The subunit of AP-3 is required for efficient transport of VSV-G from the trans-Golgi network to the cell surface

Abstract

Vesicular stomatitis virus glycoprotein (VSV-G) is a transmembrane protein that functions as the surface coat of enveloped viral particles. We report the surprising result that VSV-G uses the tyrosine-based di-acidic motif (-YTDIE-) found in its cytoplasmic tail to recruit adaptor protein complex 3 for export from the trans-Golgi network. The same sorting code is used to recruit coat complex II to direct efficient transport from the endoplasmic reticulum to the Golgi apparatus. These results demonstrate that a single sorting sequence can interact with sequential coat machineries to direct transport through the secretory pathway. We propose that use of this compact sorting domain reflects a need for both efficient endoplasmic reticulum export and concentration of VSV-G into specialized post-trans-Golgi network secretory-lysosome type transport containers to facilitate formation of viral coats at the cell surface.

Full text

The
␦
subunit of AP-3 is required for efficient
transport of VSV-G from the trans-Golgi
network to the cell surface
Noriyuki Nishimura
†
, Helen Plutner
†
, Klaus Hahn
†
, and William E. Balch
†‡§
Departments of †Cell and ‡Molecular Biology and the Institute for Childhood and Neglected Diseases, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, CA 92037
Communicated by David D. Sabatini, New York University School of Medicine, New York, NY, March 14, 2002 (received for review June 18, 2001)
Vesicular stomatitis virus glycoprotein (VSV-G) is a transmembrane
protein that functions as the surface coat of enveloped viral
particles. We report the surprising result that VSV-G uses the
tyrosine-based di-acidic motif (-YTDIE-) found in its cytoplasmic tail
to recruit adaptor protein complex 3 for export from the trans-
Golgi network. The same sorting code is used to recruit coat
complex II to direct efficient transport from the endoplasmic
reticulum to the Golgi apparatus. These results demonstrate that a
single sorting sequence can interact with sequential coat machin-
eries to direct transport through the secretory pathway. We
propose that use of this compact sorting domain reflects a need for
both efficient endoplasmic reticulum export and concentration of
VSV-G into specialized post-trans-Golgi network secretory-lyso-
some type transport containers to facilitate formation of viral coats
at the cell surface.
The mechanism(s) by which diverse types of cargo exit the
sorting environment of the trans-Golgi network (TGN) to
multiple destinations remains to be clarified. At least three major
types of cargo exit the TGN: (i) those destined for the apical or
basolateral cell surface through a constitutive pathway, (ii) those
directed to the endosomal兾lysosomal system, and (iii) those
directed into regulated, post-Golgi secretory compartments
(1–4). One set of pathways makes use of adaptor protein (AP)
complexes to direct sorting. Four different APs (AP1– 4) of
distinct subunit composition are currently recognized (2, 5, 6).
Interaction with APs can involve both tyrosine-based sorting
motifs (YXX⌽; where X can be polar residues and ⌽is a bulky
hydrophobic residue) (7) as well as di-leucine兾acidic residue-
containing motifs found in the cytoplasmic tail of transmem-
brane cargo (2). AP-2 complexes mediate endocytosis through
clathrin coats at the cell surface (2), and A P-1A adaptor
complexes contain ing the
␮
1A subunit direct clathrin-dependent
sorting of soluble enzymes from the TGN to the endosomal兾
lysosomal pathway (8). A novel AP-1 complex (AP-1B) con-
taining the
␮
1B isoform restricted to polarized epithelial cells
facilitates the sorting of the low density lipoprotein receptor
from the TGN to the basolateral surface through the tyrosine-
based sorting motif NPXY (9, 10). AP-4 has recently been
suggested to promote the basolateral targeting of a restricted set
of proteins (11). Finally, AP-3 complexes direct the transport of
transmembrane cargo from the TGN to the vacuole in yeast (5).
In mammalian cells, AP-3 directs cargo to lysosomes (12) and
specialized secretory lysosome-type compartments that include
melanosomes and platelets (13, 14).
Given the complexity of selective export of cargo from the TGN,
we used a two-hybrid approach to identify sorting factors兾coat
complexes that interact with the tyrosine-based sorting motif
(YTDIE) found in the cytoplasmic tail of vesicular stomatitis virus
glycoprotein (VSV-G), a type 1 transmembrane protein. VSV-G is
transported from the TGN to cell surface by using both vesicular
and tubular containers (15–17). We now report that AP-3, but not
AP-1 or AP-2 coat complexes, interact with the YTDIE export
motif and that AP-3 is involved in transport of VSV-G to the cell
surface in vivo. Binding to A P-3 involves a novel interaction with the
␦
subunit of AP-3 coats. Our results demonstrate that VSV-G
contains a compact sorting domain that uses overlapping codes to
recruit sequential cytosolic coat machineries that dictate movement
from the endoplasmic reticulum (ER) to the cell surface.
Materials and Methods
Materials. C57BL兾6J and Mocha mice were obtained from The
Jackson Laboratory. Rabbit antibodies against
␣
-,
␥
-, and
␦
-adaptin
were provided by M. S. Robinson (University of Cambridge,
Cambridge, U.K.). VSV-G antibodies (T25I and 8G5) have been
described (18, 19). The H4A3 mAb was obtained from the Devel-
opmental Studies Hybridoma Bank (University of Iowa, Iowa City).
DNA Constructs and Two-Hybrid Screening. pAR VSV-G-YxDxE,
-AxDxE, and -YxAxA have been described (20). Two-hybrid bait
vectors were constructed by standard techniques using the 29-
residue cytoplasmic tail of VSV-G sequences with the indicated
mutations (21). The Human HeLa MATCHMAKER cDNA li-
brary was obtained from CLONTECH, and two-hybrid screens of
a HeLa cell cDNA library in pGAD-GH were performed and
evaluated according to the manufacturer’s instructions.
Transfection and Transport. HeLa cells were transfected with pAR
vector encoding either VSV-G-YxDxE, AxDxE, or YxAxA, and
cells were lysed and immunoprecipitated with a mouse antibody
against VSV-G as described (22). Where indicated, cells were
pulse-labeled with [
35
S]methionine (0.1 mCi兾ml) at 37°C and
chased for the indicated times at 20°C or 37°C and analyzed as
described (22). To detect surface VSV-G, biotinylation was per-
formed by incubating the cells with 0.5 mg兾ml Sulfo-NHS-Biotin
(Pierce) in PBS at 4°C (30 min) and quenched with 50 mM NH
4
Cl
in PBS. Cells were lysed with 1% Triton X-100 in TBS-150 (50 mM
Tris兾150 mM NaCl, pH 7.5). Total VSV-G was immunoprecipi-
tated (22, 23), washed and eluted with 0.1 ml of 1% SDS in TBS-150
at 90°C for 3 min, and diluted with 1 ml of 1% Triton X-100 in
TBS-150. Biotinylated VSV-G was subsequently recovered with
streptoavidin-agarose and surface VSV-G was eluted with 2⫻
SDS兾PAGE sample buffer.
Results
The Cytoplasmic Tail of VSV-G Binds the
␦
Subunit of AP-3. To identify
the machinery involved in transport of VSV-G from the TGN to the
cell surface, we screened a cDNA librar y by using a yeast two-hybrid
selection for proteins that bind the wild-type sequence. Subse-
quently, we evaluated the ability of positive clones to interact with
Abbreviations: TGN, trans-Golgi network; AP, adaptor protein; VSV-G, vesicular stomatitis
virus glycoprotein; ER, endoplasmic reticulum; endo H, endoglycosidase H; PM, plasma
membrane; HA, hemaggulutinin; BHK, baby hamster kidney.
§To whom correspondence should be addressed. E-mail: webalch@scripps.edu.
The publication costs of this article were defrayed in part by page charge payment. This article
must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely
to indicate this fact.
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CELL BIOLOGY

VSV-G tail mutants in which the YTDIE motif was mutated to
either YxAxA (to remove the di-acidic code involved in ER export)
(18, 20) or AxDxE (to interfere with the function of the critical Tyr
residue involved in TGN sorting to the basolateral surface) (24). In
this way, we could focus on prey clones that interact only with the
complete YTDIE motif. Of 91 clones recovered in the first screen,
five clones were recovered in the second screen, two of which
encoded a fragment (residues 578–825) of
␦
-adaptin, a subunit of
the AP-3 complex (Fig. 1A).
To determine whether the interaction of
␦
-adaptin with the
VSV-G cytoplasmic tail was important physiologically, we exam-
ined whether they could be coprecipitated in vivo. HeLa cells were
transfected with VSV-G containing either wild-type VSV-G
(YxDxE) or the YxAxA or AxDxE mutants, lysed, and immuno-
precipitated by anti-VSV-G antibody. The resulting immunopre-
cipitates were examined by using quantitative immunoblotting.
Whereas AP-3 was found to interact strongly w ith wild-type VSV-G
(YxDxE) with ⬇7% of the total AP3 pool recovered in the
immunoprecipitate, the AxDxE mutant bound with nearly 4- to
5-fold less efficiency based on quantitative immunoblotting (Fig. 1
Band C). A similar level of reduced binding was detected with the
YxAxA mutant lacking the acidic residues (Fig. 1 Band C).
Although consistent with the two-hybrid results, the differences
observed in retention of partial binding in vivo by the AxDxE and
YxAxA mutants could reflect the contributions of other compo-
nents of the AP3 complex, in particular, the
␮
subunit known to be
involved in recognition of tyrosine-based motifs. The latter result is
consistent with previous observations that acidic residues are
critical for AP-3 binding to other cargo proteins (25–29). The
interactions with AP-3 were specific, because neither AP-1A nor
AP-2 could be detected in the VSV-G immunoprecipitate (Fig. 1B).
Thus, our results show that the
␦
subunit of AP-3 specifically
recognizes the YxDxE motif in nonpolarized cells.
AP-3 Facilitates Export of VSV-G from the TGN. The above results
suggest that the interaction of VSV-G with AP-3 requires both Tyr
and di-acidic resides. As both have also been implicated in coat
complex II recruitment (18, 20, 30), which step(s) in the secretory
pathway use the AP-3 coat to promote cargo selection? Given that
the AP-3 prey clone isolated in the two-hybrid screen encoded only
a fragment (residues 578–825) of the full-length
␦
-adaptin (1,153
aa), this fragment must contain the VSV-G binding region and
therefore may behave as a competitive inhibitor of normal AP-3
function in vivo (31). To test this possibility, the AP-3 fragment
(AP-3*) and VSV-G were cotransfected into HeLa cells and its
effect on the transport of VSV-G was determined. For this purpose,
we used a variant form tsO45 VSV-G (VSV-G
ts
) that has a
temperature-sensitive transport phenotype. When cells are trans-
fected with VSV-G
ts
at 39.5°C (the restrictive temperature), the
protein is retained in the ER because of a folding defect. Transfer
of cells to the permissive temperature (32°C) rapidly reverses this
defect, causing VSV-G
ts
to be synchronously exported from the ER
(32, 33). Delivery to the Golgi was determined by measuring
the extent of processing of VSV-G
ts
to endoglycosidase H
(endo H)-resistant forms by Golgi-associated
␣
-mannosidases and
glycosyltransferases.
As shown in Fig. 2A Lower Inset, overexpression of the AP-3* did
not have any affect on the extent of processing of VSV-G
ts
to endo
H-resistant forms. Next, we examined whether the transport of
VSV-G
ts
from the TGN to the plasma membrane (PM) was
affected by overexpression of the AP-3* by using cell surface
biotinylation (34). To only measure transport from the TGN to the
cell surface, transfected cells were labeled with [
35
S]Met at 39.5°C
and chased for 3 h at 20°C, a temperature that leads to the
accumulation of VSV-G in the TGN (35). Subsequently, cells were
incubated for the indicated time period at 37°C, transferred to ice,
and biotinylated, and cell surface VSV-G
ts
was recovered on
streptavidin beads. In contrast to the lack of effect of the AP-3* on
Fig. 1. Specific interaction of VSV-G and AP-3. (A) Two-hybrid analysis of
the interaction of
␦
-adaptin (residues 578– 825) with wild-type (YxDxE),
AxDxE, and YxAxA sorting motifs found in the VSV-G cytoplasmic tail.
␤
-Galactosidase activity of Y190 strains coexpressing the indicated VSV-G
tail and
␦
-adaptin constructs was performed in triplicate. (B) Coimmuno-
precipitation of VSV-G and APs. HeLa cells were transfected with either pAR
wild-type VSV-G or the AxDxE or YxAxA mutants, and VSV-G was immu-
noprecipitated by using anti-VSV-G antibody. The VSV-G containing im-
munoprecipitates (Left) or 10% of total lysate used for immunoprecipita-
tion (Right) were immunoblotted by using
␥
-,
␣
-, and
␦
-adaptin antibodies.
(C) Quantitation of recovery in Badjusted for total AP-3 in lysate.
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ER to Golgi transport, the rate of TGN to PM transport (Fig. 2A
Upper Inset) was significantly (30–40%) inhibited (Fig. 2 A). These
results are consistent with the localization of AP-3 to the TGN
(36–38). We conclude that the transport step in which VSV-G uses
AP-3 for delivery to the cell surface is initiated at the TGN.
Mutant Sorting Codes Show Reduced Kinetics of TGN to PM Transport.
If AP-3 is specifically required for transport of VSV-G from the
TGN to the cell surface, then the YxAxA and AxDxE VSV-G
ts
mutants, which have reduced affinity for AP-3 (Fig. 1B), should
exhibit reduced kinetics of TGN to PM transport compared with
wild-type protein. Because the YxAxA mutant is transported from
the ER to the Golgi at a much slower rate than the wild-type protein
(20), we first measured the kinetics of ER to Golgi transport of
VSV-G
ts
wild type (YxDxE) and the YxAxA and AxDxE mutants
during incubation of cells at 20°C to determined the time point at
which 50% of the protein acquired endo H resistance. These values
were 100 min for the YxDxE and AxDxE mutants and 240 min for
YxAxA (Fig. 2B), consistent with previous results (20). After
accumulation of 50% of the total VSV-G in the TGN, we then
determined the rate of TGN to PM transport of endo H-resistant
forms by cell surface biotinylation. We analyzed only the initial
40-min time period when no more than 50% of the wild-type
VSV-G had reached the cell surface to exclude any contribution of
cell surface labeling from newly synthesized VSV-G exiting the ER.
Both the YxAxA and AxDxE mutants showed significantly im-
paired rates of TGN to PM transport compared with wild-type
VSV-G
ts
(Fig. 2C). Importantly, the efficiency of TGN to PM
transport (YxDxE⬎AxDxE⬎YxAxA) was consistent with the rel-
ative affinity of VSV-G for AP-3 (YxDxE⬎AxDxE⬎YxAxA)
(Figs. 1Band 2C).Taken together, these data reinforce the con-
clusion that AP-3 functions in TGN to PM transport of VSV-G.
Transport of VSV-G Is Markedly Reduced in Mocha Mice Fibroblasts
Lacking AP-3. Analyses of hereditary deficiencies have now clearly
shown that loss of AP-3 function results in defective biosynthesis of
the secretory lysosomes including melanosomes and platelet gran-
ules (5, 13, 31, 39–45). For example, mutations in both
␦
-adaptin
(Mocha mouse) and
␤
3A-adaptin (Hermansky Pudlak syndrome
and Pearl mouse) cause defects in the biogenesis of the melano-
some pigment melanin as a consequent of failure to efficiently sort
enzymes required for melanin synthesis from the TGN to this
regulated pathway (41, 42, 45).
To further characterize the role of AP-3 in VSV-G transport, we
established a primary skin fibroblast culture from both
␦
-adaptin
deficient (Mocha) and control (C57BL兾6J) mice (45). Mocha and
control fibroblasts were transfected with wild-type VSV-G
ts
and
transport from the TGN after accumulation at 20°C was measured
with surface biotinylation. Consistent with the above results, TGN
to PM transport of VSV-G
ts
in the Mocha fibroblast was signifi-
cantly slower than control fibroblast (Fig. 3A). The reduced rate of
transport in Mocha fibroblasts was specific for the TGN to PM step,
as the rate of transport of VSV-G from the ER to the cis兾medial
Golgi compartments (based on the rate of processing of VSV-G to
endo H-resistant forms) was identical to control fibroblasts (Fig. 3B
Inset).
To examine whether impaired TGN to PM transport in Mocha
fibroblasts was specific for cargo directed to the basolateral surface,
we also measured the rate of cell surface transport of the apical
marker protein influenza virus hemaggulutinin (HA) (46 –48). In
contrast to VSV-G, whose transport to the PM was reduced in
Mocha mice, HA transport to cell surface was unaffected (Fig. 3B).
Thus, Mocha fibroblasts recapitulate the TGN sorting activities that
separate basolateral and apical targeted cargo in polarized epithe-
lial cells (47).
VSV-G Overexpression Interferes with Segregation of the AP-3-De-
pendent Cargo Protein LAMP1 into the Regulated Pathway. If VSV-G
is an authentic cargo for an AP-3-dependent transport carrier, it is
likely that transport through this specific pathway is saturable. Thus,
overexpression of VSV-G may compete with other cargo molecules
that normally use AP-3 for export from the TGN. For example, the
cell surface levels of the lysosomal proteins CD63兾Lamp3, LAMP1
and Lamp2, but not M6PR or transferrin receptor, are increased in
primary fibroblasts from Hermansky Pudlak syndrome patients
defective in the (AP-3)
␤
3A subunit (41). Moreover, disruption of
AP-3 function results in the delivery of the lysosomal marker
Lgp120 to the cell surface (12).
Fig. 2. AP-3can facilitate TGN-PM transport of VSV-G. (A) The AP-3 fragment inhibits TGN to PM transport of VSV-G. Baby hamster kidney (BHK) cells were transfected
with the pAR wild-type VSV-G and either the pCR3.1 (
䊐
) (mock) or pCR3.1-AP-3 fragment (AP-3*) (
E
). Cell surface (S) and intracellular forms are indicated (I). (Inset)
AP-3* does not inhibit ER to Golgi transport of VSV-G. BHK cells were transfected with wild-type VSV-G and either pCR3.1 (mock) or the AP-3*, labeled, and processed
for endo H resistance. (B) ER to Golgi transport of VSV-G at 20°C. Appearance of endo H-resistant forms of wild-type (YxDxE) (
䊐
) or the AxDxE (
‚
) and YxAxA (
E
) mutants
in BHK cells was measured for the indicated time at 20°C. (C) TGN to PM transport of VSV-G. Appearance of biotinylated forms of wild-type (YxDxE) (
䊐
) or the AxDxE
(
‚
) and YxAxA (
E
) mutants in BHK cells was measured for the indicated time at 37°C after either 100 min (YxDxE and AxDxE) or 240 min (YxAxA) at 20°C.
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To address this possibility, we followed the effect of VSV-G
expression on disrupting the normal transport of LA MP1 to the
lysosome by infecting HeLa cells with wild-type VSV. Dislocation
of LAMP1 to the cell surface can be detected by incubation of cells
in the presence of a LAMP1-specific mouse monoclonal IgG. If
transport to the cell surface occurs, binding of plasma membrane-
localized LAMP1 leads to internalization and the accumulation of
the antibody in the lysosome. Internalization can be quantitated by
using a mouse-specific secondary antibody and indirect immu-
nofluoresence of fixed, permeabilized cells. As shown in Fig. 4A,
HeLa cells infected with VSV for4hexpressVSV-G in the
secretory pathway including the Golgi and cell surface. HeLa cells
have an abundant population of LAMP1-containing lysosomes as
shown by antibody labeling of endogenous LAMP1 in fixed, per-
meabilized cells (Fig. 4B). In uninfected, control cells that do not
express of VSV-G, incubation in the presence of the LAMP1-
specific antibody in the medium for 45 min did not result in
internalization and labeling of the endogenous lysosome population
(Fig. 4C). In contrast, after incubation of infected cells for 45 min
with anti-LA MP1 IgG, punctate structures containing the LA MP1-
specific mouse IgG were readily detected (Fig. 4C). These results
suggest that either biosynthetic LAMP1 was displaced to the cell
surface from its normal AP3-dependent pathway during the time
course of infection and兾or that a recycling LAMP1 pool between
endosome兾lysosome compartments and the TGN was misdirected
to the cell surface. Quantitation of these results indicate that
whereas ⬎95% of infected cells showed striking internalization of
the LAMP1-specific IgG to the lysosome (300 cells counted), less
than 2% of control cells showed comparable levels of LAMP1
Fig. 3. VSV-G but not HA uses AP-3 for its transport to cell surface. (A)
Appearance of biotinylated forms of VSV-G in C57BL兾6J (
䊐
) and Mocha (
E
)
fibroblasts was measured at the indicated time at 37°C after a 100-min
preincubation at 20°C. (Inset) Appearance of endo H-resistant forms of
VSV-G in C57BL兾6J and Mocha fibroblasts was measured after a 100-min
preincubation at 20°C. (B) C57BL兾6J (
䊐
) and Mocha (
E
)fibroblasts were
transfected with HA, labeled for 20 min with [35S]Met, chased with unla-
beled Met for the indicated time at 37°C, digested with trypsin to cleave cell
surface HA, and lysed as described (64). Total HA including uncleaved (HA0
representing intracellular HA) and cleaved (HA1 and HA2 representing cell
surface HA) forms was immunoprecipitated with anti-HA antibody. The
values for surface delivery of HA are expressed as a percent of HA1 ⫹
HA2兾HA0 ⫹HA1 ⫹HA2.
Fig. 4. VSV-G can compete with LAMP1 for AP-3-mediated packaging in the
TGN. Uninfected (Band C) or infected HeLa cells (Aand D) with VSV at 32°Cas
described (33) were incubated in the presence of mouse anti-LAMP1 mAb (Cand
D) for 45 min at 32°C, fixed, permeabilized, and processed for indirect immuno-
fluorescence microscopy with the indicated antibodies as described (33). Images
typical of ⬎300 cells were examined for each indicated condition. Arrows in A
indicate VSV-G in Golgi elements, arrowheads in Band Dindicate distribution of
LAMP1 containing punctate structures. Asterisked line indicates location of den-
sity profile (pixels) shown in Cand D Insets. (Magnifications: ⫻63.)
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antibody internalization. The efficiency of internalization of
LAMP1-specific antibody in VSV-G-expressing cells is illustrated
by the ratio of the pixel intensity of antibody-containing punctate
structures to background cytoplasmic staining. This difference was
generally 5- to 10-fold that observed in control cells (Fig. 4 Cand
D Insets). We conclude that VSV-G can usurp the endogenous
TGN-derived AP-3 trafficking pathways to promote its efficient
delivery to the cell surface.
VSV-G Recruits AP-3 to the TGN. VSV infection of cells leads to
reduction in host–protein synthesis. After4hofinfection VSV-G
is a major form of cargo transiting the secretory pathway. Indeed,
we have previously demonstrated that retention of VSV-G
ts
in the
ER during infection at 39.5°C not only prevents VSV-G export, but
results in inhibition of coat complex II assembly and budding from
the ER, reflecting lack of available cargo (49). Transfer to 32°C
results in a synchronous burst of vesicles containing VSV-G bud-
ding from the ER, demonstrating that cargo can modulate coat
complex II assembly (49). Similarly, the mannose-6-phosphate
receptor can stimulate recruitment of AP-1 and GGA adaptors to
the TGN for clathrin-mediated export (50–53), and lysosomal
glycoproteins can promote recruitment of AP-3 coats to the TGN
(12). If VSV-G indeed recruits AP-3 for transport to the cell
surface, a specific prediction is that release of VSV-G
ts
from the ER
in virus-infected cells should lead to localization of A P-3 to the
TGN. This process should occur after a brief lag ref lecting the time
period (t
1/2
⬇5–10 min) for VSV-G to reach terminal Golgi
compartments (54, 55).
In infected BHK cells held at 39.5°C (Fig. 5A) or noninfected
cells at either 32°C or 39.5°C (data not shown),AP-3 had a nearly
random punctate distribution, reflecting association of AP-3 with
endosomal compartments (37). This finding is in contrast to the
diffuse distribution of VSV-G in the ER (Fig. 5B). After transfer to
32°C, there was a striking time-dependent recruitment of AP-3 to
the perinculear Golgi region (Fig. 5 A,C,E, and G) corresponding
to the typical kinetics of transport of VSV-G from the ER to the
Golgi compartments (Fig. 5 B,D,F, and H). By 10 min of incubation
where VSV-G populates the entire Golgi apparatus, 70–80% of
infected cells show striking overlap with A P-3 in the Golgi region
(Fig. 5 Gand H). Thus, movement of a single type of cargo results
in the sequential recruitment of the coat complex II (49) and AP-3
coat machineries to promote transport through the secretory
pathway.
Discussion
We have provided evidence that ex port of VSV-G from the TGN
can use AP-3 adaptor complexes through an interaction with the
␦
-subunit: (i) we observed specific binding of the VSV-G tail to
the
␦
subunit with two-hybrid analysis, (ii) VSV-G binding to
AP-3 but not A P-1 or AP-2 could be readily detected in vivo,(iii)
the AP-3 fragment inhibited TGN to cell surface, but not ER to
Golgi transport, (iv) cell surface transport of VSV-G transport
was reduced in Mocha mice fibroblasts lacking the AP-3 com-
plex, (v) overexpression of VSV-G displaced the AP3 substrate
LAMP1 to the cell surface, and (vi) synchronized release of
VSV-G from the ER in viral-infected cells results in the recruit-
ment of AP-3 to the TGN. Thus, our data suggests a new role for
AP-3 and the
␦
subunit in transport of basolateral-sorted pro-
teins containing an acidic tyrosine-based cytoplasmic sorting
motif from the TGN.
Heterotetrameric APs (1–4) have a similar structure and are
composed of two large chains (
␣
兾
␥
兾
␦
兾␧and
␤
1–4), a medium chain
(
␮
1–4), and a small light chain (
␴
1–4). The
␣
兾
␥
兾
␦
兾␧chains have
been shown to bind accessory factors (56 –58). Our data now suggest
that the
␦
subunit has sorting function. It is intriguing that the
578–825
␦
subunit fragment recovered in the two-hybrid screen
spans a putative region of the
␦
subunit that includes the hinge and
the ear, regions implicated in the interaction with Eps15, epsin,
amphiphysin, and
␥
-synergin (56–58). Although the function of
these accessory factors is unknown, we now suggest that VSV-G
may also modulate AP-3 function through the
␦
subunit to promote
rapid TGN export by AP-3.
The
␮
chains are now generally thought to recognize signals
conforming to the Yxx⌽motif found in the VSV-G cytoplasmic tail
(2). Although we have not investigated directly the ability of the
VSV-G YTDIE sorting motif to bind
␮
1–4 subunits, other studies
have clearly demonstrated that closely related peptide sequences
show strong interaction (2, 59). The structure of
␮
2 suggests that the
Tyr and ⌽residues fit into a hydrophobic pocket and that the
identity of the ⌽residue and residues flanking the critical Tyr play
an important role in recognition of specific
␮
subunits. For exam-
ple,
␮
1,
␮
2,
␮
3A, and
␮
4 prefer Leu, Leu, Ile, and Phe residues at
the ⌽position, and neutral, basic, acidic, or basic residues at the Tyr
⫹2 position, respectively (2). Interestingly, the YTDIE motif of
Fig. 5. VSV-G recruits AP-3 to the TGN. BHK cells were infected with the tsO45
VSV variant at 39.5°C for 4 h and subsequently transferred to ice (Aand B)or
incubated at 32°Cfor1(Cand D),5(Eand F)or10(Gand H) min before transfer
to ice. The distribution of VSV-G and AP-3 (
␦
-adaptin) were visualized by Texas red
and Alexa 488, respectively. (Magnifications: ⫻63.)
Nishimura et al. PNAS
兩
May 14, 2002
兩
vol. 99
兩
no. 10
兩
6759
CELL BIOLOGY

VSV-G conforms best to the
␮
3A consensus recognition motif with
an acidic residue in the Tyr ⫹2 position and Ile in the ⌽position.
Indeed, we found that mutation of these residues to Ala reduces the
rate of TGN export. Acidic stretches associated with di-leucine
motifs of the vam3 SNARE protein are also important in binding
to
␮
3 (25). Thus, export of the homotrimeric VSV-G from the TGN
may involve association with the AP-3 complex through both the
␦
and
␮
3 subunits. Such multivalent interactions with A P-3 subunits
could mediate the assembly of the coat lattice to ensure highly
efficient sorting and concentration of VSV-G for export from the
TGN.
Why is VSV-G transported to the cell surface rather than being
targeted to lysosomes or secretory lysosomes? One possibility is that
AP-3 could direct the recruitment of all classes of AP-3 interacting
proteins into a common vesicle兾tubular carrier budding from the
TGN, and that these different cargo molecules are segregated from
one another in a post-TGN compartment such as a recycling
endosome. However, the absence of VSV-G in endosomal com-
partments (16) argues against this possibility. A second possibility
stems from the observation that cargo sequestered into regulated
secretory pathways emerging from the TGN are diverted from the
constitutive pathway by the specific removal of SNAp receptors that
modulate constitutive fusion to the plasma membrane (60). Given
the ability of VSV-G to usurp the function of the putative AP-3-
regulated pathway, we suggest that it is both the type of cargo and
the type of coat that dictates the targeting information recruited to
or retained in carrier intermediates exiting the TGN. Indeed, two
distinct classes of AP-3-coated structures, clathrin-associated and
clathrin-independent AP-3s, have been reported (61), and a third
may involve a novel form of clathrin (62). Thus, a combination of
cargo, AP-3, and other coat components may favor coassembly w ith
specific vesicle targeting proteins that direct the fate of the transport
container to either the lysosome (yeast vacuole) (i.e., LAMP1),
regulated secretory lysosome (i.e., tyrosinase), or constitutive path-
ways (VSV-G, other?). Although VSV-G can use AP-3 to promote
export from the TGN, it is not the only pathway to the surface. We
found only 50% inhibition of transport in the absence of AP-3
function in the Mocha mouse fibroblast lacking A P-3. Thus, VSV-G
as well as other forms of cargo may be able to use other coat
machineries in the absence of normal AP-3 function (9, 17). Given
the recent report of the involvement of AP-4 in the basolateral
sorting of tyrosine-motif containing proteins (11), A P-4 could serve
as an alternative trafficking pathway, a possibility that remains to be
explored.
Importantly, we have discovered that the di-acidic motif in the
cytoplasmic tail of VSV-G participates in at least two sequential
selection events in the secretory pathway—the first being efficient
export from ER (18, 20) and the second, transport from the TGN
to the cell surface by a AP-3-dependent mechanism. Thus, the
YTDIE code appears to have the properties of a compact, multi-
functional sorting domain that can be used to transport cargo
through the entire secretory pathway. Because the Tyr residue, but
not the acidic residues (DxE), have previously been shown to direct
the fidelity of sorting of VSV-G from the TGN to the basolateral
surface (63) it is now apparent that the DxE motif may supplement
the specific role of Tyr in sorting by improving the efficiency of
capture into AP-3-coated structures (18, 20). The use of a multi-
functional sorting domain may reflect a need for both efficient ER
export and prepackaging of VSV-G into specialized secretory
lysosome-type transport containers to promote efficient formation
of the dense VSV-G-containing viral coat at the cell surface.
N.N. was a Senior Postdoctoral Fellow of American Cancer Society-
California Division. This work was supported by National Institutes of
Health Grant GM42336 and National Cancer Institute Grant CA58689,
Imaging Core C.
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www.pnas.org兾cgi兾doi兾10.1073兾pnas.092150699 Nishimura et al.