University of Cambridge, Department of Clinical Biochemistry, Cambridge CB2 2QR, United Kingdom
We have recently shown that two proteins
related to two of the adaptor subunits of clathrincoated vesicles, p47 (µ3) and -NAP (
3B), are part
of an adaptor-like complex not associated with clathrin (Simpson, F., N.A. Bright, M.A. West, L.S. Newman, R.B. Darnell, and M.S. Robinson, 1996. J. Cell
Biol. 133:749-760). In the present study we have
searched the EST database and have identified, cloned,
and sequenced a ubiquitously expressed homologue of
-NAP,
3A, as well as homologues of the
/
and
adaptor subunits,
and
3, which are also ubiquitously
expressed. Antibodies raised against recombinant
and
3 show that they are the other two subunits of the
adaptor-like complex. We are calling this complex
AP-3, a name that has also been used for the neuronalspecific phosphoprotein AP180, but we feel that it is a
more appropriate designation for an adaptor-related
heterotetramer. Immunofluorescence using anti-
antibodies reveals that the AP-3 complex is associated with
the Golgi region of the cell as well as with more peripheral structures. These peripheral structures show only
limited colocalization with endosomal markers and
may correspond to a postTGN biosynthetic compartment. The
subunit is closely related to the protein
product of the Drosophila garnet gene, which when mutated results in reduced pigmentation of the eyes and
other tissues. Because pigment granules are believed to
be similar to lysosomes, this suggests either that the
AP-3 complex may be directly involved in trafficking to
lysosomes or alternatively that it may be involved in another pathway, but that missorting in that pathway may
indirectly lead to defects in pigment granules.
The first step in the trafficking of proteins from a donor to an acceptor membrane compartment is the
formation of a transport vesicle. This process is mediated by coat proteins, which are recruited from the cytosol onto the donor membrane. Here they form a scaffold
that drives vesicle budding while at the same time choosing the vesicle cargo by interacting with the cytoplasmic domains of selected transmembrane proteins. The first coated
vesicles to be described were the clathrin-coated vesicles.
These are coated with clathrin and adaptor (or AP) complexes, and they bud from the TGN and the plasma membrane where they concentrate cargo proteins for delivery
to endosomal compartments. The clathrin associated with
these two membranes is the same, but the adaptors are
different: the AP-1 complex is associated with the TGN,
while the AP-2 complex is associated with the plasma membrane. More recently, two other types of coated vesicles
have been identified: COPI-coated vesicles, which bud from
the Golgi stack and the intermediate compartment; and
COPII-coated vesicles, which bud from the ER. Although
clathrin, COPI, and COPII coats are assembled from different components, there are certain features that are shared
by all coated vesicles. In all cases the membrane needs to
be primed by the binding of a small GTP-binding protein
(ARF or Sar1p) before the coat proteins can be recruited,
and in all cases the coat appears to be required not only
for vesicle budding but also for cargo selection (for review
see Schekman and Orci, 1996 There are many other membrane traffic pathways in the
cell in addition to those mediated by the three well characterized types of coats, and this has led to the suggestion
that there must be additional coat proteins to carry out
these pathways (Robinson, 1991 Immunoprecipitation experiments indicated that the
complex consists not only of
Cloning and Sequencing
The EST database was searched for proteins with homology to the The
A similar strategy was used in the cloning of the Two small chain homologues were initially identified in the EST database (GenBank/EMBL/DDBJ clones R23892 and R87391; Image consortium clone ID 131033 and 166044), cloned from human placenta and human brain, respectively, which encoded closely related but distinct
proteins, and these were used for initial sequencing and for antibody production. Although neither clone is full length (R23892 encodes amino acids 79-193 of Northern Blotting
Human multiple tissue Northern blots were purchased from Clontech
Laboratories, Inc. and probed according to the manufacturer's instructions, except that higher stringency washing conditions were used. Three
of the probes consisted of 60-mer antisense oligonucleotides that had been
end labeled with 32P. The Antibody Production and Coimmunoprecipitation
Antibodies were raised in rabbits against GST fusion proteins using PCR
to amplify the appropriate sequences that were then cloned into the expression vector pGEX-3X (Pharmacia Biotech, Piscataway, NJ). For the Immunoprecipitations of both AP-3 and AP-1 complexes were carried
out under nondenaturing conditions. Pig brain cytosol was prepared in
PBS (Seaman et al., 1993 Immunofluorescence
NRK cells and MDBK cells were grown on multiwell test slides, fixed with
either 2% paraformaldehyde followed by 0.1% NP-40 or with methanol
and acetone, and prepared for immunofluorescence as previously described (Robinson, 1987 Preparation of Drosphila Eye Sections
Wild-type (Barton strain) and mutant Drosophila were obtained from the
Bloomington Drosophila Stock Center (Bloomington, IN). Their eyes
were dissected out and fixed in glutaraldehyde followed by osmium
tetroxide as described by Seaman et al. (1993) Identification of New Adaptor-related Proteins
To find the missing subunits of the AP-3 complex we
searched the EST database for likely candidates. Both µ3
and
Fig. 2 shows the sequences of the candidate The The Both µ3 and
Composition of the Adaptor-related Protein
Complex, AP-3
To characterize
The Thus, we have correctly identified the two missing subunits of the adaptor-related complex. Like a conventional
adaptor, the complex consists of an Localization of AP-3 in Nonneuronal Cells
In our previous study we localized the AP-3 complex by
immunofluorescence and immunogold EM using antibodies against Fig. 6 shows the normal distribution of the complex in
NRK cells (a-c, and g) and in MDBK cells (h). Like the
newly recruited exogenous complex, endogenous AP-3 is
mainly perinuclear, with punctate labeling extending out
towards the cell periphery. Such punctate labeling is reminiscent of the pattern seen with markers for endosomal
compartments, so we carried out double labeling with antibodies against the transferrin receptor (Fig. 6 d), as a
marker for the early and/or recycling endosomal compartment, and against lgp120 (Fig. 6 e), as a marker for late endosomes and lysosomes. Both antibodies produced similar
types of patterns to those seen with the
Cells were also double labeled with the We also compared the distribution of
An AP-3 Mutant in Drosophila
When the database was searched for homologues of the
AP-3 subunits, we found that Fig. 8 shows sections through the eyes of both wild type
(a and d) and mutant (b, c, e, and f) flies. Two different
garnet alleles are illustrated: g3 (Fig. 8, b and e), which results in reduced pigmentation throughout the fly's body,
including dull brownish eyes; and g53d (Fig. 8, c and f),
which appears to be tissue specific, giving rise to pale orange eyes and colorless Malpighian tubules but normal
pigmentation of the testis sheath. Both phase contrast (Fig.
8, a-c) and bright field (Fig. 8, d-f) views are shown.
The repeating units, or ommatidia, of the compound eye
consist of photoreceptor cells surrounded by pigment cells
containing prominent pigment granules. In phase contrast
micrographs of eyes from wild type flies (Fig. 8 a), the pigment granules appear as bright structures surrounded by
dark rims. They are also easily visible in bright field micrographs (Fig. 8 d) because of their intense color. In g3 flies,
the pigment granules tend to be dark in phase contrast micrographs (Fig. 8 b) and are much less visible in bright
field micrographs (Fig. 8 e). This is consistent with reports
that both red and brown pigments are reduced in flies with
this allele (Nolte, 1954 These observations indicate that the garnet gene is required for pigment granule biogenesis. However, it is
likely to be involved in other pathways as well, since in situ
hybridization indicates that it is also expressed in nonpigment cells (Lloyd, V., personal communication), and its
mammalian homologue, There are many membrane traffic pathways for which no
coats have yet been identified, and this is what prompted
us initially to look for adaptor-related proteins that might
be components of novel types of coats. In a previous report we showed that two recently described proteins, p47
(µ3) and The identification of the missing subunits of the AP-3
complex relied upon the availability of random cDNA sequences in the EST database. Based on the degree of homology between µ3 and µ1/µ2 and between Both the µ and the In our previous study we were able to localize the neuronal-specific complex using antibodies against An important clue is provided by the finding that the The most likely explanation for the garnet phenotype is
a defect in the delivery of proteins to pigment granules.
The pigments themselves are small molecules, but the
granules must also contain a specific set of proteins for
synthesizing, transporting, and/or storing the pigments, and
it is possible that the sorting of these proteins may be impaired in the garnet flies. But what role might the complex
play in nonpigment cells? In mammalian cells, pigment granules have been shown to be like modified lysosomes.
Thus, patients with Chediak Higashi syndrome, or mice
with the beige mutation, have not only giant lysosomes but
also giant melanosomes (Burkhardt et al., 1993 Taken together, these observations suggest that the AP-3
complex may play a role in trafficking from the TGN to
the lysosome. However, a coat already exists for this pathway: the AP-1 and clathrin-containing coat. One possibility is that both coats are involved in this pathway but that
they act at different stages or sort different types of cargo
molecules. Alternatively, the role of the AP-3 coat in lysosomal biogenesis may be less direct. For instance, it may
be involved in a different pathway, such as trafficking to
the plasma membrane or recycling back to the TGN, but
defective sorting may result in a certain amount of "scrambling" of proteins and interfere with other pathways as well.
Specialized organelles such as pigment granules may be
particularly susceptible to such missorting and may also be
especially sensitive to problems at earlier stages of the
secretory pathway (e.g., ER to Golgi), as has been shown
for the specialized secretory granule-like trichocysts of
Paramecium (Gautier et al., 1994 Although the garnet phenotype provides important information about the role of the AP-3 complex, further
studies will be required to establish this role definitively.
One promising lead comes from the observation that another genetically tractable organism, Sarcharomyces cerevisiae, contains genes encoding homologues of all four
types of AP subunits, including some (e.g., SCYPL195W [).
). This possibility is supported by the observation that tyrosine-based sorting signals, which promote internalization of plasma membrane proteins by binding to AP-2 adaptor complexes, can function in other pathways as well, suggesting that the coats associated with such pathways may contain components related to adaptors (Matter and Mellman, 1994
; Ohno et al.,
1995
). Recently we described a novel adaptor-related protein complex that has many of the properties expected for
a component of a new type of coat (Simpson et al., 1996
).
Two of the subunits of the complex are proteins that have previously been identified: p47, a homologue of the adaptor medium chains, or µ subunits (Pevsner et al., 1994
); and
-NAP, a homologue of the adaptor
subunits (Newman
et al., 1995
). p47 exists as two isoforms: p47A, which is expressed ubiquitously, and p47B, which is specifically expressed in neuronal tissues (Pevsner et al., 1994
).
-NAP
is also neuronal specific (Newman et al., 1995
), which suggests that in nonneuronal cells there may be another, ubiquitously expressed isoform of
-NAP to form complexes
with p47A. Using a heterologous in vitro system we showed
that the p47/
-NAP complex can be recruited from brain
cytosol onto membranes prepared from tissue culture cells
or liver fractions. Recruitment is enhanced by GTP
S and
inhibited by brefeldin A, indicating that it is ARF dependent. Cell fractionation, immunofluorescence, and immunogold electron microscopy suggested that the complex binds to a subcompartment of the TGN, and this was confirmed by EM labeling of endogenous complex in primary
cultures of neuronal cells. Both the newly recruited exogenous complex and the endogenous complex were found to
be associated with buds and vesicles that were coated on
the cytoplasmic side, but these coats were thinner than a
clathrin coat. In addition, the complex did not colocalize with clathrin at either the light or the electron microscope
level and did not copurify with clathrin-coated vesicles. Thus,
we proposed that the complex is required for a nonclathrin-mediated budding event from the TGN (Simpson et al.,
1996
).
-NAP and p47, but also of
two other proteins of ~160 (p160) and ~25 kD (p25),
making it a heterotetramer like an adaptor complex. A
model for this complex is shown in Fig. 1, together with
models of the AP-1 and AP-2 complexes. We are calling
this complex AP-3, by analogy with AP-1 and AP-2. The name AP-3 has also been used for AP180, a clathrin assembly-promoting phosphoprotein specifically expressed
in neurons (Keen, 1987
; Morris et al., 1993
). However,
AP180 is a monomer that does not show any sequence homology with the adaptor subunits, so it seems more appropriate to use this name for an adaptor-related heterotetrameric complex. Both AP-1 and AP-2 consist of four
subunits:
- or
-adaptin;
1- or
2-adaptin (also called
and
); µ1 (AP47) or µ2 (AP50); and
1 (AP19) or
2
(AP17). The two subunits of AP-3 that have already been
identified, p47 and
-NAP, are labeled µ3 and
3 on the
diagram. The other two proteins that coimmunoprecipitate with µ3 and
3, p160 and p25, are labeled
and
3, respectively. We predicted that
would turn out to be a homologue of the
- and
-adaptins and that
3 would turn
out to be a homologue of
1 and
2 (Simpson et al., 1996
).
In the EST database, there are candidates for both of
these proteins as well as a candidate for a ubiquitously expressed isoform of
-NAP. We have now raised antibodies
against the
and
3 candidates expressed as fusion proteins, and here we show by coimmunoprecipitation that
they are indeed the missing subunits of the complex. To
learn more about the function of the AP-3 complex we
have used the antibodies to localize endogenous AP-3 in
nonneuronal cells and to compare its distribution with that
of other proteins. In addition, we have found a close homologue of the
subunit in Drosophila, which is encoded by
the garnet gene. Studying the mutant phenotype of the gene
provides additional insights into the role of the complex.
Fig. 1.
Diagrams of the two conventional adaptor complexes,
together with the adaptor-related complex, AP-3. The AP-1 complex is associated with the TGN, the AP-2 complex is associated
with the plasma membrane, and the AP-3 complex also appears
to be associated with the TGN as well as with more peripheral
membranes. Each complex consists of four subunits, belonging to
four different families. ,
, and
are related;
1 (
),
2 (
), and
3 (
-NAP/
3B and
3A) are related; µ1 (AP47), µ2 (AP50),
and µ3 (p47A/µ3A and p47B/µ3B) are related; and
1 (AP19),
2 (AP17), and
3 (A and B) are related. EM studies of the AP-2
complex have revealed that it has a structure resembling a head
flanked by two ears connected by flexible hinges (Heuser and
Keen, 1988
), and although such studies have not yet been carried
out on AP-1 or AP-3, the sequence homologies suggest that all
three complexes have a similar structure.
[View Larger Version of this Image (25K GIF file)]
Materials and Methods
- and
-adaptins, to
-NAP, and to the adaptor small chains
1 and
2. Clones
were then obtained from the appropriate source, sequenced, and expressed as recombinant fusion proteins for antibody production. Most molecular biology procedures were carried out as described by Sambrook et al.
(1989)
.
subunit of the complex was first identified as GenBank/EMBL/
DDBJ clone T30164. This is a cDNA cloned from human uterus encoding
a protein with homology to both
- and
-adaptins, available from the
American Type Tissue Culture (Rockville, MD). Sequencing of the clone
indicated that both the 5
and 3
ends of the cDNA were missing (the
clone encodes amino acids 22-756 of the full length
subunit; Fig. 2). The 3
end of the clone was found to be the human homologue of a bovine cDNA
previously cloned as a candidate bovine leukemia virus receptor (Ban et
al., 1993
). Although it is not clear why the
subunit was cloned in this
way, it is possible that its unusually high content of charged amino acids caused it to bind the probe nonspecifically (the screen was carried out using an expression library). We then searched the EST database with the
bovine sequence and identified another human EST (GenBank/EMBL/ DDBJ clone R54523; Image consortium clone ID 39286) encoding the COOH-terminal end of the
subunit (amino acids 865-1112). To obtain
the 5
end and the middle part of the cDNA, two library screens were carried out using a human heart cDNA library (Clontech Laboratories, Inc.,
Palo Alto, CA). A 60-mer oligonucleotide (antisense for amino acids 22-
81) was used for the 5
end, while two restriction fragments, one from
clone T30164 encoding amino acids 485-756 and one from clone R54523
encoding amino acids 865-925, were used on duplicate filters to isolate a
clone overlapping both of the database clones. Sequencing was carried out
by John Lester (University of Cambridge, Cambridge, UK) on an automated ABI sequencer using oligonucleotide primers to "walk out" along
the DNA. The entire coding sequence was read in both directions.
Fig. 2.
Sequences of novel
components of the AP-3
complex. cDNAs encoding
the four components were
originally identified as ESTs
encoding homologues of the
known AP subunits. The
complete sequences of the and
3A subunits were obtained by library screening.
Both the cDNA and the protein sequence data are available from GenBank/EMBL/ DDBJ under accession numbers U91930 (
), U91931
(
3A), U91932 (
3A), and
U91931 (
3B).
[View Larger Version of this Image (71K GIF file)]
-NAP homologue,
3A. Two cDNAs were initially found in the EST database (GenBank/
EMBL/DDBJ clones R02669 and T98538; Image consortium clone ID
124011 and 123092), encoding a protein or proteins homologous to
-NAP
but without the neuronal specificity (both were cloned from a human liver
and spleen cDNA library). Sequencing revealed that they did not overlap,
but a preliminary Northern blot indicated that they were derived from the
same mRNA. Thus, duplicate filter lifts were taken from the human heart
cDNA library and screened with probes prepared from each of the two
database clones. One of the clones that hybridized to both probes contained an insert of 3.6 kb, and when sequenced in its entirety it was found
to encode the complete open reading frame of the protein.
3A, and R87391 encodes amino acids 85-193 of
3B), subsequent database searches identified full length clones for both (GenBank/
EMBL/DDBJ clones N30960 and W97614; Image consortium clone ID
266095 and 422534), from human melanocytes for
3A and from mouse
embryo for
3B, respectively. The mouse and human
3B protein sequences are 100% identical in the region of overlap. Moreover, while this
work was in progress, Dell'Angelica et al. (1997)
independently isolated a
full length human clone encoding
3B that also shows 100% identity at
the protein level to mouse
3B.
probe was the same oligonucleotide used for
library screening; the two
3 probes both corresponded to the last five
amino acids, the stop codon, and 42 bases of 3
UTR. The probe used for
3A was the insert from clone T98538, encoding amino acids 946-1093, plus ~500 bp of 3
UTR, labeled with 32P by random priming.
subunit, the entire insert of clone T30164 was expressed, resulting in a fusion protein containing amino acids 22-756. This construct was insoluble
and was purified from inclusion bodies (Page and Robinson, 1995
). For
the two
subunits, only their COOH-terminal domains, amino acids 113-
193 were expressed, since previous studies on
1 and
2 indicated that this
region was likely to be the most antigenic part of the protein (Page and
Robinson, 1995
). These constructs were soluble and were purified by glutathione-Sepharose affinity chromatography. Immunization and affinity
purification of the resulting antisera were carried out as described by Page
and Robinson (1995)
. After affinity purification, the antisera were tested
on Western blots of whole brain homogenate. The
antiserum gave a very
clean signal, strongly labeling an ~160-kD band and much more weakly
labeling an ~120-kD band, possibly a breakdown product. The two
antisera were less specific, both labeling high molecular weight bands as well
as bands of ~20-25 kD. However, when these antisera were used for immunoprecipitations (see below) and then tested on blots of the proteins
they brought down, the low molecular weight bands were found to be specifically precipitated.
) and precleared by the addition of 100 µl of 50%
protein A Sepharose for each ml of cytosol. 10 µl of affinity-purified antibody were then added to 100 µl aliquots of the precleared cytosol, and the
samples were incubated for 1 h at room temperature. The antibodies that
were used were the
,
3A, and
3B antibodies described above, anti-
NAP (Simpson et al., 1996
), and anti-
-adaptin (Seaman et al., 1996
). After the antibody incubations, 70 µl of 50% protein A Sepharose were added and the samples incubated for an additional h at room temperature, after which the Sepharose was collected by centrifugation and washed five
times with PBS containing 0.1% NP-40. The samples were then boiled in
sample buffer, run on SDS polyacrylamide gels, and subjected to Western
blotting. The
subunit was found to elute very poorly in the absence of
carrier proteins, so in some experiments yeast cytosol was added to the
immunoprecipitates to improve the blotting efficiency. Blots were probed
with the various antibodies followed by 125I-protein A, as previously described (Robinson and Pearse, 1986
).
). For some experiments the cells were allowed to
internalize rhodamine-conjugated wheat germ agglutinin before fixation
(Seaman et al., 1993
). Other experiments involved treating the cells with 5 µg/ml brefeldin A (Sigma Chemical Co., St. Louis, MO) for 2 min before
fixation. The cells were then labeled with rabbit anti-
, either alone or together with a mouse monoclonal antibody. The monoclonal antibodies included anti-transferrin receptor (Chemicon International Inc., Temecula, CA), anti-lgp120 (Grimaldi et al., 1987
; generously provided by Paul Luzio, University of Cambridge, Cambridge, UK), anti-p200 (Narula et al.,
1992
; generously provided by Jenny Stow, University of Brisbane, Brisbane, Australia), anti-
-adaptin (Sigma Chemical Co.), and anti-clathrin
heavy chain (X22; generously provided by Frances Brodsky, University of
California at San Francisco, CA; Brodsky, 1985
). Secondary antibodies
were fluorescein-conjugated donkey anti-rabbit IgG and Texas red-conjugated sheep anti-mouse IgG, both obtained from Amersham Life Science (Arlington Heights, IL). The cells were viewed either using an Axioplan fluorescence microscope (Zeiss, Inc., Thornwood, NY) or using a
confocal microscope (MRC1000; BioRad Laboratories, Hercules, CA).
. The tissue was infiltrated
and embedded in Spurrs resin, and sections were cut for both light and
electron microscopy. The pigment granules tended to lose their contents
in thin sections, so most studies were carried out on thick (1-µm) sections,
using phase contrast and bright field microscopy.
Results
3 are more distantly related to µ1/µ2 and
1/
2 than
µ1 and µ2 or
1 and
2 are to each other (Fig. 1 and see
Fig. 3); thus, we looked for proteins with a similar type of
relationship to
- and
-adaptin and to
1 and
2, as candidates for the
and
3 subunits. We also looked for a
nonneuronal-specific homologue of
-NAP. cDNAs with
the appropriate properties were then obtained and sequenced. In some cases the cDNAs were not full length,
and it was necessary to carry out library screens to obtain
the complete coding sequence.
Fig. 3.
Diagon plots comparing related proteins in the AP-1,
AP-2, and AP-3 complexes. The sequences of all four types of
subunits were compared using the "SIP" program (Staden, 1990),
which was also used to calculate the percent identity. The
subunit shows the least similarity with its counterparts in the AP-1
and AP-2 complexes, while the
subunits (
3A and
3B) show
the most. The protein product of the Drosophila garnet gene is
also shown, compared with the
subunit.
[View Larger Version of this Image (43K GIF file)]
subunit, a
homologue of
-NAP (
3A), and two closely related
1/
2 homologues (
3A and
3B), while Fig. 3 shows diagon
plots illustrating the relationship between these proteins
and other members of the same families. The
subunit has
a predicted size of 125 kD, which is less than the apparent
molecular weight (~160 kD) of the large protein that
coimmunoprecipitates with µ3 and
3, but its unusual amino acid content (see below) may make it run anomalously on SDS polyacrylamide gels. It shares the least sequence identity of any of the novel sequences with its
counterparts in the AP-1 and AP-2 complexes, being only
~15% identical to
- and
-adaptins, and this homology is
mainly restricted to the extreme NH2-terminal portion of
the protein. However, it does have a "WIIGEY" consensus sequence, WICGEF, at the appropriate position (amino
acids 396-401). This is a motif of unknown function found
not only in the
- and
-adaptins (Robinson, 1989
, 1990
)
but also in all three types of
subunits (Kirchhausen et al.,
1989
; Ponnambalam et al., 1990
; Newman et al., 1995
) and
even in the very distantly related
-COP, a subunit of the
COPI complex (Duden et al., 1991
). The protein is also
adaptin like in that it appears to have a three domain structure consisting of an NH2-terminal domain of ~650
amino acids, a highly hydrophilic linker or hinge domain
(normally particularly rich in acidic residues, prolines, and
alanines, although the
subunit has a high proportion of
basic residues as well), and a COOH-terminal ("ear") domain.
-NAP homologue,
3A, is much more closely related to
3B (
-NAP; 61.4% identity) than to either
1 or
2 (22.6% and 21.4% identity, respectively). Its homology
to
1 and
2 is restricted to its NH2-terminal domain of
~600 amino acids, while its hinge and ear domains appear
to be unrelated. Similarly,
3B only appears to be related
to
1 and
2 in its NH2-terminal domain (Newman et al.,
1995
). The major difference between
3A and
3B lies
within their hinge domains, including amino acids 644-
814. Both have a similar amino acid composition in this region, with a high concentration of acidic residues and
serines, but their sequences diverge considerably. Interestingly, this is precisely the domain that was used for raising
3B antibodies (Newman et al., 1995
), which probably explains why the antibodies are neuronal specific. While this
paper was under review we raised a similar antibody
against the hinge domain of
3A, which immunoprecipitates the other AP-3 subunits in nonneuronal cells (data
not shown).
3A and
3B subunits are the most similar to their
counterparts in the AP-1 and AP-2 complexes, with over
30% identity. They mainly differ from the other members
of the
family in that they extend farther at their COOHterminal ends, giving them predicted molecular weights of
21.7 and 22 kD, as compared with 19 kD for
1 and 17 kD
for
2. These subunits have been independently cloned and sequenced by Dell'Angelica et al. (1997)
, using the
same approach of searching the EST database for
1/
2
homologues.
3 have neuronal-specific isoforms, so we
probed multiple tissue Northern blots to find out whether
any of the novel sequences also show tissue-specific expression. Fig. 4 shows that
,
3A,
3A, and
3B are all
expressed ubiquitously. Thus,
3A is indeed a nonneuronal-specific isoform of
3; the only known isoform of
is
expressed in all tissues examined; and although there are
two isoforms of
3, they have similar expression patterns.
Fig. 4.
Expression patterns of ,
3A,
3A, and
3B. Northern
blots were probed with either oligonucleotides or cDNAs specific
for each of the four sequences. All four genes are expressed ubiquitously. The relative weakness of the signal obtained with
3A
probe is probably a consequence of the blot already having been
probed several times.
[View Larger Version of this Image (54K GIF file)]
and
3 further and to find out whether
they are in fact associated with µ3 and
3, portions of
them were expressed as fusion proteins and used to raise
antibodies in rabbits. The antibodies were then used in immunoprecipitation and Western blotting experiments, together with the previously described antibodies against µ3
and
3. Fig. 5 shows the results of one such experiment. Pig brain cytosol was immunoprecipitated under nondenaturing conditions with anti-
3, anti-
, and anti-
3 (using
pooled antibodies raised against both the A and B isoforms). As a control, cytosol was also immunoprecipitated
with anti-
-adaptin to bring down the AP-1 adaptor complex. Strips were then cut from Western blots and probed
with antibodies against
,
3, µ3,
3, and
.
Fig. 5.
Coimmunoprecipitation of AP-3 subunits. Pig brain cytosol was immunoprecipitated under nondenaturing conditions
with affinity-purified polyclonal antibodies against 3B,
,
3
(crossreacting with both the A and B isoforms), and
. Gels were
blotted, and the appropriate region was cut out and probed with
each of the above antibodies, as well as with anti-µ3 (which does
not recognize the native complex). The four subunits of the AP-3
complex,
,
3, µ3, and
3, all coimmunoprecipitate; the
subunit of the AP-1 complex does not coimmunoprecipitate with antibodies against the AP-3 components, and antibodies against
do not bring down any of these components. The doublet labeled
with anti-
3 presumably corresponds to the A and B isoforms,
one of which appears to be preferentially immunoprecipitated with this antibody.
[View Larger Version of this Image (75K GIF file)]
3 antibody brings down not only
3 and µ3, as has
been previously reported, but also
and
3 (which appears as a doublet, presumably because there are two isoforms of the protein). It does not bring down
, as expected, since
is a component of a different complex.
Similarly, the new
antibody brings down
,
3, µ3, and
3, but not
. The
3 antibodies bring down these four
subunits as well, although the signal from
,
3, and µ3 is
weaker than with the other two antibodies, and one of the
3 isoforms appears to be preferentially immunoprecipitated. The control antibody, anti-
-adaptin, brings down
, but not
,
3, µ3, or
3.
/
-like subunit (
), a
subunit, a µ subunit, and a
subunit. We propose that
by analogy with AP-1 and AP-2, the complex should be
called AP-3.
3B. However, because of the tissue specificity
of this antibody, we were only able to examine the distribution of the complex in neurons, which are not well
suited for immunofluorescence, or in our heterologous system using permeabilized cells incubated with brain cytosol (Simpson et al., 1996
). The
antibody also works well
for immunofluorescence, so we are now able to localize
the endogenous complex in nonneuronal cells.
antibody (Fig. 6 a
and b), but close examination of the more peripheral
structures revealed little if any colocalization. As a more
general marker for the endosomal system we allowed cells
to endocytose rhodamine-conjugated wheat germ agglutinin (r-WGA) for various lengths of time. The cells in Fig. 6 f
had been incubated with r-WGA for 5 min and show a small but significant amount of colocalization of r-WGApositive structures with the
antibody (Fig. 6, c and f).
Longer incubations in r-WGA did not increase the amount
of colocalization (data not shown), indicating that endocytosed proteins have access to compartments that can bind
AP-3 but only at early time points.
Fig. 6.
Immunofluorescence localization of the subunit of the AP-3 complex.
(a, b, d, and e) NRK cells
were fixed with methanol/acetone and double labeled
with anti-
(a and b) and
anti-transferrin receptor (d)
or anti-lgp120 (e). Although
all three antibodies show
perinuclear labeling, many
organelles in the cell are concentrated in this region, and
there is little colocalization
of the more peripheral
elements. (c and f) Cells
were allowed to endocytose rhodamine-conjugated wheat
germ agglutinin (f) for 5 min
before fixation in methanol/
acetone and were then labeled with anti-
(c). Again,
there is little colocalization
of the more peripheral elements, although some structures do coincide (arrowheads). (g and j) NRK cells
were fixed with paraformaldehyde and permeabilized with
NP-40 and then double labeled with anti-
(g) and antip200 (j), a putative TGN coat.
Both antigens have a perinuclear distribution, but the
fine details are different, indicating that they are not components of the same
coat. (h, i, k, and l) MDBK
cells were fixed with methanol/acetone, either with (i and
l) or without (h and k) prior
incubation for 2 min in 5 µg/
ml brefeldin A and then double labeled with anti-
(h and
i) and anti-
(k and l). Both
antigens are brefeldin A sensitive, and both have a similar perinuclear distribution in
control cells, but little if any
colocalization of more peripheral structures. Bar, 20 µm.
[View Larger Version of this Image (123K GIF file)]
antibody and
antibodies against TGN markers, since our earlier study
indicated that at least some of the AP-3 complex is associated with the TGN. The cells in Fig. 6, g and j, were double
labeled with anti-
(g) and anti-p200 (j), a TGN-associated
protein that may be a component of a nonclathrin coat
(Narula et al., 1992
; Narula and Stow, 1995
). Although
both show the same type of perinuclear localization, the
two patterns are quite distinct, indicating that the two proteins are not associated with each other and thus are unlikely to be components of the same coat. The cells in Fig.
6, h and k, were double labeled with anti-
(h) and anti-
adaptin (k), a component of the AP-1-containing coat that
is also associated with the TGN. Again, although they are
concentrated in the same perinuclear region they have distinct labeling patterns. In the final pair of panels the cells
were treated with 5 µg/ml brefeldin A for 2 min before
double labeling with anti-
(Fig. 6 i) and anti-
-adaptin (Fig. 6 l). Both proteins can be seen to have completely redistributed to the cytoplasm. Thus, the membrane localization of the AP-3 complex, like that of the AP-1 complex, appears to be ARF dependent, not only in our in
vitro system but in vivo as well.
with that of clathrin heavy chain. Because of the large number of clathrincoated pits and vesicles in the cell, it was necessary to use
confocal microscopy to assess the degree of colocalization.
Fig. 7 shows
labeled in green and clathrin labeled in red.
Although occasionally the labeling coincides, especially in
the perinuclear region, for the most part the two patterns
are distinct. The small amount of colocalization at the light
microscope level is consistent with our earlier observations using immunogold electron microscopy, which revealed that
3B and clathrin were often in close proximity, although they were found on different budding profiles
(Simpson et al., 1996
). Thus, these results, together with
the finding that AP-3 subunits are not enriched in clathrincoated vesicles prepared from brain (Simpson et al., 1996
)
or from liver (data not shown), indicate that the AP-3
complex is a component of a novel, nonclathrin coat.
Fig. 7.
Confocal micrographs of an NRK
cell double labeled for and clathrin. NRK
cells were fixed with methanol/acetone and
labeled with anti-
(a, shown in green) and a
monoclonal antibody against clathrin heavy
chain (b, shown in red). The merged images
(c) show numerous dots that are positive for
but not for clathrin, and the limited
amount of overlap is consistent with the two
being found on the same membranes but
different populations of coated buds. Bar,
20 µm.
[View Larger Version of this Image (25K GIF file)]
is closely related to the
protein product of the Drosophila garnet gene (GenBank/
EMBL/DDBJ DMU31351). The garnet (g) locus was first
described in 1916 as an eye color gene (Bridges, 1916
) and
was cloned by P element tagging and partially sequenced
by V. Lloyd in 1995. The homology between the two proteins is strongest near their NH2-terminal ends, including a
stretch of >100 amino acids (178-287 in
) where human
and Drosophila garnet are >96% identical (Fig. 3). Thus,
garnet is almost certainly Drosophila
, and an analysis of
the garnet mutant phenotype should help to establish the
function of the AP-3 complex.
Fig. 8.
Phenotype of Drosophila with mutations in the garnet gene, which encodes the subunit. Sections were cut from the eyes of both wild type (a and d) and mutant (b, c, e, and f) flies. Two alleles were examined: g3 (b and e) and g53d (c and f). a-c are phase contrast
micrographs; d-f show brightfield views of the same images. Each ommatidium consists of photoreceptor cells surrounded by pigment
cells. In wild-type flies the pigment granules in the pigment cells can be readily seen in brightfield as well as phase contrast micrographs
(a and d). The granules are still visible in the g3 flies but contain less pigment and thus are less prominent when viewed by brightfield (b
and e). They are essentially undetectable in the g53d flies (c and f). Bar, 10 µm.
[View Larger Version of this Image (97K GIF file)]
, 1959
). However, the granules are
of normal size and shape, and the number of granules surrounding each ommatidium is not appreciably different in
g3 and wild-type flies. A much more severe phenotype is
seen in the g53d flies. In phase contrast micrographs (Fig. 8 c),
the ommatidia can be seen to be well developed, implying
that the pigment cells are present; but no pigment granules
are detectable, and the sections are nearly invisible in
bright field micrographs (Fig. 8 f).
, is also ubiquitously expressed,
as shown in Fig. 4. Thus, the AP-3 complex may play a
fundamental role in all types of cells, but mutations in the
subunit appear to have a particularly strong effect on
pigment granules.
Discussion
-NAP (
3B), are associated with each other in
an adaptor-like complex (Simpson et al., 1996
). We have
now identified the remaining subunits of the complex and have further investigated its function. We have named the
complex AP-3, by analogy with AP-1 and AP-2.
3 and
1/
2
we predicted that the
and
3 subunits would be relatively distant homologues of
/
and of
1/
2, respectively. Because the complex is fairly abundant, it was easy
to find entries in the database for both types of subunits;
indeed, a recent search revealed over 50 mammalian ESTs
for the
subunit alone. Interestingly, there are now additional homologues of the
, µ, and
subunits in the database, suggesting that there is likely to be at least one more
type of AP complex.
subunit have neuronal-specific as
well as ubiquitously expressed isoforms; however, no neuronal-specific isoforms have been found for either of the
other two subunits,
and
. Although we cannot rule out
the possibility that such isoforms may exist, candidates for
them do not appear to be present in the EST database, in
spite of the fact that many of the entries are from brain
and that both µ3B and
3B can be found as multiple "hits." Why are there neuronal-specific isoforms of two of
the subunits? Is µ3B always associated with
3B and µ3A
with
3A, or can the subunits "mix and match"? We do
not yet know the answers to either of these questions, but
studies on the µ and
subunits of the AP-1 and AP-2
complexes may provide some clues. µ1 and µ2 have been
shown to bind to tyrosine-based sorting signals (Ohno et al.,
1995
), and recently µ3 has also been shown to bind such sequences (Dell Angelica et al., 1997). Possibly µ3A and
µ3B recognize different repertoires of signals.
1 and
2
have been shown to bind to clathrin, an activity that involves the hinge domain of the protein (Gallusser and
Kirchhausen, 1993
; Shih et al., 1995
). The AP-3 complex is
not clathrin associated (Simpson et al., 1996
) but may interact with another type of scaffolding protein, and such
an interaction would be likely to involve the
3 subunit, with the two different
3 isoforms possibly interacting
with different types of scaffolds. Alternatively, it may be
that only one of the two proteins has a neuronal-specific
role. The µ and
subunits of the AP-1 and AP-2 complexes show a strong interactaction with each other (Page
and Robinson, 1995
; Seaman et al., 1996
), and it is possible
that neuronal-specific isoforms of µ3 and
3 may have coevolved because there is an obligatory coupling between them.
3B (Simpson et al., 1996
). The present study confirms and extends
our earlier findings by localizing the endogenous AP-3
complex in nonneuronal cells. We have shown that the
complex is brefeldin A-sensitive in vivo as well as in vitro
and have confirmed that it is associated with both perinuclear and more peripheral membranes. What do these peripheral membranes correspond to? There is only very
limited colocalization of either
or
3B with endosomal
markers, suggesting that the complex is more likely to be
involved in a biosynthetic pathway than in an endocytic
one. Our earlier study indicated that some of the complex
is associated with the TGN (Simpson et al., 1996
), and the
more peripheral labeling may represent a post-TGN compartment. This compartment may also be able to receive
endocytosed proteins without being a conventional type of
endosome. The presence of a µ subunit in the complex
indicates that it plays a role in the sorting of proteins containing tyrosine-based signals (Ohno et al., 1995
; Dell'Angelica et al., 1997
). Such signals have been shown to function in the post-Golgi biosynthetic pathway as well as in
the endocytic pathway and may be used to send proteins
to any one of several destinations: the (basolateral) plasma
membrane, endosomes, lysosomes, or back to the TGN
(Humphrey et al., 1993
; Matter and Mellman, 1994
). Which
of these pathways might the complex mediate?
subunit is the mammalian homologue of the Drosophila
garnet gene product. The garnet mutant alleles that have
been described only appear to affect pigmentation, yet the
gene is expressed ubiquitously, not just in pigment cells. In
addition, although a number of alleles have been identified, so far none of those tested by Northern blotting have
been found to be nulls; thus, the protein may be essential
(Lloyd, V., personal communication). Of the two alleles shown in Fig. 8, g53d is tissue specific, suggesting that the
mutation is in the 5
upstream regulatory region, while g3
contains an insertion within the coding portion of the gene
(Lloyd, V., personal communication). Thus, the g3 mutation is likely to affect all the cells in the flies' bodies, yet
the animals are viable and in particular do not appear to have any neurological problems. This is in contrast to flies
with conditional mutations in two other genes encoding
membrane traffic proteins, dynamin and NSF, where incubation at a nonpermissive temperature causes a block in
neurotransmission (Chen et al., 1991
; Van der Bliek and
Meyerowitz, 1991; Pallanck et al., 1995
). Although more
information is needed about the activity of the mutant
protein, the g3 phenotype appears to be inconsistent with a
role previously proposed for both the
3 and µ3 subunits
in synaptic vesicle biogenesis (Pevsner et al., 1994
; Newman et al., 1995
).
). Less is
known about the formation of pigment granules in flies,
but it seems likely that they too are lysosome-like in origin. This possibility is supported by the discovery that
VPS18, a yeast gene involved in the sorting of proteins
to the lysosome-like vacuole (Robinson et al., 1991
), is
homologous to another fly eye color gene, deep orange
(Reider, S., and S. Emr, personal communication).
). There are numerous
Drosophila eye color genes, many of which have not yet
been cloned, and it seems likely that some of these other
genes will also be found to encode proteins involved in
membrane traffic.
] and SCYJL024C [
3]) whose protein products are much
more closely related to components of the AP-3 complex
than to components of either the AP-1 or the AP-2 complexes. Targeted gene disruptions may indicate whether
the complex is involved in trafficking to the vacuole, to the
plasma membrane, or in some other pathway. Microinjection and/or immunodepletion experiments, using the antibodies we have generated that recognize the nonneuronal
complex in its native form, may also help to define the role
of the complex. AP-3 is not the only novel coat component
that has recently been identified. Database searches indicate that there is at least one other AP-type complex, and
there may be more. Monoclonal antibodies have identified
p200 as yet another possible coat component (Narula and
Stow, 1995
), and electron microscopy has revealed the existence of lace-like coats associated with the TGN (Ladinsky et al., 1994
). Thus, there are pathways in the cell that
require coats and coats that require pathways, and the
next step will be to fit the two together.
Received for publication 23 December 1996 and in revised form 6 March 1997.
Fiona Simpson and Andrew Peden contributed equally to this paper.We are grateful to Vett Lloyd for communicating unpublished information about the garnet gene, to Matthew Seaman, Scott Emr, and Stephanie Reider for telling us about the VPS18-deep orange connection, to Paul Luzio, Jenny Stow, and Frances Brodsky for antibodies, and to Matthew Freeman for introducing us to Drosophila. We also thank Rainer Duden, Matthew Freeman, John Kilmartin, Vett Lloyd, Paul Luzio, and members of the Robinson lab for comments on the manuscript and for helpful discussions.
This work was supported by grants from the Medical Research Council, the Wellcome Trust, and the Human Frontier Science Program.