins to mammalian early endosomes,
and that these domains are multifunctional, depending
on the differing activities of the effector proteins re-
endocytosis • Rab5 • hVPS45 • EEA1 •
In eukaryotic cells, trafficking of membrane and proteins
through the biosynthetic and endocytic pathways is subject
to regulation by Rab GTPases (Mellman, 1996). Different
members of the Rab GTPase family have been identified
to localize to distinct compartments of the endomembrane
system (Simons and Zerial, 1993). In several cases, these
Rab GTPases have been demonstrated to regulate diverse
functions that are associated with membrane trafficking,
such as vesicle formation (Jones et al., 1993; McLauchlan
et al., 1998), vesicle docking, and fusion events (Salminen
and Novick, 1987; Stenmark et al., 1995b; Mayer and
Wickner, 1997; Simonsen et al., 1998; Christoforidis et al.,
1999a). Recently, in addition to their role in regulation of
membrane tethering and docking of organelles, Rab GTP-
ases have been implicated in the regulation of organelle
association with, or movement upon, cytoskeletal net-
works within eukaryotic cells (Echard et al., 1998; Nielsen
et al., 1999).
In the case of vesicle transport, a complex series of pro-
tein interactions ensures the coupling between the vesicle
tethering, which is regulated by Rab GTPases through their
effector proteins (Mayer and Wickner, 1997; Christoforidis
et al., 1999a; Guo et al., 1999), and membrane fusion, which
occurs through priming and proper pairing of soluble
-ethylmaleimide–sensitive factor (NSF)
tein (SNAP) receptor (SNARE) molecules. However, the
mechanism underlying the coupling of these two processes
is still poorly understood. For the small GTPase Rab5,
which regulates membrane traffic into and between early
endosomes in mammalian cells, the coordination between
vesicle tethering and SNARE function has been proposed
to involve the organization of a specialized membrane do-
Address correspondence to Marino Zerial, Max-Planck-Institute for Mo-
lecular Cell Biology and Genetics, Pfotenhauerstrasse, 01307 Dresden,
Germany. Tel.: 49-6221-387-232. Fax: 49-6221-387-512.
S. Christoforidis’ present address is Laboratory of Biological Chemis-
try, Medical School, University of Ioannina, 45110 Ioannina, Greece.
early endosome antigen 1; EGFP, enhanced GFP; EH, E15 homology; EST,
expressed sequence tag; GFP, green fluorescent protein; GST, glutathione
-ethylmaleimide–sensitive factor; P, phosphate; PC,
phosphatidylcholine; PI, phosphatidylinositol; PVC, prevacuolar compart-
ment; SNAP, soluble NSF attachment protein; SNARE, SNAP receptor.
Abbreviations used in this paper:
CCV, clathrin-coated vesicle; EEA1,
The Journal of Cell Biology, Volume 151, 2000
main on early endosomes (McBride et al., 1999; Sonnichsen
et al., 2000). Consistent with this hypothesis, morphological
studies have shown that Rab5 occupies a restricted mem-
brane domain on endosomes that displays distinct bio-
chemical features compared with the neighboring subcom-
partments occupied by Rab4 and Rab11 (Sonnichsen et al.,
2000). The Rab5 GTPase forms this domain by recruit-
ing the specific phosphatidylinositol (PI)-3-kinase isoform
hVPS34, causing localized production of PI-3-phosphate
(PI-3P) (Christoforidis et al., 1999b). The concomitant
presence of Rab5 and PI-3P is necessary for the efficient re-
cruitment of the Rab5 effector protein early endosome an-
tigen 1 (EEA1) on early endosomes. EEA1, which serves
as a membrane-tethering molecule, binds PI-3P through
the interaction of a specialized zinc finger, called a FYVE
finger (Simonsen et al., 1998). However, the recent demon-
stration that at least 20 proteins specifically interact with
active Rab5 highlights the complexity of the downstream
regulation of this GTPase (Christoforidis et al., 1999a), and
raises the possibility that other effector proteins could be
involved in regulation of endosomal fusion events.
Here, we identify and characterize a novel Rab5 effector
complex that plays an important role in endosome fusion
events. This complex contains two proteins. One is the
previously described Sec1-like protein, hVPS45. The sec-
ond is a novel protein that interacts directly with Rab5.
We have named this protein Rabenosyn-5 to highlight its
role as a link between the Rab5 GTPase and the syntaxin
binding protein hVPS45: Rab
syn (syntaxin-binding protein hVPS45). As with
EEA1, Rabenosyn-5 contains a FYVE zinc finger, indicat-
ing that the generation of PI-3P is an important character-
istic for the recruitment of multiple Rab5 effector proteins
to the endosome.
enono (“to link” in
Materials and Methods
Antibodies, Plasmids, and Other Reagents
Human anti-EEA1 serum (1:10,000) was a gift from Ban Hok Toh (Mo-
nash Medical School, Adelaide, Australia). Secondary antibody conju-
gates (HRP and fluorescently labeled) were purchased from Dianova.
pGEM-Rab5Q79L (Stenmark et al., 1994), pGEX-syntaxin 7, and pGEX-
syntaxin 13 (McBride et al., 1999) have been described previously.
pGEX-syntaxin 6 was a gift from R. Piper (University of Iowa, Iowa City,
IA) and pGEX-hVPS45 was a gift from R. Scheller (Stanford University,
Stanford, CA). pCDNA3-syntaxin4
tal for Sick Children, University of Toronto, Toronto, Canada). pGEX-
syntaxin 4 was constructed by insertion of the syntaxin4
from a pCDNA-syntaxin4
TM into the BamHI and EcoRI sites of
pGEX-4T1 (Amersham Pharmacia Biotech). Full-length Rabenosyn-5 as
well as fragments of Rabenosyn-5 were PCR amplified using primers
against the corresponding cDNA sequence, and cloned into the EcoRI
and HindIII restriction sites of the pGEM-myc4 vector (Stenmark et al.,
1994). hVPS45 was PCR amplified and cloned into the XbaI–XhoI sites
of pBluescript II KS (Stratagene).
TM was a gift from A. Klip (Hospi-
Production of Antipeptide Antibodies against
Rabenosyn-5 and hVPS45
A peptide, CRELKHTLAKQKGGTD, corresponding to the Rabenosyn-5
COOH-terminal sequence (Genosys Inc.), and two peptides, CQGRN-
WDPAQLSRTTQ and CSRESSQATSRSASRR, corresponding to inter-
nal and COOH-terminal sequences, respectively, were synthesized (Euro-
gentec). These peptides were conjugated to keyhole limpet hemocyanin
(and mixed, in the case of the two hVPS45 peptides), and injected into
rabbits. Antipeptide pAbs were affinity purified using the respective pep-
tides immobilized on Sulfolink beads (Pierce Chemical Co.). Antibodies
were eluted from the affinity column following standard procedures and
equilibrated in PBS.
Gel Overlay Assay
This assay was a modification of the procedure of Horiuchi et al. (1997).
Proteins were separated by two-dimensional SDS-PAGE, transferred to
nitrocellulose (BA 85; Schleicher and Schuell), renatured by incubation at
C overnight, and washed as described previously (Horiuchi et al., 1997).
The blot was incubated in binding buffer (12.5 mM Hepes/KOH, pH 7.4,
1.5 mM magnesium acetate, 75 mM potassium acetate, 1 mM DTT, 2 mg/
ml BSA, 0.005% Triton X-100, 4 mM
ence of 10 mg/ml glutathione
-transferase (GST)-Rab5 loaded with ei-
S or GDP (Christoforidis and Zerial, 2000). After washing with
binding buffer, the blot was incubated for 1 h with 2.5
tibodies (Amersham Pharmacia Biotech) in binding buffer, washed again
with binding buffer, and incubated for 1 h in binding buffer in 1:5,000
anti–sheep HRP-conjugated antibodies. After washing with binding
buffer, the filter was incubated in chemiluminescence buffer (NEN Life
Sciences Products) and exposed to x-ray film.
-octylglycopyranoside) in the pres-
g/ml anti-GST an-
Amino Acid Sequence Determination and
Proteins were excised from gels and enzymatically digested (Schevchenko
et al., 1996; Wilm et al., 1996). The tandem mass spectroscopy protein se-
quencing procedure was performed as described previously (Wilm et al.,
1996; Wilm and Mann, 1996). Peptides determined from Rabenosyn-5
were from bovine brain, and were used to identify corresponding ex-
pressed sequence tags (ESTs) using BLAST similarity searches (available
at http://www.ncbi.nim.nih.gov/BLAST/). Four peptides (APEYIR,
PPHPSNLR, YSATLFVQEK, and EQFEELK) were contained in an
EST (sequence data available from EMBL/GenBank/DDBJ under acces-
sion no. W02080) that corresponded to the 5
gene. The remaining 3
end of Rabenosyn-5 was then cloned and se-
quenced by screening a random primed HeLa cDNA plasmid library
(Stenmark et al., 1995b) using a biotinylated primer, 5
, with a ClonCapture cDNA selection
Kit (CLONTECH Laboratories, Inc.).
end of the Rabenosyn-5
Cells, Transfection, and Cathepsin D Trafficking
HeLa cells were grown in MEM containing 5% heat-inactivated FCS, 5%
FCS, 100 U/ml penicillin, 100
g/ml streptomycin, 2 mM
nonessential amino acids. Stable transformed green fluorescent protein
(GFP)-Rab5 A431 cells (Nielsen et al., 1999) were grown in DMEM con-
taining 10% FCS, 100 U/ml penicillin, 100
tamine, and 0.5
g/ml G-418. For transient expression studies, HeLa cells
were infected for 30 min with T7 RNA polymerase recombinant vaccinia vi-
rus and then transfected with plasmids containing cDNAs of interest, as de-
scribed in Stenmark et al. (1995a). For cathepsin D trafficking studies,
HeLa cells were allowed to express cDNA constructs for 2 h, starved for 15
min, and then labeled with [
S]methionine for 30 min, and chased with cold
methionine for 0–4 h. Immunoprecipitation and analysis of
cathepsin D were performed as described previously (Press et al., 1998).
g/ml streptomycin, 2 mM
Confocal Immunofluorescence Microscopy
Cells grown on glass coverslips were processed for immunofluorescence as
described previously (Stenmark et al., 1995b). Cells were mounted in mo-
viol and examined on a confocal microscope (Microsystems LSM-510;
ZEISS) using an Axioplan2 microscope with 63
lens (ZEISS). Fluorescent images were collected at 2
ZEISS LSM software package, and processed using Adobe Photoshop
v5.0. Quantification of the signal overlap was performed as described pre-
viously (Sonnichsen et al., 2000).
zoom using the
In Vitro Endosomal Fusion and Recruitment Assays
Early endosomes labeled with biotinylated transferrin or antitransferrin an-
tibodies and clathrin-coated vesicles (CCVs) labeled with biotinylated
transferrin were prepared from HeLa cells (Rubino et al., 2000). In vitro fu-
sion assays were performed as in Horiuchi et al. (1997) and quantified using
the ECL-Analyzer system from IGEN Inc. Recruitment of cytosolic pro-
teins to early endosomes and liposomes was performed as described previ-
ously (Christoforidis et al., 1999b), using early endosomes from HeLa cells,
or with liposomes (98% phosphatidylcholine [PC], 2% phosphoinositides; 1
Nielsen et al.
Rabenosyn-5: A Novel Rab5 Effector Protein
mg/ml final concentration in 50 mM Hepes-KOH, pH 7.4, 110 mM KCl, 2
mM EGTA, 2 mM MgCl
) prepared as in Otter-Nilsson et al. (1999).
In Vitro Binding Assays
The GST-Rab5 affinity chromatography, and subsequent Superose-6 size-
exclusion chromatography of Rab5 effector proteins, was performed as in
Christoforidis and Zerial (2000). [
transcribed and translated in vitro using a TnT™ coupled transcription–
translation kit (Promega). For Rab5 effector recruitment assays, in vitro–
translated proteins were incubated with glutathione-sepharose beads
complexed with GST-Rab5-GTP
S or GST-Rab5-GDP, as described
in Christoforidis et al. (1999b), and then eluted using procedures as in
Christoforidis and Zerial (2000). For hVPS45 recruitment to syntaxins, in
vitro–translated hVPS45 was incubated with glutathione-sepharose beads
complexed to GST-syntaxins (5 mg/ml) in binding buffer (50 mM Hepes/
KOH, pH 8, 150 mM NaCl, 5 mM
Tween 20) overnight at 4
C. After incubation, beads were isolated and
washed in binding buffer. GST-syntaxins, and associated proteins, were
eluted with binding buffer containing 25 mM glutathione.
S]Methionine-labeled proteins were
-mercaptoethanol, 0.05% [vol/vol]
Identification of a Novel Rab5 Effector Protein
Although some previously characterized Rab5-interacting
proteins bind to Rab5 directly (e.g., the PI-3-kinase cata-
lytic subunit p110
), others (e.g., the regulatory subunit
) interact indirectly through binding to other Rab5 ef-
fectors (Christoforidis et al., 1999b). To identify those pro-
teins in the Rab5-effector protein fraction that were capa-
ble of directly interacting with Rab5, we used a previously
characterized gel-overlay assay (Horiuchi et al., 1997). In
brief, proteins eluted from the Rab5 affinity column
(Christoforidis et al., 1999b; Christoforidis and Zerial,
2000) were separated by two-dimensional gel electro-
phoresis, transferred to nitrocellulose blots, and then
probed for their ability to interact specifically with GST-
Figure 1. Rabenosyn-5 is a novel Rab5 effector protein with
FYVE finger and C2H2 zinc finger domains. (A) Specific binding
of Rab5 effector proteins to GST-Rab5-GTP?S. Rab5-interact-
ing proteins purified by GST-Rab5 affinity chromatography were
subjected to two-dimensional SDS-PAGE followed by silver
staining (left), or transferred to nitrocellulose and gel-overlay
assay with GST-Rab5-GTP?S (middle), or GST-Rab5-GDP
(right). Positions of the known Rab5 effector proteins, EEA1
(arrowhead), and Rabaptin-5? (arrow) were determined by im-
munoblotting. The position of a novel 110-kD Rab5 effector, Ra-
benosyn-5, is marked with an asterisk. (B) Alignment of the
FYVE finger domain of Rabenosyn-5 with other Rab effector
proteins. The FYVE finger domains of human Rabenosyn-5 (se-
quence data available from EMBL/GenBank/DDBJ under acces-
sion no. AY009133), S. cerevisiae Vac1p (accession no. P32609),
S. pombe Vac1p homologous protein (accession no. Z99162), and
human EEA1 (accession no. S44243) were aligned using CLUST-
ALW. (C) Domain organization of Rabenosyn-5, Vac1p, and
EEA1. Each protein is represented as a line; the relative lengths
are proportional to the length of the coding sequence. Positions
of C2H2, RING, and FYVE zinc fingers, and the NPF motif–con-
taining domains are indicated. (D) The five NPF-containing mo-
tifs of Rabenosyn-5, and their consensus sequence. (E) Sche-
matic diagram of the truncation mutations of Rabenosyn-5.
The Journal of Cell Biology, Volume 151, 2000
Rab5 in the presence of GTP
periments are displayed in Fig. 1 A. A significant fraction
of the proteins from the Rab5 column eluate were capable of
interacting directly with Rab5. Representative positions of
the three most abundant Rab5-interacting proteins were
determined by silver staining (Fig. 1 A, left). Two proteins,
indicated by an arrowhead and an arrow, corresponded to
EEA1 and Rabaptin-5, respectively. A third protein, indi-
cated with an asterisk, corresponded to an unidentified
protein with a molecular mass of 110 kD. This protein, as
well as EEA1 and Rabaptin-5, interacted specifically with
the GTP-associated form of GST-Rab5 (Fig. 1 A, middle),
but not the GDP-associated form (Fig. 1 A, right). We iso-
lated this protein band, subjected it to trypsin digestion,
and sequenced the resultant peptide mixtures by nano-
electrospray tandem mass spectrometry (Wilm et al., 1996;
Wilm and Mann, 1996).
Several of the peptides from this 110-kD protein
matched the deduced amino acid sequence of an EST (se-
quence data available from EMBL/GenBank/DDBJ un-
der accession no. W02080). Using primers derived from
end of this insert, the entire coding region of the
110- kD protein was isolated from a random primed HeLa
cDNA library (sequence data available from EMBL/Gen-
Bank/DDBJ under accession no. AY009133; see Materials
and Methods). Computer predicted structural analysis of
the open reading frame indicated that the protein was
hydrophilic with no signal peptide or potential trans-
membrane domains. When we searched the GenBank
nonredundant database using the BLAST program, we
determined that this protein showed highest homology to
Vac1 homologue from
man protein EEA1. However, in all cases, homology to
the 110-kD protein, which we called Rabenosyn-5, was
largely restricted to two predicted zinc finger domains, an
-type finger, and an internal FYVE fin-
ger domain (Fig. 1 B) (Stenmark et al., 1996; Stenmark
and Aasland, 1999). Although the domain organization
within the NH
-terminal half of Rabenosyn-5 was more
similar to Vac1p than EEA1, Rabenosyn-5 also showed
features that distinguished it from Vac1p. Vac1p con-
tained an additional RING zinc finger domain between
-type zinc finger and the FYVE finger (Fig. 1 C).
Additionally, Rabenosyn-5 contains a significantly larger
COOH-terminal region, displaying no apparent homology
to Vac1p, that contains five copies of the amino acid motif
NPF (Fig. 1 D). NPF-containing motifs have recently been
identified as the core of a binding site for proteins contain-
ing Eps15 homology (EH) domains (Salcini et al., 1997)
and are considered protein–protein interaction motifs.
Therefore, Rabenosyn-5 is a novel protein and the second
mammalian protein, after EEA1, that directly interacts
with Rab5 and contains a FYVE finger domain (Mu et al.,
1995; Simonsen et al., 1998).
S. The results from these ex-
protein Vac1p, a putative
and the hu-
Rabenosyn-5 Colocalizes with EEA1 on
Because the FYVE finger domain plays an important role
in targeting EEA1 to endosomes (Simonsen et al., 1998),
we wanted to determine whether Rabenosyn-5 was local-
ized to the same endosomes as EEA1. We performed tri-
ple labeling–confocal microscopy analysis to compare the
localization of endogenous Rabenosyn-5 and EEA1 with
each other and with Rab5 in A431 cells, which have stable
expression of enhanced GFP (EGFP)-Rab5 (Nielsen et
al., 1999). Cells were processed for immunofluorescence
for Rabenosyn-5 and EEA1 (Fig. 2 A). Both Rabenosyn-5
and EEA1 showed significant overlap with one another
and with EGFP-Rab5. When overlap of these proteins
was quantitated (see Materials and Methods),
EGFP-Rab5–positive structures colocalized with EEA1 or
95% of EEA1 structures colocalized
with Rabenosyn-5. We concluded that Rabenosyn-5 colo-
calized with Rab5-positive endosomes in vivo, as well as
interacting with Rab5 in vitro (Fig. 1 A), and that these en-
dosomes contain both EEA1 and Rabenosyn-5.
Rabenosyn-5 Is Targeted to Early Endosomes in a
PI-3-kinase–dependent Manner through a
Because EEA1 is coordinately recruited to endosomes by
the action of Rab5 and the interaction of PI-3P with its
FYVE domain (Simonsen et al., 1998), we wanted to de-
termine if the FYVE finger domain of Rabenosyn-5 was
responsible for targeting this protein to early endosomes
in a PI-3-kinase–dependent manner. First, we tested the
effect of the PI-3-kinase inhibitor, wortmannin, on recruit-
ment of Rabenosyn-5 to endosomes using an in vitro re-
cruitment assay. As for EEA1 (Simonsen et al., 1998;
Christoforidis et al., 1999b), efficient recruitment of Ra-
benosyn-5 to endosomes was cytosol and ATP dependent
(Fig. 2 B, lanes 1, 2, and 8). In the presence of wortmannin,
Rabenosyn-5 was no longer efficiently recruited to endo-
somes (Fig. 2 B, compare lanes 2 and 3). Addition of anti-
hVPS34 inhibitory antibodies, but not anti-110
specific IgG, inhibited Rabenosyn-5 recruitment (Fig. 2 B,
compare lanes 3, 4, and 5). This indicates that Rabeno-
syn-5 requires PI-3P to translocate from the cytosol to
early endosomal membranes. To provide further support
for the conclusion that Rabenosyn-5 binds endosomal
membranes specifically via PI-3P, we prepared liposomes
containing phosphatidylcholine and phosphoinositides
(Fig. 2 C). Both cytosolic and in vitro–translated Rabeno-
syn-5, as well as EEA1, were efficiently recruited to lipo-
somes containing PI-3P (Fig. 2 C). The recruitment of Ra-
benosyn-5 and EEA1 to PI-3P–containing liposomes was
specific, as no significant association was observed with li-
posomes containing PI, PI-4P, or PI-4,5P
The FYVE finger of EEA1 has been demonstrated to
overlap with one of two Rab5 interaction domains found
in EEA1 by yeast two hybrid screening methods (Simon-
sen et al., 1998). Additionally, this domain is capable of re-
cruitment to endosomes in vivo (Stenmark et al., 1996). To
determine the regions of the Rabenosyn-5 protein respon-
sible for its localization to endosomes, we constructed a se-
ries of truncation mutations of Rabenosyn-5 (see Fig. 1 E)
and coexpressed them with Rab5Q79L in HeLa cells (Fig.
3). Full-length Rabenosyn-5 (see scheme in Fig. 1 E), and
, and the FYVE finger trunca-
tion mutants all efficiently colocalized with Rab5Q79L en-
larged endosomes, whereas the
mutant did not. We conclude that the region (amino acids
100–263) containing the FYVE finger domain is capable of
, or non-
(Fig. 2 C).
Nielsen et al.
Rabenosyn-5: A Novel Rab5 Effector Protein
Figure 2. Localization of Rabenosyn-5 with early endosomes is
PI-3-kinase dependent. (A) Rabenosyn-5 colocalizes with EEA1 on
early endosomes in A431 cells. A431 cells expressing EGFP-Rab5
were processed for immunofluorescence and analyzed by laser scan-
ning confocal microscopy to detect the extent of colocalization of
EGFP-Rab5 fluorescence (top left), EEA1 was detected with hu-
man antiserum (top middle), and Rabenosyn-5 was detected with af-
finity-purified rabbit antibodies (top right). EGFP-Rab5 (green)
colocalized significantly with Rabenosyn-5 (red; bottom left) and
EEA1 (red; bottom middle); EEA1 (green) and Rabenosyn-5 (red)
displayed complete colocalization (bottom right). (B) Recruitment of Rabenosyn-5 on early endosomes. Reactions containing early endosomes,
cytosol (3 mg/ml), and an ATP-regenerating system were incubated for 30 min at 37?C (?CYT, lane 2), membranes were recovered by centrifu-
gation, resuspended in SDS-PAGE buffer, and analyzed by immunoblotting with antibodies against Rabenosyn-5. Reactions were carried out
in the absence of cytosol (?CYT, lane 1) or an ATP-regenerating system (?ATP, lane 8). Other reactions were carried with both cytosol and an
ATP-regenerating system (lanes 2–7): alone (?CYT, lane 2); in the presence of 100 nM wortmannin (WM 100 nM, lane 3); function blocking
antibodies against p110? (anti-110?, lane 4); hVPS34 (anti-hVPS34, lane 5); control nonspecific IgG (IgG, lane 6); or control concentration of
DMSO (DMSO, lane 7). (C) Recruitment of Rabenosyn-5 on artificial liposomes. Reactions containing cytosol (5 mg/ml), or in vitro–trans-
lated, [35S]methionine-labeled Rabenosyn-5 were incubated for 15 min at room temperature with liposomes (100 ?g total lipid) consisting of PC
alone (100% total lipid), or PC mixed with PI (2% total lipid), PI-3P (2%), PI-4P (2%), or PI-4,5P2 (2%). Supernatants (S) and membrane pel-
lets (P) were separated by centrifugation, resuspended in SDS-PAGE buffer (10% of total supernatants), and analyzed by immunoblotting with
antibodies specific to Rabenosyn-5, EEA1, and hVPS45, or by fluorography to detect [35S]methionine-labeled Rabenosyn-5.
The Journal of Cell Biology, Volume 151, 2000
Figure 3. The Rabenosyn-5 FYVE domain is sufficient to target Rabenosyn-5 to early endosomes. HeLa cells coexpressing
Rab5Q79L and myc-tagged truncation mutants of Rabenosyn-5 were processed for immunofluorescence and analyzed by laser scan-
ning confocal microscopy for the extent of colocalization of myc-tagged truncations of Rabenosyn-5 (red, Merge) with Rab5Q79L-
positive endosomal structures (green, Merge).
Nielsen et al.
Rabenosyn-5: A Novel Rab5 Effector Protein
targeting Rabenosyn-5 to endosomes. These results sug-
gest that, as for EEA1, both Rab5 interaction and PI-3P
are required for localization of Rabenosyn-5 to early en-
dosomes (Lawe et al., 2000).
Rabenosyn-5 Associates with the Sec1
The data presented so far imply that Rab5-dependent en-
docytic membrane transport requires two FYVE finger–
containing Rab5 effectors. We next sought to explore func-
tional differences between these two proteins. In
, Vac1p is thought to act in membrane trafficking as a
complex with the Sec1 homologue Vps45p (Burd et al.,
1997; Peterson et al., 1999; Tall et al., 1999). Given the sim-
ilarities in domain order observed between the NH
mini of Vac1p and Rabenosyn-5, and the large number of
proteins recruited to the Rab5 affinity column (Christofo-
ridis et al., 1999a), we wanted to determine if Rabenosyn-5
could be found in complex with other proteins, perhaps
Sec1-like proteins. When Rab5 effector proteins were sep-
arated by size-exclusion chromatography, we observed
that Rabenosyn-5 coeluted with a 65-kD protein (Fig. 4
A). Because this protein also eluted earlier than other pro-
teins with higher apparent molecular weight, we suspected
it might form a complex with Rabenosyn-5. Using mass
spectroscopy protein sequencing techniques, we identified
this protein as hVPS45, the human homologue of yeast
Vps45p, and a Sec1-related protein (Pevsner et al., 1996).
To determine whether Rabenosyn-5 and hVPS45 in-
deed form a complex, and if hVPS45 interacts directly or
indirectly with Rab5, we performed an affinity-capture as-
say using glutathione beads containing GST-Rab5-GTP
or GST-Rab5-GDP (Fig. 4 B). In vitro–translated Ra-
benosyn-5 alone, but not hVPS45 alone, preferentially in-
teracted with GST-Rab5 in a GTP-specific manner (Fig. 4
B). Upon cotranslation of Rabenosyn-5 with hVPS45,
hVPS45 was corecruited to GST-Rab5-GTP
firmed that Rabenosyn-5 was capable of forming a com-
plex with hVPS45, and also indicated that Rabenosyn-5
was responsible for recruitment of hVPS45 to the GST-
Rab5 column. Efficient recruitment of hVPS45, along with
Rabenosyn-5, to PI-3P–containing liposomes (see Fig. 2
C) further indicated that these proteins are corecruited to
endosomes, most likely in a complex.
S. This con-
hVPS45 Interacts with Multiple Endosomal
The observation that Rabenosyn-5 is found in complex
with hVPS45 implies that this protein serves as a link be-
tween Rab5 regulation of endosomal fusion events and reg-
ulation of endosomal SNARE complex formation. As the
yeast Vps45p can complex with different syntaxin isoforms,
e.g., Pep12p and Tlg2p (Burd et al., 1997; Nichols et al.,
1998; Abeliovich et al., 1999), hVPS45 was expected to in-
teract with multiple syntaxins present on early endosomes.
Besides syntaxin 6 (Tellam et al., 1997), hVPS45 was not
known to interact with any other syntaxin family members.
However, we have previously observed a requirement of
syntaxin 13 for homotypic endosome fusion (McBride et
al., 1999). Additionally, syntaxin 7 localizes to early endo-
somes in vivo (Prekeris et al., 1999). Therefore, we wanted
to examine if hVPS45 could interact with these syntaxins.
In vitro– translated hVPS45 was incubated with fusion pro-
teins of GST-syntaxin 4, 6, 7, and 13, or GST alone. Fig. 4 C
shows that hVPS45 interacted with GST-syntaxin 4, 6, and
13, but not GST-syntaxin 7 or GST. The lack of interaction
of hVPS45 with GST-syntaxin 7 was not simply because
this protein was inactive, since all GST-syntaxins, including
syntaxin 7, bound
-SNAP (Fig. 4 C). We conclude that
hVPS45 interacts with multiple syntaxins implicated in en-
docytic trafficking and/or TGN-endosome trafficking.
Figure 4. Rabenosyn-5 recruits the Sec1-like protein hVPS45 to
Rab5. (A) SDS-PAGE analysis and Coomassie blue staining of
Rab5-interacting proteins separated by Superose-6 size-exclusion
chromatography. Fraction numbers are indicated at the top of
each lane. MS/MS tandem mass spectroscopy sequencing identi-
fied Rabenosyn-5 and hVPS45 proteins. (B) Rabenosyn-5 recruits
hVPS45 to GST-Rab5. Glutathione-sepharose beads loaded with
GST-Rab5-GTP?S (GTP?S) or GST-Rab5-GDP (GDP) were in-
cubated with [35S]methionine-labeled in vitro–translated Rabeno-
syn-5 alone (Rabenosyn), hVPS45 alone (hVPS45), or both Ra-
benosyn-5 and hVPS45 cotranslated together (Rabenosyn ?
hVPS45). Bound proteins were eluted and analyzed by SDS-
PAGE followed by fluorography. (C) hVPS45 interacts with mul-
tiple syntaxin isoforms. Glutathione-sepharose beads loaded with
GST-syntaxin fusion proteins (GST-syntaxin 4, GST-Syn4; GST-
syntaxin 6, GST-Syn6; GST-syntaxin 7, GST-Syn7; and GST-syn-
taxin 13, GST-Syn13), or GST alone (GST), and incubated with
[35S]methionine-labeled in vitro–translated hVPS45 (top), or
?-SNAP (bottom). GST fusions and associated proteins were
eluted and analyzed by SDS-PAGE followed by fluorography.
The Journal of Cell Biology, Volume 151, 2000
Rabenosyn-5 Is Required for Fusion of Endosomes,
either Homotypically or with CCVs
An established function of Rab5 is the regulation of fusion
of plasma membrane–derived CCVs with early endosomes
and homotypic early endosome fusion. Both processes re-
quire the activity of the PI-3-kinase hVPS34, which is es-
sential for the membrane recruitment of EEA1 (Chris-
toforidis et al., 1999b). Release of EEA1 from early
endosomes after inhibition of PI-3-kinase activity was sug-
gested as the reason for inhibition of endosome fusion (Li
et al., 1995; Simonsen et al., 1998). The finding that Ra-
benosyn-5 is a Rab5 effector whose membrane association
was also mediated by its FYVE finger domain raised the
possibility that it might also play a role in endosomal fu-
sion events. Therefore, we wanted to determine if Ra-
benosyn-5 was necessary for either homotypic endosome
fusion, or fusion of endosomes with CCVs. Affinity-puri-
fied antibodies were used to quantitatively immunode-
plete Rabenosyn-5 from the cytosol (Fig. 5 A). As a con-
trol, we verified that EEA1 levels in the cytosol were
unaffected (Fig. 5 A). Additionally, hVPS45 levels were
reduced, but not quantitatively depleted, indicating that
not all cytosolic hVPS45 was associated with Rabenosyn-5.
CCV–endosome fusion and homotypic endosome fusion
were both inhibited by
80% in Rabenosyn-5 immunode-
pleted cytosol (Fig. 5 B; compare anti-Rabenosyn with
Figure 5. Requirement of Rabenosyn-5 for homotypic early endosome–early endosome, and heterotypic CCV–early endosome fusion.
(A) Immunodepletion of Rabenosyn-5 from the cytosol. 100 ?g of cytosol (cytosol), cytosol immunodepleted of Rabenosyn-5 (anti-
Rabenosyn), or cytosol immunodepleted with nonspecific IgG (IgG) were suspended in SDS-PAGE buffer, and analyzed by immuno-
blotting with antibodies specific to EEA1, Rabenosyn-5, and hVPS45. (B) Fusion of CCVs loaded with biotinylated transferrin (donor)
and early endosomes loaded with antitransferrin antibody (acceptor), or donor and acceptor loaded early endosomes was performed
under standard conditions (see Materials and Methods). Reactions were carried out either in the absence of cytosol (?Cytosol), in the
presence of untreated cytosol (Basal), in the presence of untreated cytosol but with no ATP-regenerating system (?Energy), in the
presence of cytosol immunodepleted of Rabenosyn-5 (?Rabenosyn), or in the presence of cytosol treated with nonspecific IgG (IgG).
Inhibition of fusion observed upon immunodepletion of Rabenosyn-5 could be rescued with Rab5 effector fractions containing Ra-
benosyn-5 (Fraction 33), but not with fractions containing EEA1 (Fraction 19). If Rabenosyn-5 was immunodepleted from fraction 33,
the ability of this fraction to rescue fusion was abolished (anti-Rabenosyn depleted Fraction 33).
Nielsen et al.
Rabenosyn-5: A Novel Rab5 Effector Protein
IgG lanes). Control treatment of cytosol with nonspecific
IgG did not significantly effect fusion (Fig. 5 B; compare
Basal with IgG lanes). Inhibition of endosome fusion was
rescued upon addition of a fraction from the Rab5 column
eluate containing Rabenosyn-5 and hVPS45 (Fig. 5 B,
Fraction 33). The specific presence of Rabenosyn-5 in the
cytosol was required because (a) addition of purified
EEA1 could not rescue endosome fusion (Fig. 5 B)
(Christoforidis et al., 1999a), and (b) upon immunodeple-
tion of Rabenosyn-5, fraction 33 failed to rescue the fusion
activity. Loss of Rabenosyn-5 resulted in quantitative
codepletion of hVPS45, but did not significantly reduce
the levels of other proteins present in fraction 33 (data not
shown). Because hVPS45 was not completely removed
from the cytosol upon Rabenosyn-5 immunodepletion
(Fig. 5 A), we conclude that Rabenosyn-5 is required for
fusion of plasma membrane–derived CCVs with endo-
somes, and for homotypic endosome fusion.
Rabenosyn-5, but Not EEA1, Plays a Role in
Lysosomal Trafficking of Cathepsin D
Vac1p was originally identified as a gene required for vac-
uole inheritance and vacuole protein sorting (Weisman
and Wickner, 1992; Burd et al., 1997). Since Rabenosyn-5
is similar to Vac1p and interacts with a Sec1 homologue,
hVPS45, it is possible that Rabenosyn-5 might be involved
in transport of newly synthesized lysosomal enzymes to ly-
sosomes in mammalian cells through its function on the
early endosome. To this end, we sought to examine
whether Rabenosyn-5 is involved in trafficking of cathep-
sin D from the Golgi complex to lysosomes. Cathepsin D
is synthesized as a 53-kD precursor, which is processed
into a 47-kD intermediate form in endocytic compart-
ments (Gieselmann et al., 1983). HeLa cells were trans-
fected with expression vectors containing Rabenosyn-5,
and as controls, EEA1 or empty vector. Cells were pulsed
S]methionine, chased with cold methionine for the
given times (Fig. 6 A), collected, and subjected to quanti-
tative immunoprecipitation with antibodies specific for
cathepsin D. Fig. 6 A shows that overexpression of Ra-
benosyn-5 induced a delay in the processing of proca-
thepsin D to its 47-kD intermediate. In contrast, overex-
pression of EEA1 had no effect when compared with
mock-transfected cells. It is worth noting that overexpres-
sion of Rabenosyn-5 did not induce missorting of cathep-
sin D into the culture medium (data not shown) and,
therefore, did not yield a full
One trivial possibility to explain the phenotype induced
by Rabenosyn-5 overexpression would be that the excess
of FYVE fingers sequesters the PI-3P present on early en-
dosomal membranes. However, this is unlikely given that
EEA1 has no effect. To directly clarify this point, we in-
vestigated the effect of expressing truncation mutants (see
scheme in Fig. 1 E) containing the FYVE finger and other
phenotype (Burd et al.,
Figure 6. Rabenosyn-5 overexpression impairs cathepsin D traf-
ficking. (A) Time course analysis of cathepsin D trafficking. HeLa
cells overexpressing Rabenosyn-5, EEA1, or mock transfected
were metabolically labeled with [35S]methionine for 30 min, chased
with cold methionine for the indicated times, and then cellular
cathepsin D was immunoprecipitated and relative quantities of
precursor (left) or processed intermediate (right) cathepsin D were
analyzed by SDS-PAGE followed by autoradiography. The signal
was quantified by densitometric analysis of the autoradiograms.
(B) Effect of overexpression of Rabenosyn-5 truncation mutants
upon cathepsin D trafficking. Experiments were performed as de-
scribed in A, except quantification of relative percentages of pre-
cursor cathepsin D (black bars) and processed intermediate cathep-
sin D (white bars) were performed after 4 h of chase time.
The Journal of Cell Biology, Volume 151, 2000
portions of Rabenosyn-5 on cathepsin D processing (Fig. 6
B). Neither the ?COOH-terminal truncation mutant nor
the Rabenosyn-5 FYVE finger domain (data not shown)
had any effect upon processing. We also tested the ef-
fect of the dominant-negative, COOH-terminal FYVE
finger domain of EEA1, which inhibits endosomal fusion
(EEA1-CT; Simonsen et al., 1998; McBride et al., 1999).
Overexpression of EEA1-CT had no effect upon cathep-
sin D processing (Fig. 6 B), ruling out the possibility that
titration of PI-3P and Rab5-GTP causes the cathepsin D
processing defect. However, as observed for full-length
Rabenosyn-5, the ?NH2-terminal truncation mutant inhib-
ited cleavage of cathepsin D (Fig. 6 B). In control cells, af-
ter 4 h incubation, ?58% of cathepsin D was converted to
the intermediate 47-kD form and ?42% remained as the
53-kD precursor (ratio r47kD/53kD ? 1.3), whereas in Ra-
benosyn-5 and Rabenosyn-?N overexpressing cells this
proportion was inverted, with a majority (59 and 57%, re-
spectively) remaining in the precursor form (r47kD/53kD ?
0.7). At present, the mechanism underlying this inhibition
is unclear, but interestingly involves the most divergent re-
gion between Vac1p and Rabenosyn-5 that contains NPF
motifs. In conclusion, the overexpression studies suggest
that Rabenosyn-5, but not EEA1, is somehow involved in
transport of cathepsin D from the Golgi complex to lyso-
somes, most likely at the level of the early endosomes.
Rab5 specifically interacts with PI-3-kinases in a GTP-
dependent manner (Christoforidis et al., 1999b), resulting
in localized synthesis of PI-3P. This mechanism is impor-
tant not only for membrane docking and fusion, but also
for the minus end–directed motility of endosomes along
microtubules (Nielsen et al., 1999). In identifying Rabeno-
syn-5, we have established that this system is exploited not
only to recruit EEA1 to the early endosome membrane
(Simonsen et al., 1998), but also to coordinate the recruit-
ment of multiple FYVE finger–containing Rab5 effectors
within the same membrane environment (Sonnichsen et
al., 2000). In support of this, Rabenosyn-5 displays almost
complete overlap of localization with EEA1. Although
these two Rab5 effectors share a role in the same transport
steps (homotypic endosome fusion and fusion of CCVs
to endosomes), they clearly perform distinct functions.
First, except for the presence of the FYVE finger and
Rab5-binding domains, Rabenosyn-5 and EEA1 possess
very different structural features (see Fig. 1 C). Second,
whereas Rabenosyn-5 associates with the Sec1 homologue
hVPS45, EEA1 does not appear to associate with Sec1-
like proteins. Third, the addition of EEA1 cannot rescue
endosome fusion inhibited by immunodepletion of Ra-
benosyn-5. Finally, Rabenosyn-5 overexpression inhibits
cathepsin D processing, though neither EEA1 nor its
COOH-terminal domain has any effect.
It is well established that the function of SNAREs in
membrane transport is subject to regulation by Rab pro-
teins and their effectors (Novick and Zerial, 1990). Rab ef-
fectors mediate initial docking of vesicles to their target
compartment, which must be synchronized with the prim-
ing of SNAREs and generation of trans-paired SNARE
complexes, ultimately resulting in lipid bilayer fusion (We-
ber et al., 1998). SNARE priming and pairing is a multi-
step process that must be coordinated with membrane
tethering. EEA1 has recently been demonstrated to asso-
ciate with oligomeric structures containing NSF and Ra-
baptin-5/Rabex-5 on endosomal membranes, and interacts
directly with syntaxin 13 in vitro (McBride et al., 1999).
Through EEA1 (Christoforidis et al., 1999a), membrane
tethering can be spatially and temporally coupled to
SNARE priming by NSF. However, SNAREs have been
shown to bind several regulatory proteins in cis that modu-
late their ability to form complexes with other SNAREs
(e.g., tomosyn) (Fujita et al., 1998). Among them, Sec1-
like proteins are thought to serve as negative regulators of
SNARE pairing by sequestering syntaxin molecules (Pevs-
ner et al., 1994; Yang et al., 2000). For syntaxins to assem-
ble into membrane fusion–competent complexes, these
proteins must first be removed. Recent studies of the
structure of the Sec1–syntaxin 1A complex raise the possi-
bility that binding of other proteins, possibly Rab effec-
tors, to Sec1 could trigger conformational changes causing
Sec1 to “present” the syntaxin molecule to other SNARE
complex members (Misura et al., 2000). We propose that
for endosomal SNAREs, this function would be contrib-
uted by Rabenosyn-5 through its interaction with hVPS45.
Rabenosyn-5 could confer similar changes to endosomal
syntaxins and present them to EEA1, or the SNARE
priming machinery, NSF and ?-SNAP. In the case of syn-
taxin 13, this would then allow for efficient pairing of this
protein to EEA1, thus leading to a transition from en-
docytic membrane docking to fusion. Sec1p has also been
shown to bind preassembled SNARE complexes, suggest-
ing that it may play an active role in SNARE-dependent
membrane docking and fusion (Carr et al., 1999). In this
case, by presenting hVPS45 to trans-paired v-t-SNAREs,
Rabenosyn-5 would thus stabilize the fusion complex. Im-
portantly, both Rabenosyn-5 and EEA1 are recruited to
PI-3P–enriched Rab5 endosomal subcompartments (Son-
nichsen et al., 2000) by FYVE finger domains. If SNAREs
flow along the pathway of membrane traffic between the
Golgi complex, plasma membrane, and endosomes mostly
in an inhibited conformation, upon arrival in the Rab5 do-
main, the concomitant presence of the two Rab5 effectors
would ensure local activation of these molecules and their
engagement in fusion-competent complexes. It is also not
excluded that Rabenosyn-5 itself may directly participate
together with EEA1 in endosome membrane docking and
In yeast, the connection between Rab GTPases and
SNARE function in prevacuolar membrane trafficking has
been attributed to the action of a single protein, Vac1p.
Vac1p was found in complex with Pep12p and Vps45,
upon isolation of these proteins from cells with mutant
NSF (Sec18) (Burd et al., 1997), and these proteins are im-
plicated in proper sorting and trafficking of proteins from
the TGN to the vacuole (Piper et al., 1994; Halachmi and
Lev, 1996; Burd et al., 1997). Vac1p also interacts with the
yeast homologue of Rab5, Vps21p/Ypt51p (Horazdovsky
et al., 1994; Singer-Krüger et al., 1994; Peterson et al.,
1999). Due to the common features between EEA1 and
Vac1p, (both are Rab5 effectors and have a FYVE finger),
EEA1 has been considered as the mammalian homologue
Nielsen et al. Rabenosyn-5: A Novel Rab5 Effector Protein
of Vac1p (Peterson et al., 1999). This view needs to be re-
examined in light of our data. With respect to domain or-
ganization, Rabenosyn-5 shares more homology to Vac1p
than EEA1 and, like Vac1p, it complexes with hVPS45.
Therefore, regarding SNAREs, Rabenosyn-5 may exhibit
similar functions as Vac1p. Indeed, we have found that
overexpression of Rabenosyn-5 results in inhibition of
cathepsin D processing, suggesting a role for Rabenosyn-5
in trafficking of newly synthesized proteins through early
endosomes en route to lysosomes. This is not due to a gen-
eral perturbation of endosome function, as recycling of
transferrin to the plasma membrane is not inhibited under
the same conditions (De Renzis, S., and M. Zerial, unpub-
Exactly which step along this transport route is affected
and by what mechanism are not clear at present. It is possi-
ble that Rab5 and Rabenosyn-5 may regulate the influx of
vesicles not only from the plasma membrane, but also
from the Golgi complex towards early endosomes, as pro-
posed for Vac1p in yeast. Given its ability to interact with
syntaxin 6 (Simonsen et al., 1999), in addition to syntaxin
13 (McBride et al., 1999), EEA1 may also participate in
the same transport reaction, despite the fact that no effect
was observed in our experiments. Alternatively, Rabeno-
syn-5 may not only participate in the biosynthetic trans-
port to endosomes, but may also carry out additional func-
tions on the early endosome that are critical for lysosomal
enzyme sorting and transport. In this respect, it is interest-
ing to note that expression of the COOH-terminal region
of Rabenosyn-5, to which Vac1p does not display homol-
ogy, inhibited cathepsin D processing. This region con-
tains several NPF motifs, which mediate interactions with
proteins containing EH domains. Many EH domain–con-
taining proteins have been implicated in endocytic traf-
ficking and signaling pathways (Di Fiore et al., 1997), rais-
ing the possibility that Rabenosyn-5 interaction with as yet
unidentified EH domain–containing partner(s) may be re-
sponsible for the observed cathepsin D trafficking defects.
There also appear to be important differences between
yeast and mammalian proteins and their site(s) of action in
the pathways. In mammalian cells, transport of lysoso-
mal hydrolases from the Golgi complex to lysosomes is
thought to intersect the endocytic pathway at the level of
the early endosome (Ludwig et al., 1991; Press et al.,
1998). In S. cerevisiae, Vac1p and Vps21p/Ypt51p are
thought to participate in transport from the Golgi complex
to a prevacuolar compartment (PVC), the postulated
equivalent of a mammalian late endosome (Vida et al.,
1993; Gerrard et al., 2000). However, direct evidence that
these proteins play a role in fusion of Golgi-derived vesi-
cles to PVCs has not been provided. Furthermore,
whereas Rab5 regulates plasma membrane to early endo-
some transport in mammalian cells, Vps21p/Ypt51p is
thought to function between early endosomes and PVCs
in yeast (Gerrard et al., 2000). Using a specific in vitro fu-
sion assay, we have shown that, like Rab5, Rabenosyn-5 is
required for fusion of plasma membrane–derived CCVs
with the early endosomes. To date, such function has not
been attributed to Vac1p. In yeast, Vps45p interacts with
Tlg2p, in addition to Pep12p (Nichols et al., 1998). These
interactions have been demonstrated to reflect involve-
ment of Vps45p in distinct trafficking pathways to vacu-
oles (Abeliovich et al., 1999). Given that hVPS45 interacts
with several different syntaxin homologues, including syn-
taxin 6, 13, and 4, the role of Rabenosyn-5 and hVPS45
may, therefore, include all trafficking steps involving these
components, e.g., sorting between the vacuole and lyso-
somes, recycling to the plasma membrane, and integration
of these trafficking pathways in highly polarized cells.
In conclusion, our results suggest that in contrast to bud-
ding yeast, which centers on Vac1p, the mammalian early
endocytic system has evolved at least two proteins, Ra-
benosyn-5 and EEA1, to cope with the regulation of
SNARE priming and complex formation in vesicular traf-
ficking on early endosomes. Future work should shed light
on the function of this machinery in the entry and sorting
of proteins in early endosomes of mammalian cells.
We are grateful to Drs. A. Klip, R. Piper, H. Stenmark, and R. Scheller for
providing antibodies and plasmids. We would also like to thank A. Giner
for technical assistance. Special thanks to Dr. H. McBride and S. De Ren-
zis for valuable discussions and critical reading of the manuscript.
E. Nielsen, S. Christofordis, and M. Miaczynska were recipients of Eu-
ropean Molecular Biology Organization (EMBO) Long-term, Max-
Planck, and Human Frontier Science Program Fellowships, respectively.
This work was supported by the Max Planck Gesellschaft and by grants
from the Human Frontier Science Program (RG-432/96), European
Union-Training and Mobility of Researchers (EU TMR) (ERB-CT96-
0020), and Biomed (BMH4-97-2410) (M. Zerial).
Submitted: 12 June 2000
Revised: 24 August 2000
Accepted: 30 August 2000
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