Molecular Biology of the Cell
Vol. 20, 4563–4574, November 1, 2009
HOPS Interacts with Apl5 at the Vacuole Membrane and
Is Required for Consumption of AP-3 Transport Vesicles
Cortney G. Angers and Alexey J. Merz
Department of Biochemistry, University of Washington, Seattle, WA 98195-3750
Submitted April 6, 2009; Revised August 25, 2009; Accepted August 27, 2009
Monitoring Editor: Sandra Lemmon
Adaptor protein complexes (APs) are evolutionarily conserved heterotetramers that couple cargo selection to the formation
of highly curved membranes during vesicle budding. In Saccharomyces cerevisiae, AP-3 mediates vesicle traffic from the
late Golgi to the vacuolar lysosome. The HOPS subunit Vps41 is one of the few proteins reported to have a specific role
in AP-3 traffic, yet its function remains undefined. We now show that although the AP-3 ? subunit, Apl5, binds Vps41
directly, this interaction occurs preferentially within the context of the HOPS docking complex. Fluorescence microscopy
indicates that Vps41 and other HOPS subunits do not detectably colocalize with AP-3 at the late Golgi or on post-Golgi
(Sec7-negative) vesicles. Vps41 and HOPS do, however, transiently colocalize with AP-3 vesicles when these vesicles dock
at the vacuole membrane. In cells with mutations in HOPS subunits or the vacuole SNARE Vam3, AP-3 shifts from the
cytosol to a membrane fraction. Fluorescence microscopy suggests that this fraction consists of post-Golgi AP-3 vesicles
that have failed to dock or fuse at the vacuole membrane. We propose that AP-3 remains associated with budded vesicles,
interacts with Vps41 and HOPS upon vesicle docking at the vacuole, and finally dissociates during docking or fusion.
Vesicle trafficking is used by eukaryotes to move cargo
throughout the cell while maintaining the steady-state mem-
brane and protein composition of each compartment. As
there are numerous vesicle trafficking pathways, each step of
trafficking (budding, transport, and fusion) should impart
specificity. During budding, cytosolic adaptor proteins se-
lect specific cargo molecules for incorporation into incipient
transport vesicles. Cargo recruitment is coupled to vesicle
formation, as many adaptors also recruit an outer-shell coat,
such as clathrin, and other proteins involved in membrane
deformation (McMahon and Mills, 2004; Owen et al., 2004).
One family of adaptors, the adaptor protein complexes
(APs), consists of four heterotetramers (AP-1, -2, -3, and -4)
involved in distinct trafficking pathways within the endo-
cytic system (Owen et al., 2004). AP-3 has an assigned role
both in higher eukaryotes and in yeast. In metazoans, AP-3
regulates trafficking from early endosomes to late endo-
somes, lysosomes, and lysosome-related organelles such as
platelet-dense granules, melanosomes, and neutrophil gran-
ules. In addition, a neuron-specific AP-3 isoform mediates
trafficking of a subset of synaptic vesicles (Newell-Litwa et
al., 2007). In Saccharomyces cerevisiae, AP-3 regulates traffic
from the late Golgi to the vacuolar lysosome through a
pathway that bypasses the late endosome (Cowles et al.,
1997a,b; Piper et al., 1997; Stepp et al., 1997).
AP complexes are composed of a small ? subunit, a me-
dium ? subunit, a large conserved ? subunit, and a large
unique subunit (?, ?,?, or ?, for AP-1, -2, -3, or -4, respec-
tively). Each of the two large subunits consists of an
N-terminal “trunk” domain that interacts with the other
subunits, a C-terminal “ear” domain, and a flexible hinge
that links the ear to the trunk (Owen et al., 2004). The AP-1
and AP-2 ear domains play key roles during budding by
recruiting clathrin and other factors necessary for formation
of the nascent vesicle. Interactions of AP-3 ears with clathrin
and other proteins during budding are not as well char-
acterized (Owen et al., 2000; Praefcke et al., 2004; Schmid
et al., 2006).
Besides AP-3, one of the few S. cerevisiae proteins reported
to have a specific function in AP-3 trafficking is Vps41.
Unlike vps41? mutants, which exhibit defects in all traffick-
ing pathways that converge upon the vacuole, two alleles of
VPS41 (vps41tsfand vps41-231) were reported to exhibit more
selective defects in transport of the AP-3–specific cargo,
alkaline phosphatase (ALP), without impairing trafficking
through the parallel carboxypeptidase Y (CPY) pathway
(Paravicini et al., 1992; Cowles et al., 1997a; Stepp et al., 1997;
Darsow et al., 2001). Both alleles encode single amino acid
substitutions within conserved domains of Vps41: domain I
(aa 99-231), an uncharacterized domain conserved among
Vps41 homologues, and a clathrin heavy chain repeat
(CHCR) domain (aa 753-901), which appears to mediate
Vps41 self-association in vitro (Darsow et al., 2001). Remi-
niscent of clathrin–AP interactions, the N-terminal region of
Vps41 is predicted to fold into a ?-propeller and interacts
with Apl5, the large ? subunit of AP-3, through its ear
domain (Rehling et al., 1999; Darsow et al., 2001; our unpub-
lished results). This interaction was hypothesized to under-
lie the ALP trafficking defect of vps41-231 cells, as the vps41-
231 mutation abrogates Vps41–Apl5 binding (Darsow et al.,
This article was published online ahead of print in MBC in Press
on September 9, 2009.
Address correspondence to: Alexey J. Merz (firstname.lastname@example.org.
Abbreviations used: ALP, alkaline phosphatase; AP, adaptor pro-
tein complex; CORVET, class C core vacuole/endosomal tethering;
CPY, carboxypeptidase Y; GEF, guanine nucleotide exchange factor;
HOPS, homotypic fusion and vacuole protein sorting; SNARE, sol-
uble N-ethylmaleimide–sensitive factor attachment protein recep-
tor; VPS-C, vacuolar protein sorting class C.
© 2009 by The American Society for Cell Biology4563
2001). Together, these findings prompted the suggestion that
Vps41 polymerizes into a clathrin-like outer shell for AP-3
vesicles (Rehling et al., 1999; Darsow et al., 2001).
Vps41 functions in vacuole fusion as a subunit of the
HOPS (homotypic fusion and vacuole protein sorting) dock-
ing complex, which is required for both homotypic and
heterotypic fusion at the vacuole (Seals et al., 2000; Brett et
al., 2008; Mima et al., 2008; reviewed by Nickerson et al.,
2009). HOPS is composed of the Vps-C core (vacuolar
protein sorting class C: Vps11/Pep5, Vps16, Vps18/Pep3,
and Vps33), and two vacuole-specific subunits (Vps41 and
Vps39/Vam6). The Vps-C core also interacts with Vps3
and Vps8 (paralogs of Vps39 and Vps41) to form the class
C core vacuole/endosomal tethering (CORVET) complex
at endosomes (Peplowska et al., 2007).
The HOPS subunit Vps39 has been identified as a guanine
nucleotide exchange factor (GEF) for the vacuole Rab
GTPase Ypt7, whereas Vps41 interacts with activated Ypt7-
GTP directly and is necessary for stable recruitment of HOPS
by Ypt7 (Wurmser et al., 2000; Brett et al., 2008; unpublished
results). Ypt7-bound HOPS promotes membrane fusion both
by mediating membrane docking and by catalyzing the as-
sembly of trans-SNARE (soluble N-ethylmaleimide–sensi-
tive factor attachment protein receptor) complexes (Rieder
and Emr, 1997; Rehling et al., 1999; Price et al., 2000; Collins
and Wickner, 2007, Brett et al., 2008). Thus, HOPS activates
Ypt7, binds (through its Vps41 subunit) to active Ypt7 and
controls Ypt7-dependent membrane docking and fusion at
the vacuole (Nickerson et al., 2009). Given the importance of
Vps41 in AP-3 trafficking and its role in the HOPS complex,
we hypothesized that Vps41 binds Apl5 in the context of the
HOPS holocomplex. We now show that this is indeed the
case, and that AP-3 and HOPS colocalize predominantly at
the vacuole. We also present evidence suggesting that Vps41
and HOPS are required for AP-3 vesicle consumption at the
MATERIALS AND METHODS
Yeast Strains and Reagents
Yeast strains are summarized in Table 1. All fusion proteins were chromo-
somally expressed unless otherwise noted. Vps16 was tagged with superglow
green fluorescent protein (GFP; Kahana and Silver, 1998). Apl5 and Vps33
were tagged with a superglow GFP that included a TTX (TEV, thrombin, and
factor X) linker. Apl5 was also tagged with mCherry (Shaner et al., 2004).
GFP-Vps41 was expressed off a pRS415 plasmid under the NOP1 promoter
(LaGrassa and Ungermann, 2005). Sec7-DsRed was expressed from a pRS316
plasmid under its endogenous promoter (Calero et al., 2003).
All reagents were purchased from Fisher Scientific (Auburn, WA), Sigma-
Aldrich (St. Louis, MO), or Invitrogen (Carlsbad, CA), except the lyticase
enzyme used in preparing yeast spheroplasts (further purified from Zymol-
yase 20T from Seikagaku, Tokyo, Japan). Rabbit polyclonal sera raised against
Vps11, Vps16, Vps18, Vps33, Vps39, Vps41, Vam3, Ypt7, Vam7, Nyv1, and
GFP were gifts from W. Wickner (Dartmouth College, Hanover, NH) and
were affinity-purified and/or cross-adsorbed to reduce cross-reactivity.
Monoclonal antibodies against ALP and CPY were purchased from Invitro-
gen. Affinity-purified rabbit polyclonal antibody against Apl5 was a gift from
R. Piper (University of Iowa, Iowa City, IA).
Cloning of GST-AP-ear
Because of low sequence homology among the C-termini of the different
adaptor proteins, we used the structural motif recognition programs Pfam
and DLP-SVM to define the AP ear domains; Apl4-ear (aa 717-832), Apl5-ear
(aa 711-932), and Apl6-ear (aa 673-809; Miyazaki et al., 2002; Finn et al., 2006).
Glutathione S-transferase (GST)-tagged AP ear domains were constructed by
amplifying the corresponding DNA sequences and ligating them into a mod-
ified GST-Parallel vector at NcoI and BamHI restriction sites (Sheffield et al.,
1999). The GST-AP-ear vectors were transformed into Escherichia coli BL21
(DE3) for protein expression. BL21 (DE3) cells containing GST-AP-ear con-
structs were grown to OD600 nm?1.5 in terrific broth (TB) at 37°C, and protein
expression was induced with 200 ?M IPTG. Cells expressing GST-Apl6-ear
were incubated at 18°C overnight, and cells expressing GST, GST-Apl5-ear,
and GST-Apl4-ear were incubated for 3 h at 37°C. One-liter cultures were
harvested by centrifugation, and the cells were resuspended in 40 ml of lysis
buffer (PBS, pH 7.4 [Maniatis et al., 1984], 1 mM DTT, 1 mM PMSF, 1 ?g/ml
leupeptin, and 1 ?g/ml pepstatin). The cell suspension was snap-frozen in
liquid nitrogen and stored at ?80°C until use.
Yeast Cell Lysates
To prepare yeast lysates, yeast were grown in YPD at 30°C to OD600 nm?1.0.
Cells were harvested by centrifugation and washed in 0.1 M Tris-Cl, pH 9.4,
and 10 mM ?-mercaptoethanol for 10 min at room temperature. Cells were
Table 1. Strains
MAT? his3?1 leu2?0 lys2?0 ura3?0
BY4742; VPS16-GFP::HIS3 APL5-mCherry::HphMX
BY4742; APL5-mCherry::HphMX pRS415-NOP1pr-GFP-VPS41
BY4742; VPS16-GFP::HIS3 pRS316-SEC7-DsRed
BY4742; pRS415-NOP1pr-GFP-VPS41 pRS316-SEC7-DsRed
MAT? leu2-3,112 ura3-52 his3-?200 trp1-?901 lys2-801 suc2-?9
SEY6210; vam3tsf APL5-ttx-GFP::TRP1
MAT? pep4::HIS3 prb1-?1.6R his3-?200 lys2-801 trp1-?101 (gal3) ura3-52 gal2 can1
BJ3505; vps39?::URA3 pRS404-GFP-VPS39
BJ3505; vps39?::URA3 pRS404-GFP-VPS39 APL5-mCherry::HphMX
BJ3505; VPS33-ttx-GFP::TRP1 APL5-mCherry::HphMX
BJ3505; VPS33-ttx-GFP::TRP1 pRS316-SEC7-DsRed
BJ3505; APL5-ttx-GFP::TRP1 pRS316-SEC7-DsRed
BJ3505; vps41?::KanMX4 APL5-ttx-GFP::TRP1 pRS316-SEC7-DsRed
BJ3505; vps1?::KanMX4 APL5-ttx-GFP::TRP1 pRS316-SEC7-DsRed
Robinson et al. (1988)
This study; Darsow et al. (2001)
Jones et al. (1982)
Wang et al. (2002)
C. G. Angers and A. J. Merz
Molecular Biology of the Cell 4564
then sedimented and incubated in spheroplasting buffer (1 M sorbitol, 50 mM
Tris-Cl, pH 7.9, and 8% YPD) with lyticase at 30°C for 25 min. The sphero-
plasts were resuspended in yeast lysis buffer (20 mM HEPES, pH 6.8, 0.2 M
sorbitol, 2 mM EDTA, 50 mM potassium acetate, 1 ?g/ml aprotinin, 1 ?g/ml
leupeptin, 1 ?g/ml pepstatin, 1 ?g/ml Pefabloc-SC, and 1 mM PMSF) and
lysed by 20–30 strokes in a Dounce homogenizer. Clarified cell lysates were
produced by sedimenting unlysed cells at 1000 ? g for 5 min.
To prepare GST-affinity ligand resins, 700 ?l of BL21 (DE3) cells expressing
GST-fusion protein were thawed, lysed by sonication in the presence of 0.5%
Triton X-100 and 10 ?g DNase I, and centrifuged at 20,000 ? g for 20 min. 500
?l of the resulting clarified lysate was then added to 50 ?l of glutathione
Sepharose 4B (Amersham Biosciences, Piscataway, NJ) beads that had been
washed with PBS. After binding overnight at 4°C on a nutator, the beads were
washed with PBS, PBS ? 350 mM NaCl, and yeast lysis buffer. The beads were
then added to 500 ?l (?150 OD600 nm? ml) of yeast detergent lysate.
Detergent lysate was produced by adding 0.5% Triton X-100 to the clarified
yeast cell lysate and sedimenting the insoluble material at 20,000 ? g for 15
min. After binding for 1 h at 4°C, the beads were washed with yeast lysis
buffer. Interacting proteins were eluted in two different ways. In the first
method, beads were incubated in SDS loading buffer (350 mM Tris-Cl, pH 6.8,
10% SDS, and 30% glycerol) for 5 min at 95°C. In the second method, the
beads were incubated twice in elution buffer (50 mM Tris-Cl, pH 7.9, 20 mM
reduced glutathione, 600 mM NaCl, and 1% Triton X-100) for 10 min at 4°C.
Immunoprecipitation and Gel Filtration of the HOPS
Yeast detergent lysates from ?3000 OD600 nm? ml of GFP-Vps39 cells were
incubated with 500 ?l glutathione-Sepharose 4B decorated with GST-Apl5-
ear. After binding at 4°C for 1 h on a nutator, the resin was washed and
poured into a column, and the column was attached to a BioLogic DuoFlow
liquid chromatography system (Bio-Rad, Hercules, CA). Bound proteins were
eluted by applying a continuous NaCl gradient (0–0.7 M) in yeast lysis buffer.
HOPS-containing peak fractions were identified by Western blotting and
pooled. Five hundred microliters of the pooled lysate was incubated with 50
?l ?-GFP antibody covalently coupled to Protein A-Sepharose beads (Pierce
Chemical, Rockford, IL; see Harlow and Lane, 1999) equilibrated with yeast
lysis buffer ? 415 mM NaCl. After binding for 2 h at 4°C on a nutator, the
beads were washed with yeast lysis buffer ? 415 mM NaCl, and proteins were
eluted by incubating the beads with SDS loading buffer for 5 min at 95°C.
Alternatively, 500 ?l of the pooled HOPS-containing eluate was loaded onto
a 30 cm Superose 6 column (Amersham Biosciences) preequilibrated with
yeast lysis buffer ? 415 mM NaCl. Five hundred-microliter fractions were
collected, and 2% of each fraction was loaded onto an SDS-PAGE gel for
Western blot analysis.
GST Pulldowns with Purified Vps41 and Vps33
Pulldowns were performed as described above, but with the following
changes. N-terminally hexahistidine-tagged Vps41 and Vps33 were expressed
from a Baculovirus vector in insect cells (Brett et al., 2008). For each pulldown,
5 ?g purified Vps41 or Vps33 was diluted into 500 ?l 20 mM PIPES-KOH, pH
6.8, 200 mM sorbitol, 125 mM KCl, 1 mM DTT, and 0.5% Triton X-100. The
GST-Apl5-ear beads were pre-equilibrated and washed with the same buffer.
Bound proteins were then eluted with SDS-PAGE loading buffer.
Subcellular fractionation of yeast cell extracts was performed by modifying a
previously described protocol (Rehling et al., 1999). Clarified yeast cell lysates
were prepared as above (see Yeast Cell Lysates) and sedimented at 13,000 ? g
for 15 min to obtain P13 (pellet) and S13 (supernatant) fractions. The S13
fraction was then centrifuged at 100,000 ? g for 45 min to obtain P100 (pellet)
and S100 (supernatant) fractions. The pellets were gently resuspended in lysis
buffer. Twenty percent of the load fraction and equivalent volumes of the
other fractions were loaded onto SDS-PAGE gels and subjected to Western
Yeast cells were grown in synthetic complete medium (SC) or in SC dropout
media at 25°C overnight in the dark. Once cells reached OD600 nm?0.5, 10 ml
of culture was harvested by centrifugation, and the cells were resuspended in
50–100 ?l of synthetic media. To observe VAM3 and vam3tsfcells at nonper-
missive temperature, cells were prepared as above, placed on a 37°C heat
block for 1 h, and immediately imaged. For FM4-64 labeling of vacuoles, 5 ml
of cells was grown to OD600 nm?0.4. The cells were pelleted, resuspended in
2 ml SC containing 10 ?M FM4-64, and incubated at 30°C for 2 h. Cells were
washed once and resuspended in SC. After an additional 20-min incubation
at 30°C, cells were pelleted and resuspended in 50 ?l of SC.
Images were captured using a 100? objective (UPlanFLN 1.30; Olympus,
Melville, NY) on a microscope (IX71; Olympus) equipped with an electron-
multiplying charge-coupled device (iXon; Andor Technology, South Windsor,
CT), and IQ software (version 184.108.40.206; Andor). For some images, a custom-
built, electronically shuttered light-emitting diode illuminator was coupled to
the microscope with an optical fiber. The optical path and CCD were matched
to satisfy the Nyquist criterion. Images at 37°C were captured at the W. M.
Keck Center for Advanced Studies in Neural Signaling. These images were
acquired using a 100? objective (UPlan S-Apo, NA 1.4; Olympus) on a
microscope (DeltaVision RT; Applied Precision, Issaquah, WA) equipped
with a EM-CCD (Cascade II; Photometrics, Tucson, AZ) and enclosed in a
Plexiglas manifold kept at 37°C. Exposure times for the fluorescent proteins
were as follows: Apl5 (1500–2000 ms), Sec7 (350 ms), and HOPS subunits
(350–2000 ms). Micrographs were processed using Image J (version 1.42c;
http://rsb.info.nih.gov/ij/; National Institutes of Health) and Adobe Photo-
shop (San Jose, CA; Version 8.0) software.
The AP-3 ? Ear Domain Interacts with the HOPS
To determine whether the HOPS complex binds to Apl5,
pulldown experiments were performed by incubating wild-
type yeast detergent lysates with glutathione resin coupled
to GST-Apl5-ear, a GST fusion to the ear domain (aa 711-932)
of Apl5. As shown in Figure 1, we found that GST-Apl5-ear
bound Vps41 and the other five HOPS subunits (Vps11,
Vps16, Vps18, Vps33, and Vps39). In addition, GST-Apl5-ear
interacted with the AP-3 cargo molecules ALP (a resident
vacuole hydrolase), Vam3, and Nyv1 (SNARE proteins that
catalyze fusion at the vacuole). Nyv1 contains a sorting
motif that interacts with the AP-3 ? subunit (Wen et al.,
2006), whereas both Vam3 and ALP contain dileucine sort-
ing motifs (Darsow et al., 1998; Vowels and Payne, 1998). It
is unknown where in the AP-3 complex dileucine motifs
bind, but both Vam3 and ALP were previously reported to
Apl5-ear resin were performed as described (see Materials and Meth-
ods) with ?150 OD600 nm? ml of BY4742 yeast detergent lysate.
Bounds proteins were eluted by incubating beads in 20 mM Tris-Cl,
20 mM glutathione, and 600 mM NaCl for 10 min at 4°C (top panel)
or SDS loading buffer and heating at 95°C for 5 min (bottom panel).
Twenty percent of the load (L) and 20% of the unbound fraction (U)
relative to the bound fraction (B) were loaded, separated by SDS-
PAGE, and analyzed by immunoblot. Asterisk (*) indicates nonspe-
cific bands detected by the antibodies. The various forms of ALP are
indicated: p, proALP; m, mature ALP; and s, soluble ALP. In wild-
type cells, a large majority of ALP exists in the mature form.
GST-Apl5-ear binds HOPS subunits. Pulldowns on GST-
AP-3 Interacts with HOPS at the Vacuole
Vol. 20, November 1, 20094565
interact with Apl5 (Rehling et al., 1999). GST-Apl5-ear did
not interact with a non-AP-3 cargo, CPY (Figure 1), suggest-
ing that these interactions were specific for substrates of the
AP-3 pathway. Importantly, GST-Apl5 bound mature mALP
but not soluble sALP, a cleavage product that lacks the
cytoplasmic AP-3–targeting signal. In addition, GST-Apl5-
ear bound Vam7, a soluble SNARE that acts with at least two
other SNAREs, Vam3 and Nyv1, in fusion at the vacuole.
We next sought to determine whether Apl5 binds HOPS
subunits individually or as a holocomplex. Detergent lysate
from yeast cells expressing GFP-Vps39 was subjected to
affinity chromatography on GST-Apl5-ear resin (Figure 2A).
The column was washed, and bound proteins were sequen-
tially eluted by applying a continuous NaCl gradient. As
shown in Figure 2A, HOPS subunits Vps16, Vps18, Vps33,
and Vps41 coeluted from the Apl5-ear column as a single
peak at ?400 mM NaCl.
To further examine the oligomeric state of Apl5-bound
HOPS subunits, peak fractions from the GST-Apl5 affinity
column were pooled and subjected either to a second affinity
step (Figure 2B) or to size-exclusion chromatography (Figure
2C). Both assays confirmed that Apl5 binds HOPS as a
holocomplex. Using affinity-purified ?-GFP antibody (Fig-
ure 2B), all subunits of HOPS, including Vps41, were found
to coprecipitate with GFP-Vps39. Each HOPS subunit was
nearly cleared from the unbound fraction, indicating that the
HOPS subunits interact with Apl5 predominantly as a com-
plex that contains GFP-Vps39. In separate IP experiments,
?-GFP did not precipitate any HOPS subunits if the GFP tag
on Vps39 was not present (unpublished results). Consistent
with Vam7 elution from the GST-Apl5-ear resin at lower
ionic strength (Figure 2A), Vam7 did not coprecipitate with
To gauge the size of the HOPS complex bound to GST-
Apl5-ear, pooled eluates from the GST-Apl5-ear resin were
subjected to size exclusion chromatography (Figure 2C). The
HOPS complex has a calculated mass of 630 kDa (660 kDa
with GFP-Vps39). Eluted HOPS subunits migrated between
thyroglobulin (670 kDa) and blue dextran size standards (?2
MDa; the exclusion limit of Superose 6 is ?40 MDa). No
HOPS subunits were detected in lower molecular weight
fractions. The slightly larger-than-expected apparent mass
of HOPS may reflect a nonunitary subunit stoichiometry, the
presence of additional unidentified subunits, or deviation of
the complex from a spherical shape. Together, these results
demonstrate that the GST-Apl5-ear binds not only Vps41,
but the entire HOPS holocomplex.
An Intact Holocomplex Is Required for Efficient Binding of
HOPS to Apl5
We next asked whether the two HOPS-specific subunits,
Vps39 and Vps41, are required for binding of the remaining
HOPS subunits to GST-Apl5-ear. To answer this question,
GST-Apl5-ear pulldown studies were performed using de-
tergent lysates from vps39? or vps41? null mutant cells.
Stable HOPS-like complexes are present in each strain, as
GST-Apl5-ear glutathione resin. The resin was washed, and interacting proteins were eluted by a linear gradient of 0–0.7 M NaCl. Fractions
were immunoblotted for the presence of HOPS. (B) Peak HOPS fractions were pooled, and 500 ?l of the pooled peak was subjected to
immunoprecipitation by ?-GFP antibodies immobilized on Protein A-Sepharose. Note that the unbound material is depleted of all HOPS
subunits but not of Vam7, which binds to AP-3 independently of HOPS. (C) The pooled HOPS peak was also subjected to size exclusion
chromatography on a Superose 6 column. In calibration runs, blue dextran (2 MDa) eluted at fraction 17 (not shown), and thyroglobulin (670
kDa) eluted at fraction 26, as indicated. The exclusion limit of Superose 6 is Mr?4 ? 107.
GST-Apl5-ear interacts with the HOPS holocomplex. (A) Cell lysate from BJ3505 GFP-Vps39 was incubated for 1 h with
C. G. Angers and A. J. Merz
Molecular Biology of the Cell4566
paralogs of either Vps39 (Vps3) or Vps41 (Vps8) bind to the
remaining HOPS subunits in the absence of either subunit
(Peplowska et al., 2007). The results (Figure 3A) show that
the remaining HOPS subunits bound GST-Apl5-ear with
reduced efficiency when either Vps41 or Vps39 was absent.
HOPS binding was attenuated to a greater extent with a
vps41? cell lysate versus a vps39? cell lysate. Thus, both
Vps39 and Vps41 contribute to Apl5:HOPS interactions.
Because Vps39 and Vps41 are HOPS-specific subunits, we
next asked whether a Vps-C core subunit is needed for
efficient binding of the HOPS subunits to Apl5. In a vps16?
mutant, Vps33 is not associated with HOPS, but the remain-
ing subunits are still intact (Rieder and Emr 1997). As ex-
pected, the level of Vps33 binding to GST-Apl5-ear was
severely reduced, whereas Vps11 and Vps18 binding were
slightly attenuated (Figure 3B). Vps41 still bound GST-Apl5-
ear at levels comparable to wild type. We also tested a
CORVET mutant, vps8? and detected HOPS binding com-
parable to that of wild type.
As both Vps39 and Vps41 are known to bind Ypt7 di-
rectly, and both proteins are important for binding of HOPS
to GST-Apl5-ear, we assessed whether Ypt7 is required for
this interaction (Wurmser et al., 2000; Brett et al., 2008). In
ypt7? mutants, GST-Apl5-ear was able to bind HOPS at
levels similar to wild-type cells (Figure 3C). Furthermore,
binding of the SNARE Vam7 to the GST-Apl5-ear was un-
affected by deletion of all proteins tested, suggesting that
Vam7 and HOPS bind to the GST-Apl5-ear independently.
Taken together, these results indicate that Apl5 preferen-
tially binds HOPS as an intact holocomplex.
GST-Apl5-Ear Binds Purified Vps41 Directly
Genetic data, yeast two-hybrid assays, and GST pulldowns
of HOPS mutants suggest that Vps41 mediates binding of
HOPS to Apl5 (Figure 3, A and B; Rehling et al., 1999;
Darsow et al., 2001). However, all previous studies were
performed under conditions where additional yeast proteins
could potentially mediate the Apl5–Vps41 interaction. The
C-terminal ear domain of Apl5 is able to bind Vps41 from
yeast extracts, and this interaction requires the N-terminus
of Vps41. Previous experiments have not clearly resolved
whether the N-terminus of Vps41 binds Apl5 directly or if
this domain simply bridges the binding between Apl5 and
another HOPS subunit. Yeast two-hybrid experiments indi-
cate that Vps41 also interacts with the remaining HOPS
subunits through its N-terminus (unpublished results). To
test whether Apl5 and Vps41 interact directly, we performed
pulldown experiments using GST-Apl5-ear and purified
Vps41 and Vps33 expressed as full-length His6-tagged fu-
sions in insect cells. We found that purified His6-Vps41
bound to GST-Apl5-ear (Figure 4) but not to two other AP
ears, Apl4 (? subunit of AP-1) and Apl6 (? subunit of AP-3).
Unlike His6-Vps41, His6-Vps33 was not selectively enriched
in any of the AP-ear pulldowns. These results demonstrate
that Apl5 directly and selectively interacts with Vps41.
However, as shown above, the interaction is most efficient
when Vps41 is a constituent of the HOPS holocomplex.
Apl5 Transiently Colocalizes with HOPS at the Vacuole
To assess where in the cell AP-3 and HOPS might interact,
we prepared double-tagged strains that produce Apl5-
mCherry and various HOPS subunits marked with GFP. At
steady state, adaptor proteins exist in cytosolic and mem-
brane-associated pools. APs from the cytosolic pool are
recruited onto donor membranes during budding and are
recycled into the cytosolic pool during uncoating (Bonifacino
and Glick, 2004). Tagging of Apl5 with mCherry or GFP did
not impair the transport, processing, or subcellular distribu-
tion of ALP (Figure 5A). Consistent with its function as part
of an adaptor complex that cycles on and off membranes,
Apl5-mCherry localized to the cytosol and to small mobile
puncta (Figure 5B and Supplemental Movies 1 and 2).
binding by HOPS subunits. Pulldowns on GST-Apl5-ear were per-
formed using detergent lysate from BY4742 (WT) and the deletion
mutants (A) vps39? and vps41?, (B) vps16? and vps8?, or (C) ypt7?
as described for Figure 1. Interacting proteins were eluted by incu-
bating beads twice in 20 mM Tris-Cl, pH 7.9, 20 mM glutathione, 600
mM NaCl, and 1% Triton X-100 for 10 min at 4°C. The load (L) and
unbound (U) fractions correspond to 20% of the bound (B) fraction.
Asterisk (*) indicates a nonspecific band detected by the Ypt7 anti-
body. Note that the Vps16 antibody also detects a nonspecific band
that is enriched on the GST-Apl5 resin but not the GST resin, as seen
in the vps16? eluates, resulting in a higher apparent Vps16 signal.
(D) AP-3 preferentially binds Vps41 as part of the HOPS complex.
The organization of the HOPS subunits is a composite model from
yeast two-hybrid assays and immunoprecipitation from the Merz
Lab (unpublished results) as well as published literature (Rieder et
al., 1997; Wurmser et al., 2000). Although Vps41 interacts with the
remaining HOPS subunits in the absence of Vps39 (unpublished
results), the exact subunit interactions have not been mapped.
An intact holocomplex is required for efficient Apl5
micrograms of purified Vps41 or Vps33 was incubated with each of
the GST-AP-ear constructs. After incubation at 4°C for 1 h, the beads
were washed, and bound protein was eluted with SDS loading
buffer. The load fraction corresponds to 2% of the bound fractions.
Purified GST-Apl5-ear binds to purified Vps41. Five
AP-3 Interacts with HOPS at the Vacuole
Vol. 20, November 1, 20094567
Fluorescence microscopy and differential centrifugation
experiments indicate that Vps-C subunits are found in the
cytosol, at the vacuole as part of the HOPS complex, at
endosomes as part of the CORVET complex, and possibly at
other organelles as well (Rieder and Emr 1997; Seals et al.,
2000; Peplowska et al., 2007; this study). Consistent with
these previous reports, all tagged HOPS subunits were ob-
served on the vacuole membrane, but localization of HOPS
subunits to other sites varied. GFP-Vps39 was found almost
exclusively at the vacuole, whereas GFP-Vps41 exhibited
both vacuolar and cytoplasmic staining (Figures 5B and 6B
and Supplemental Movies 1 and 2; LaGrassa et al., 2005).
Because GFP-Vps41 was supplied from a plasmid, we veri-
fied that this fusion protein functionally complements a
vps41? null mutation and confirmed that the localization
pattern of GFP-Vps41 is identical when expressed in wild-
type VPS41 and in vps41? mutant cells (unpublished re-
sults). In marked contrast to Vps41 and Vps39, the Vps-C
core subunits Vps16 and Vps33 were found both on the
vacuole and in small punctate structures usually associated
with the vacuole. As these core Vps-C subunits are constit-
uents of CORVET as well as HOPS, the nonvacuolar Vps16
and Vps33 puncta presumably correspond to late endo-
somes. Despite the punctate distribution of Vps16 and
Vps33, we were not able to detect any HOPS subunits on
AP-3 puncta (Figure 5B; Supplemental Movies 1 and 2).
We also considered the possibility that HOPS subunits
might interact with AP-3 at the late Golgi. Exit sites on the
late Golgi are marked by Sec7, an activator of the small
G-protein Arf1, which is required for recruitment of AP-1
and AP-3 to incipient vesicles (Franzusoff et al., 1991;
Stamnes and Rothman, 1993; Traub et al., 1993; Faundez et
al., 1998; Ooi et al., 1998; Sata et al., 1998). Consistent with this
functional relationship, ?40% of AP-3 puncta colocalized
with Sec7 (Figure 6A and Supplemental Movie 4). In con-
trast, Vps41 and other HOPS subunits did not substantially
colocalize with Sec7-DsRed in time-lapse studies or at steady
state (Figure 6B; Supplemental Movies 5–7). We cannot ex-
clude the possibility that Vps41 or other HOPS subunits
associate with early AP-3 transport intermediates, either at
very low concentrations or very transiently. Nevertheless,
our results strongly suggest that Vps41 and HOPS do not
substantially concentrate at AP-3 budding sites at the late
Golgi or with post-Golgi AP-3 transport vesicles before their
arrival at the vacuole.
Although Apl5-mCherry did not show extensive colocal-
ization with HOPS subunits, we did observe transient coin-
cidence of Apl5-mCherry puncta with HOPS at the vacuole
membrane (Figure 5B, arrows; Supplemental Movies 1 and
2). Averaging multiple video frames also demonstrated
Apl5-mCherry fluorescence at the vacuole membrane in
some cells (Figure 5B, bottom panel). We also observed
transient vacuolar localization of Apl5-GFP after labeling the
vacuole with FM4-64 (Figure 5C; Supplemental Movie 3).
Taken together, these results suggest that AP-3 likely re-
mains on the vesicle throughout trafficking and that uncoat-
ing may occur during or after docking and possibly after
fusion of AP-3 vesicles at the vacuole target membrane.
Deletion of Vacuole Docking and Fusion Factors Results
in Apl5 Accumulation on Membranes
Because AP-3 binds to the HOPS holocomplex and the two
complexes transiently colocalize at the vacuole membrane,
we asked whether Vps41, HOPS, and other docking factors
regulate the subcellular distribution of AP-3. Two HOPS
deletion strains (vps16? and vps41?), a CORVET deletion
mutant (vps8?), and two strains lacking other docking and
vacuole membrane. Apl5 was C-terminally tagged with either GFP
or mCherry. (A) Strains containing these Apl5 fusions were ana-
lyzed for ALP maturation using differential centrifugation (see
Materials and Methods). (B) Strains containing Apl5-mCherry and a
GFP-tagged HOPS subunit (Vps16-GFP, Vps33-GFP, GFP-Vps41,
and GFP-Vps39) were grown to mid-log phase and analyzed by
fluorescent microscopy. Arrows indicate sites of colocalization be-
tween Apl5 puncta and HOPS at the vacuole membrane. For the
GFP-Vps39 time average (part B, bottom panels), both GFP and
mCherry fluorescence were averaged across 10 frames (?24 s). (C)
Wild-type yeast expressing Apl5-GFP were labeled with FM4-64 to
stain the vacuole membrane. For the 10-frame average, both GFP
and FM4-64 channels were averaged across 10 frames (?17 s). Bar,
2 ?m. See Supplemental Movies 1–3.
Apl5 transiently colocalizes with HOPS subunits at the
C. G. Angers and A. J. Merz
Molecular Biology of the Cell4568
fusion factors (ypt7? and vam3?) were subjected to differ-
ential centrifugation analysis. Lysates of spheroplasted cells
were centrifuged at 13,000 ? g to obtain P13 pellet and S13
supernatant fractions. The P13 fraction contains almost all of
the vacuole membrane; in wild-type cells, where ALP is
correctly localized and undergoes proteolytic maturation,
almost all of the mature ALP (mALP) was found with vacu-
oles in the P13. Likewise, almost all the vacuole Rab GTPase
Ypt7 was found in the P13. The S13 fraction was then cen-
trifuged at 100,000 ? g, yielding P100 pellet and S100 super-
natant fractions. The P100 fraction is enriched in Golgi mem-
branes and small vesicles, whereas the S100 fraction contains
cytosolic molecules including cleaved soluble ALP (sALP)
released from the lumens of ruptured vacuoles. Consistent
with the fluorescence localization of Apl5-GFP (Figure 6A),
in wild-type BY4742 cells, 30–50% of the Apl5 sedimented in
the P100 membrane fraction (Figure 7), with most of the
remainder found in the S100 cytosolic fraction.
As expected, in cells lacking proteins required for docking
or fusion at the vacuole, ALP was recovered largely in its
uncleaved pro-form (pALP), much of which accumulated in
the P100 membrane pellet fraction. Concomitantly, Apl5
redistributed almost entirely into the same P100 fraction.
This was the case for cells lacking the vacuole Rab GTPase
Ypt7, the vacuole SNARE Vam3, the HOPS/Vps-C core
subunit Vps16, or the HOPS-specific subunit Vps41. Similar
fractionation results were reported by Rehling et al. (1999)
for cells harboring temperature-sensitive alleles of vps41 and
In contrast, deletion of the CORVET-specific subunit Vps8
did not alter the subcellular distribution of Apl5, consistent
with the interpretation that AP-3 vesicles do not traverse the
endosome en route to the vacuole. We did detect a mild
defect in ALP maturation in the vps8? strain. This defect
could result from defective sorting of the Pep4 and Prb1
proteases, both of which contribute to ALP processing (Chen
and Stevens, 1996; Merz and Wickner, 2004; Anand et al.,
2009). The redistribution of Apl5 to the P100 fraction in
vacuole docking and fusion mutants cannot be explained by
a change in vacuole fractionation behavior resulting from
altered vacuole morphology. Although each of the tested
mutants (with the exception of vps8?) has fragmented vacu-
oles, the vacuole marker Ypt7 is found almost exclusively in
the P13 fraction in every mutant, and little Apl5 is found in the
P13 in any strain tested. Altogether, these results indicate that
blocking docking or fusion at the vacuole target membrane
promotes AP-3 recruitment to membranes, prevents AP-3 dis-
sociation from membranes, or both.
Post-Golgi AP-3 Vesicles Accumulate When Docking and
Fusion Are Impaired
We next asked whether the increased amount of membrane-
associated AP-3 in docking and fusion deficient cells resides
on the late Golgi or on post-Golgi vesicles. In repeated
attempts to separate these membrane populations using
equilibrium sucrose gradient fractionation, AP-3 dissociated
from the membranes and sedimented as a free complex
(unpublished data). We therefore examined AP-3 dynamics
in vivo using fluorescence microscopy.
Cells harboring the mutant SNARE allele vam3tsfdisplay
relatively normal vacuole morphology, and biochemical as-
says suggest that these cells accumulate AP-3 transport in-
termediates at nonpermissive temperatures (Darsow et al.,
1998; Rehling et al., 1999). At room temperature, vam3tsf
with GFP-HOPS subunits. Cells expressing Sec7-DsRed and (A)
Apl5-GFP or (B) GFP-HOPS subunits were analyzed by fluorescence
microscopy. Bar, 2 ?m. See Supplemental Movies 4–7.
Sec7-DsRed partially colocalizes with Apl5-GFP but not
factors results in AP-3 redistribution. Differ-
ential centrifugation of BY4742 (WT), ypt7?,
vam3?, vps16?, vps41?, and vps8? mutant
yeast lysates was performed as described in
Materials and Methods. The load lane corre-
sponds to 20% of the other fractions. A weak
nonspecific band was detected by the Ypt7
antibody (*). For clarity, this band is indicated
only for the ypt7? mutant.
Deletion of docking and fusion
AP-3 Interacts with HOPS at the Vacuole
Vol. 20, November 1, 2009 4569
mutants exhibited a wild-type distribution of Apl5-GFP
(Figure 8A). However, when incubated at nonpermissive
temperature (37°C; Figure 8A; Supplemental Movie 8), the
number of AP-3 puncta in vam3tsfcells significantly in-
creased (median ? 9 puncta/cell; n ? 50) compared with
wild-type VAM3 control cells (median ? 3 puncta/cell; n ?
50). These results are consistent with published differential
centrifugation results (Rehling et al., 1999) and with our
fractionation results for vam3? null mutants (Figure 7).
Similar to the vam3tsfcells, vps41? null mutants also dis-
played an increase in the number of Apl5-GFP puncta (me-
dian ? 12 puncta/cell; n ? 50) in comparison to wild-type
VPS41 cells (median ? 6 puncta/cell; n ? 50; Figure 8B).
These findings are remarkably consistent with our differential
localization was analyzed in VAM3 and vam3tsfcells at permissive (RT) and nonpermissive temperature (37°C). To determine the effect of
mutations in (B) vps41? and (C) the dynamin homolog, vps1?, on AP-3 trafficking, these mutants were created in strains containing Apl5-GFP
and Sec7-DsRed. For each figure, Apl5-GFP puncta were manually counted for each strain, with the results shown by box plot. Each box
indicates the central 50% of the data (25th to 75th percentile), with the median denoted by a horizontal bar. Bars indicate the range. n ? 50
cell profiles (A and B) or 100 cell profiles (C), using pooled data from two independent experiments. Significance values were calculated using
the Mann-Whitney U test. Bar, 2 ?m. See Supplemental Movies 8–10.
Disruption of vacuole SNARE function or Vps41 deletion results in accumulation of post-Golgi AP-3 puncta. (A) Apl5-GFP
C. G. Angers and A. J. Merz
Molecular Biology of the Cell4570
centrifugation studies (Figure 7) and the studies of Rehling et
al. (1999), which indicated that in vam3tsf, vam3?, vps41tsf, and
vps41? mutant cells, the membrane-associated fraction of
AP-3 approximately doubles from ?50% to ?90%. More-
over, we found that in vps41? cells there was a significant
decrease in the fraction of AP-3 puncta at Golgi exit sites
marked by Sec7-DsRed (median ? 22% in vps41? cells vs.
40% in VPS41 controls; Figure 8B). This finding strongly
suggests that most of the additional AP-3 puncta found in
vps41? cells are post-Golgi AP-3 transport vesicles.
As a point of comparison, the localization of Apl5-GFP
was examined in mutant cells lacking the dynamin homolog
Vps1. Cells lacking Vps1 or expressing mutant forms of
Vps1 exhibit strong defects in ALP trafficking, most likely
manifesting at a late Golgi compartment (Rothman et al.,
1990; Nothwehr et al., 1995; Anand et al., 2009). Unlike
vam3tsfand vps41? mutant cells, vps1? mutants did not
contain increased numbers of Apl5-GFP puncta (median ? 4
puncta/cell; n ? 100) compared with wild-type cells (me-
dian ? 4 puncta/cell; n ? 100; Figure 8C; Supplemental
Movie 10). These findings strongly suggest that post-Golgi
AP-3 vesicle accumulation occurs selectively when docking
and fusion at the vacuole are blocked, and not as a general
consequence arising from defects in AP-3 traffic.
In this study, we show that the ear domain of the AP-3 ?
subunit, Apl5, binds to Vps41 in context of the HOPS holo-
complex and present evidence that consumption of post-
Golgi AP-3 vesicles at the vacuole requires Vps41/HOPS
and other vacuole docking and fusion factors. We propose
that AP-3 remains on the vesicle at least until docking or
fusion, and that the major function of HOPS within the AP-3
trafficking pathway is to tether AP-3 vesicles to the vacuole.
A working model based on these observations is shown in
Purified Vps41 binds Apl5-ear (Figure 4), indicating that
Vps41 directly mediates the primary interaction between
Apl5 and HOPS. However, efficient binding of Vps41 to the
Apl5-ear requires the HOPS-specific subunit Vps39, but not
the Vps-C core subunit Vps16 (Figure 3). Vps16 is still re-
quired for efficient binding of the remaining HOPS subunits
to Apl5-ear (Figure 3B). These results are perhaps best ex-
plained by the subunit arrangement of the HOPS complex
(Figures 3D and 9). Vps39 associates with Vps41 directly,
whereas Vps16 does not appear to directly interact with
Vps41 (Rieder and Emr, 1997; Wurmser et al., 2000; our
unpublished results). The absence of Vps39 might trigger a
conformational change in Vps41, resulting in a reduced
affinity for Apl5. Alternatively, it is possible that HOPS
contains additional low-affinity AP-3–binding sites. Using
yeast two-hybrid assays, we detected a strong interaction
between Apl5 and Vps41 and weak interactions between
Apl5 and several of the remaining HOPS subunits (unpub-
lished results). This may explain why binding of Apl5 to
Vps11 and Vps18 is attenuated in vps16? cells (Figure 3B).
Despite the known interactions of Vps39, Vps41, and the
vacuole Rab Ypt7 (Wurmser et al., 2000; Brett et al., 2008), we
found that Ypt7 was not required for AP-3 binding to HOPS
in vitro (Figure 3C). However, differential centrifugation
experiments (Figure 7) suggest that Ypt7 is likely to play a
role in the docking and fusion of AP-3 vesicles at the vacu-
ole. We also observe that GST-Apl5-ear binds Vam7, a sol-
uble vacuole SNARE, independently of HOPS (Figures 2
and 3). This result was somewhat unexpected as Vam7 has
been reported to interact with HOPS (Stroupe et al., 2006).
Although AP-3 binds two other vacuole SNAREs (Vam3 and
Nyv1) through their cargo sorting motifs, it is possible that
these SNARE–AP-3 interactions also promote docking and
fusion at the vacuole.
Rehling et al. (1999) proposed that AP-3 binds Vps41 to
mediate AP-3 vesicle formation at the late Golgi. However,
using fluorescence microscopy to examine HOPS and Apl5
localization, we were unable to detect colocalization of
HOPS subunits on AP-3 vesicles (Figure 5B and Supplemen-
tal Movies 1 and 2). Similarly, we were unable to detect
HOPS subunits on Sec7-positive late Golgi compartments
(Figure 6B and Supplemental Movies 5–7). In contrast, we
readily detected foci enriched in Apl5 at late Golgi compart-
ments marked by Sec7 (Figures 6A and 8, B and C, and
Supplemental Movies 4 and 9). The signal-to-noise ratios in
our microscopy experiments are sufficiently high that if
Vps41 were to form a clathrin-like outer-shell matrix on
AP-3 vesicles, then Vps41 (and perhaps other HOPS sub-
units) should have been readily detected on AP-3 vesicles,
late Golgi compartments, or both.
Also arguing against a strict requirement for Vps41 in
AP-3 vesicle formation, we found that a vps41? null mutant
accumulates Apl5-GFP puncta that do not colocalize with
the late Golgi marker, Sec7 (Figure 8B), and displays a
corresponding decrease in cytosolic Apl5-GFP (Figures 7
and 8B). These phenotypes are shared by vam3tsfcells, which
have been reported to accumulate vesicles at nonpermissive
temperature (Figure 8A; Rehling et al., 1999). The accumu-
lation of Apl5 vesicles is also consistent with differential
centrifugation studies of vam3 and vps41 conditional and
null mutants (Figure 7; Rehling et al., 1999). These results
suggest that both proteins are required for docking of AP-3
vesicles at the vacuole, and this docking event must take
place before AP-3 can uncoat from the vesicle and be re-
leased back to the cytosol. Although the extent of the Apl5
puncta accumulation in both strains occurs to a similar
extent, we noticed decreased mobility of some Apl5-GFP
See text for discussion.
Working model for the AP-3 pathway in budding yeast.
AP-3 Interacts with HOPS at the Vacuole
Vol. 20, November 1, 2009 4571
puncta in the vam3tsfstrain at nonpermissive temperatures
(Supplemental Movie 8). However, both the accumulation of
puncta and the decrease in puncta mobility rapidly disap-
pear upon shifting the cells to permissive temperature (un-
published results). Similar to vam3tsfmutants, cells express-
ing the dominant-negative vacuole SNARE Vam7-Qc5?
(Schwartz and Merz, 2009) also accumulate Sec7-negative
AP-3 vesicles (our unpublished results). This argues that
AP-3 vesicle accumulation is a consequence of defective
docking or fusion at the vacuole.
In marked contrast to the docking and fusion mutants, we
found that cells lacking the dynamin homolog Vps1 do not
accumulate Apl5-GFP puncta (Figure 8C), suggesting that
Apl5-GFP puncta accumulation is not a general consequence
of perturbations to AP-3 trafficking. Although our data
strongly suggest that Vps41 is not required for AP-3 vesicle
budding, we cannot rule out the possibility that AP-3 bud-
ding sites at the late Golgi, or post-Golgi AP-3 vesicles,
contain substoichiometric amounts of HOPS at levels below
the detection threshold, or that Vps41 associates with bud-
ding AP-3 vesicles briefly and then very rapidly dissociates.
Such a role would be similar to findings that although
clathrin is not essential for endocytosis in yeast, it has an
assistive role (Baggett and Wendland, 2001).
Consistent with vesicle accumulation in strains that con-
tain nonfunctional vacuole fusion machinery, we are able to
detect transient Apl5 and HOPS colocalization at the vacuole
membrane in wild-type cells (Figure 5B; Supplemental Mov-
ies 1 and 2). Localization of Apl5 puncta at the vacuole is
seen in nearly all Apl5-tagged strains, albeit to varying
extents depending on strain background. We were also able
to see vacuolar Apl5 fluorescence when with the vital stain
FM4-64 was used to mark the vacuole (Figure 5C; Supple-
mental Movie 3).
Experiments in metazoans imply that physical and func-
tional interactions between HOPS and AP-3 are evolutionarily
conserved. Mice with mutations in AP-3 subunits ? (pearl) and
? (mocha) as well as in the HOPS subunit VPS33A (buff) display
hypopigmentation of the fur as well as blood clotting defects
(Kantheti et al., 1998; Feng et al., 1999; Suzuki et al., 2003).
Similarly, in Drosophila melanogaster, mutations in both HOPS
and AP-3 result in defective pigment granule biogenesis; the
eye pigment mutations deep orange, carnation, and light corre-
spond to VPS18, VPS33A, and VPS41, whereas garnet, ruby,
carmine, and orange correspond to the AP-3 ?, ?, ?, and ?
subunits (Ooi et al., 1997; Warner et al., 1998; Mullins et al., 1999;
Sevrioukov et al., 1999; Kretzschmar et al., 2000; Mullins et al.,
2000). Small interfering RNA knockdown of VPS16A also re-
sults in defective pigment granule biogenesis (Pulipparacharuvil
et al., 2005). Furthermore, carnation, light, and deep orange show
genetic interactions with the AP-3 ? subunit garnet (Lloyd et al.,
1998), consistent with a functional interaction between AP-3
and HOPS. Finally, AP-3 and HOPS subunits were shown to
coimmunoprecipitate from cultured mammalian cells (Salazar
et al., 2008). This interaction was detected only in the presence
of cross-linker, consistent with our finding that AP-3 interac-
tions with HOPS appear to be evanescent. Thus, experimental
results from many systems support the idea that AP-3-HOPS
interactions are broadly conserved. Because multiple traffick-
ing pathways converge on the vacuole, HOPS may also tether
other vesicle adaptors and serve as a general docking nexus at
the vacuole, an idea supported by pleiotropic phenotypes in
cells lacking HOPS subunits (reviewed in Nickerson et al.,
2009). In this context, it is intriguing that a mammalian HOPS
subunit, hVps18, was reported to bind and ubiquitylate GGA3
(Yogosawa et al., 2006).
AP-3–HOPS interactions during docking at the vacuole
are somewhat unexpected, because coat proteins generally
are thought to be shed either during or immediately after
budding and as a prerequisite to docking and fusion (re-
viewed in Bonifacino and Glick, 2004). However, the COPII
inner-shell subunit Sec23 has been shown to bind the
TRAPPI (transport protein particle) docking complex at the
Golgi (Cai et al., 2007). Like HOPS, TRAPPI has GEF activity
toward its cognate Rab (Jones et al., 2000; Cai et al., 2007).
Thus, some vesicle adaptors may remain associated with
transport vesicles to impart specificity throughout the bud-
ding and fusion cycle.
We thank Braden Lobingier (University of Washington, Seattle, WA) for
purified Vps33 and Vps41, Christian Ungermann (University of Osnabr¨ uck,
Osnabr¨ uck, Germany) for GFP-Vps41 plasmid, and Bill Wickner and Rob
Piper for generous gifts of antisera. We also thank Greg Payne, Esteban
Dell’Angelica, Greg Odorizzi, and members of the Merz lab for helpful
discussions. This work was supported by National Institutes of Health Grant
GM077349 and funds from the University of Washington. C.A. was supported
in part by Public Health Service NRSA T32 GM07270 from the National
Institute of General Medical Science.
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