From the Zentrum für Biochemie und Molekulare Zellbiologie, Dept. Biochemie II, Universität Göttingen, Gosslerstrasse 12D, 37073 Göttingen, Germany
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ABSTRACT |
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Intracellular protein transport and sorting by
vesicles in the secretory and endocytic pathways requires the formation
of a protein coat on the membrane. The heterotetrameric adaptor protein complex 1 (AP-1) promotes the formation of clathrin-coated vesicles at
the trans-Golgi network. AP-1 interacts with various
sorting signals in the cytoplasmic tails of cargo molecules, thus
indicating a function in protein sorting. We generated mutants of the
At several sites along the secretory and the endocytic routes the
transport of membrane proteins and luminal cargo depends on the
formation of carrier vesicles. The formation of these transport vesicles is facilitated by coat proteins. Budding from the Golgi and
endoplasmic reticulum membranes involves the heptameric COP-I and
COP-II complexes. Budding from the trans-Golgi network
(TGN),1 the plasma membrane,
and also probably from membranes of the endosomal/lysosomal system
involves heterotetrameric adaptor protein (AP) complexes of which three
types are known at present and which show homology to the COP-I
subunits (1-3).
AP-1 and AP-2 have been implicated in the formation of clathrin-coated
vesicles at the TGN and the plasma membrane, respectively, but may
participate also in vesicle formation at other sites (3-5). AP-3-mediated vesicle formation may take place at the TGN and/or endosomes (6-8).
Although most of our knowledge on AP function is derived from studies
on their subcellular localization and interaction with membranes or
cytoplasmic domains of membrane proteins, little is based on in
vivo functional studies or genetic approaches. Deletion of genes
of AP subunits in yeast did not reveal the function of AP-1 and AP-2
but was informative in the case of AP-3. Mutations of the four AP-1 and
AP-2 subunits did not result in a mutant phenotype and only the
combination of AP-1 mutations with a temperature-sensitive clathrin
heavy-chain mutation enhanced the reduced growth rate observed in the
clathrin heavy-chain mutant
(9-11).2 No biochemical data
for cargo-AP-1 interactions are available in the yeast system. The AP-3
is needed for an alternative pathway from the TGN to the vacuole, which
is utilized by the vacuolar alkaline phosphatase and the vacuolar
t-SNARE Vam3p and does not depend on clathrin (12). In Drosophila
melanogaster AP-3 is involved in the formation of pigment
granules, as indicated by garnet, an eye color mutant caused by
mutations in one of the AP-3 subunits (8, 13). Further support for a
role of AP-3 in pigment granule formation comes from the naturally
occurring mouse mutants mocha and pearl, which belong to a larger group of mouse mutants with abnormal pigmentation, prolonged bleeding time,
and altered lysosomal sorting. Mocha as well as pearl mice carry
mutations in AP-3 subunits (14, 15). In an in vitro system,
synaptic vesicle formation from endosomes of PC12 cells is dependent on
the neuronal isoform of AP-3 (7).
The AP-1 has been found in clathrin-coated buds on the TGN. Binding of
AP-1 to the TGN depends on the GTPase ARF1 (16). The AP-1 consists of
two 100-kDa adaptins, The significance of the AP-1-mediated packaging into clathrin-coated
vesicles at the TGN for the trafficking of these membrane proteins and
their biological function is not clear. Most of the cargo proteins are
found at the plasma membrane as well as in endosomal/lysosomal
membranes. Disruption of their interaction with the AP-1 must not
necessarily prevent the targeting to their destination, as has been
demonstrated for LAMP 1 (23, 28). Replacing the glycine residue in the
GYxxI sorting signal by alanine, interferes with binding to AP-1, but
not to AP-2, and allows transport to endosomes/lysosomes via the cell
surface, albeit at the expense of an increased steady-state
concentration at the plasma membrane (29).
To obtain insight into the in vivo role of AP-1, we rendered
the Isolation of Chromosomal
Adtg MspI polymorphisms were detected between Mus
musculus and Mus spretus with a 1-kb PstI
cDNA probe encoding the C-terminal half of the protein and with a
chromosomal DNA probe (see Fig. 1A, probe 2). Hybridization
of a 3.8-kb MspI fragment of M. musculus and a
6.7-kb MspI fragment of M. spretus was seen with
the cDNA as a probe. The chromosomal DNA probe hybridized with a
5.6-kb fragment from M. musculus and with a 5-kb fragment
from M. spretus. DNA from 94 animals, produced by crossing
M. spretus with animals having a M. musculus/M. spretus genetic background, were hybridized with both probes after MspI incubation. The hybridization
pattern was analyzed using a computer program (Jackson Laboratory, Maine).
Mutation of Embryonic Stem Cells, Generation of Mutated
Animals--
The 5.5-kb PstI fragment that contained the
123-bp exon was subcloned into the cloning plasmid pBluescript SKII+
(Stratagene). A BglII-restriction endonuclease site was
introduced into the exon by oligonucleotide site-directed mutagenesis
(5'-GCCAATGCGTAGATCTGTGAATTTTTG-3'). Mutation
was confirmed by DNA sequencing. The neoR
expression cassette from pMC1neopA (Stratagene) was cloned into the
BglII site of the exon as a 1.2-kb BamHI
fragment, and the construct with opposing open reading frames was used.
To select against nonhomologues recombination events the thymidine
kinase gene was introduced in the polylinker, flanking the
"knock-out" construct. Constructs with and without the thymidine
kinase gene were linearized by NotI digest and
electroporated into the mouse 129SV/J embryonic stem cell line ES-14-1.
ES cells were cultured as described previously (31, 32). Forty-five
G418-resistant ES cell clones derived from the targeting vector without
the thymidine kinase gene were tested for the homologous recombination
event. Four positive clones were found.
Mutated ES cells were injected into day 3.5 pc blastocysts, which were
then transferred into the uteri of pseudopregnant foster mothers. Three
male chimeras were borne and mated with C57/Bl6 (outbred) and 129SV/J
(outbred) (31, 32). Mice strains were purchased from Biological
Research Laboratories, Basel. Colonies were kept in a central animal
facility at the University of Göttingen.
Superovulation, Blastocysts Isolation and
Genotyping--
Superovulation was done by injecting 5 units of serum
from pregnant mares (Sigma) at midday intraperitoneally into
6-8-week-old females and by a second injection of 10 units of human
chorionic gonadotropin (Sigma) 46 h later. Animals were
immediately mated thereafter, and seminal plaques were checked the next
morning. The mouse was sacrificed at day 3.5 in the early afternoon.
The isolated uteri were washed with PBS and flushed with 1 ml of M2 medium (31, 32). On average, 25 blastocysts were collected per animal.
Genotyping: blastocysts were washed twice with PBS and then transferred
into 0.5-ml Eppendorf cups containing 5 µl of water. After adding 5 µl of PBS they were frozen on dry ice for 10 min followed by 10 min
of incubation at 95 °C. After the addition of 2 µl of proteinase K
(Boehringer Mannheim; stock 10 mg/ml) they were incubated at 56 °C
for 30 min and cooled on ice. A second incubation was done at 95 °C
for 10 min and samples were frozen at
In a first polymerase chain reaction amplification, two exon primers,
5'-GCCATAGGAATGCAAAC-3' and 5'-GTAAGTAGCTACTCTTAC-3', were used to
amplify wild-type and mutagenized exons. 30 µl were loaded onto a 2%
agarose gel and further analyzed by Southern hybridization using probe
2 (see Fig. 1A), and 40 µl were purified via a spin column
by a Qiagen kit according to the manufacturer's manual. All of the
purified material was used for neoR polymerase
chain reaction using the oligonucleotides 5'-CGGATCAAGCGTATGCAGCCG-3' and 5'-CAAGATGGATTGCACGCAGG-3'. Polymerase chain reaction products were
separated in a 2% agarose gel and further analyzed by Southern analysis using the 1.2-kb neomycin BamHI fragment.
Preparation of Protein Extracts, Western-, and Northern Blot
Analysis--
Organs were homogenized in 0.1 M MES buffer,
pH 6.5, 1 mM EGTA, 0.5 mM MgCl2,
0.02% NaN3, 2 µg/ml pepstatin, 2 µg/ml leupeptin A, 1 mM phenylmethylsulfonyl fluoride, and 5 mM
iodoacetamide. Tissue fragments and unbroken cells were removed by
centrifugation at 1000 × g over 10 min. The
supernatant was separated into a membranous and cytosolic fraction by
centrifugation at 100,000 × g over 45 min. Gel
filtration was done on a Superdex-200 column (Smart-System, Amersham
Pharmacia Biotech) at a flow rate of 40 µl/min and a sample volume of
50 µl with 5 µg/µl protein. Fraction volume was 30 µl. Column
was calibrated with blue dextran, thyroglobulin, ferritin, catalase,
aldolase, bovine serum albumin, ovalbumin, and cytochrome C. Proteins
(50 µg of crude cell extract and the entire gel filtration fractions)
were resolved on a Laemmli gel and transferred onto nitrocellulose
membranes (Sartorius). Adaptins were detected with anti-
Total fibroblast RNA was isolated using a kit (Qiagen) according to the
manufacturer's protocol. 10 µg of RNA were separated on a 1%
agarose gel, transferred onto a nylon membrane (Amersham), and UV
cross-linked. cDNAs were labeled with [ Immunolabeling and Flow Cytometry Analysis--
Animals from a
litter were analyzed in parallel when they were 5-7 weeks old. They
were kept in the same or neighboring cages in the central animal
facility of the university. Thymus and spleen were isolated, washed in
HEPES/Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
and broken up using a Tenbroek homogenizator (Tecnomara AG,
Hüttenberg). Cells were counted and washed twice in PBS.
106 cells were transferred to fluorescence-activated cell
sorter tubes and labeled with antibodies for 1 h on ice:
anti-CD4-FITC, anti-CD8-PE, anti-CD3 Isolation of Chromosomal
The Mutation of
Cells from two targeted embryonic stem cell clones were injected into
blastocysts. Cells from one clone integrated in the embryos. Three
chimeric male mice were obtained that transmitted the targeted
adtg allele to about 25% of the offspring from C57BL/6J (outbred) and 129SV/J (inbred) females. When
Shortly before nidation at day 4.5 pc to 5 pc, blastocysts hatch from
their zona pellucida and are characterized by an elongated shape.
Blastocysts with these characteristics were found also among the day
3.5 pc blastocysts because of the asynchrony of the early stages in
development. None of the 29 embryos with this morphology turned out to
be of the
To establish
Cytosolic proteins of cultured +/
Pulse-chase experiments were performed with the fibroblasts to analyze
the MPR-dependent sorting of cathepsin D to lysosomes. No
differences were observed between the +/+ and +/ Phenotype of Heterozygous Animals--
We observed differences in
size between 4-week-old +/+ and +/
We analyzed thymocytes and splenocytes of 5-7-week-old littermates for
T- and B-lymphocyte development by flow cytometry, because we expected
this developmental system to be sensitive to alterations in receptor
trafficking. Thymocytes were analyzed using antibodies against CD4,
CD8, and CD3. Antibodies against B220/CD45R and IgM were used to
analyze B-lymphocytes. The total number of lymphocytes in spleen and
thymus was not affected. In the thymus of +/ The AP-1 complex of clathrin-coated vesicles at the TGN has a
function in protein sorting, but its role in post-Golgi vesicular transport and its biological significance are poorly understood (3). We
rendered the This phenotype is probably not caused by absence of just the
We tried to establish It was surprising to see a phenotype in This study has shown that the AP-1 complex has a unique and essential
role in mice at the TGN or on endosomes, which cannot be fullfilled by
known or unknown adaptor complexes. That Application of antisense technology to cell lines from -adaptin subunit of AP-1 in mice to investigate its role in
post-Golgi vesicle transport and sorting processes.
-Adaptin-deficient embryos develop until day 3.5 post coitus and die
during the prenidation period, revealing that AP-1 is essential for
viability. In heterozygous mice the amount of AP-1 complexes is reduced
to half of controls. Free
1- or µ1 chains were not detectable,
indicating that they are unstable unless they are part of AP-1
complexes. Heterozygous mice weigh less then their wild-type
littermates and show impaired T cell development.
INTRODUCTION
Top
Abstract
Introduction
References
and
1, a 47-kDa adaptin, µ1, and a
19-kDa adaptin,
1. Of the four AP-1 subunits,
-adaptin as well as
1-adaptin interact with clathrin (3). The C-terminal 511 of 822 amino acids of
-adaptin and the
1-adaptin subunit are dispensable
for TGN binding (17). AP-1 recruits clathrin on to the membranes by
binding to the clathrin heavy chain and to the cytoplasmic tails of
membrane (cargo) proteins. The µ1 adaptin interacts with
tyrosine-based sorting sequences in the cytoplasmic tails of cargo
proteins (18). Leucine-based sorting signals in the cytoplasmic tails
bind to µ1 or
1 adaptin (19, 20). AP-1 binding cargo molecules are
the mannose-6-phosphate receptors, MPR46 and MPR300, the lysosomal
membrane protein, LAMP 1, the invariant chain of the major
histocompatibility complex class II receptor, the CD3
subunit of the
TCR
·CD3 receptor complex and the varicella zoster virus
glycoprotein I (21-27).
-adaptin gene of AP-1 nonfunctional in mice. AP-1-deficient mouse embryos die before nidation, and even heterozygous animals display a reduced growth rate during nursing. Although the life cycle
of yeast does not depend on AP-1 function, it is essential for
development of a higher eukaryotic organism such as mice.
EXPERIMENTAL PROCEDURES
-Adaptin Sequences and Gene
Mapping--
A 1.3-kb PstI fragment of the mouse
-adaptin cDNA corresponding to the N-terminal half of the
protein was used to screen a mouse phage DNA library in EMBL3
(30).3 Seven library clones
were isolated that contained two different chromosomal DNA fragments.
80 °C. All of the material
was used for polymerase chain reaction under standard conditions.
- and
anti-
-adaptin mouse monoclonal antibodies (both 1:5,000 dilution)
(Transduction Laboratories) and an anti-mouse µ1 polyclonal rabbit
antiserum (1:500 dilution) raised against amino acids 291-310.
Anti-LAMP1 (1D4B) was a rat monoclonal antibody (1:1,000 dilution)
(Developmental Studies Hybridoma Bank, University of Iowa).
Protein-antibody complexes were made visible by chemiluminescence
(Super-SignalTM, Pierce) and x-ray film exposure. X-rays
were scanned and quantified (Wincam software). Membranes were stripped
by incubation in 0.2 M NaOH over 5 min and blocked again
with 5% milk powder and 0.1% Tween 20 in PBS.
-32P]dCTP
using a labeling kit (Amersham). Membranes were stripped by boiling in
0.1% SDS for rehybridization of the membrane. Signals were quantified
by phosphoimager analyses (Fujitsu).
-FITC, anti-CD4-PE,
anti-B220/CD45R-PE, anti-IgM-FITC, anti-rat IgG2a-PE (all
PharMingen), and anti-CD8-QR (Sigma). Cells were washed twice with
ice-cold PBS and analyzed using a FACScan and Lysis II software (Becton Dickinson).
RESULTS
-Adaptin Sequences and Genomic
Mapping--
A genomic mouse phage DNA library was screened with a
mouse
-adaptin cDNA fragment. Two different chromosomal DNA
fragments, each about 16-kb in length, were identified in seven
purified phage clones. On each fragment only a single exon was found
using cDNA as a probe. One clone contained a 123-bp exon, encoding
amino acids 68-108 of 822 (see Fig.
1A). The other clone contained
an exon of 75 bp (not shown) encoding amino acids 189-214.
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Fig. 1.
A, chromosomal -adaptin sequence and
cloning of the targeting vector. The position of the 123-bp exon is
indicated by a black bar. The 6-kb EcoRV and the
2.4-kb HindIII fragments of the wild-type locus used for
genotyping as well as the hybridization probes 1 and 2 are indicated in
the upper part of panel A. The lower part shows the
targeting construct with the inserted neoR gene
and the BglII site used for neoR
insertion. Arrows indicate orientation of the
neoR gene open reading frame (top)
and of the exon open reading frame (bottom). The position of
the exon in the
-adaptin cDNA is indicated by a black
bar. B, Southern blots of HindIII- and
EcoRV-digested chromosomal DNA hybridized with probe 2 are
shown, recognizing the wild-type 6-kb EcoRV and above the
mutated 7.2-kb fragment or the wild-type 2.4-kb HindIII
fragment, and the mutated 3.6-kb fragment.
-adaptin gene Adtg could be localized on mouse
chromosome 8 at 52 cM between the markers D8Bwg1112e and
D8Bir26 using two different Adtg MspI
polymorphisms, distinguishing M. musculus and M. spretus (see Materials and Methods, data accessible at mouse
genomic data base, Jackson Laboratory, Maine). Neither pseudogenes nor
homologues of Adtg were found in the mouse genome.
-Adaptin--
The exon encoding amino acids 68-108
was choosen to construct the targeting vector (see Fig. 1A).
A 5.5-kb PstI fragment containing the 123-bp exon was
subcloned into the plasmid pBS. A BglII site was introduced
into the exon 45 bp after the start of the exon corresponding to bp 245 of the cDNA by oligonucleotide-directed mutagenesis, and the
neomycin-resistant (neoR) gene was cloned into
the BglII site as a 1.2-kb BamHI-fragment. To
select against nonhomologous recombination events the thymidine kinase
gene was introduced into the targeting construct at the 3'-site of the
-adaptin sequences. The constructs with and without the thymidine
kinase gene were electroporated into mouse embryonic stem cells. Four
homologous recombination events with the targeting construct lacking
the thymidine kinase gene were detected in 45 ES cell clones (probe 2;
see Fig. 1, A and B).
-adaptin heterozygotes were intercrossed, no
-adaptin
/
animals were observed among the
108 newborns genotyped (Table I),
indicating that absence of
-adaptin causes embryonic lethality.
Statistics of the genotyping of newborn mice and embryos at
different stages of development
-Adaptin-deficient Embryos Die before Nidation--
To
determine the stage where embryonic development ceases, pregnancies
were terminated starting at day 13.5 pc, the last day of organogenesis
in mouse development, to 8.5 pc. None of 20 embryos was homozygous for
the targeted Adtg allele (see Table I). Twelve of 58 blastocysts isolated at day 3.5 pc were homozygous and 31 were
heterozygous for the targeted Adtg allele, revealing an
almost mendelian distribution of the
-adaptin mutation (see Table I and Fig. 2). The blastocysts displayed a
normal morphology with a zona pellucida, trophectoderm, inner cell
mass, and a blastocoele.
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Fig. 2.
Genotyping of day 3.5 pc blastocysts.
Chromosomal DNA was isolated and neoR
(top) and exon (bottom) sequences were amplified
in two separate polymerase chain reactions. Shown is a representative
experiment. Lane 1, size standard 1-kb DNA ladder;
lane 2, no sample loaded; lanes 3-12,
amplification products; lane 13, no sample loaded;
lane 14, negative control (no chromosomal DNA); lane
15, positive control (DNA from a heterozygous animal). Resulting
genotypes are given below each lane.
/
genotype, suggesting that
-adaptin deficiency is not
compatible with development to day 4.5 pc.
/
cell lines from the inner cell mass, day 3.5 pc
blastocysts were transferred onto feeder cell cultures and cultivated
for 2 days in the presence of leukemia inhibitory factor, which
stimulates growth and preserves the pluripotent state of cells of the
inner cell mass. Sixteen of 23 blastocysts attached to the feeder cells
and formed colonies. They were transferred into feeder cell-free
plastic Petri dishes and allowed to attach and expand. Twelve of these
could be successfully genotyped. The inner cell mass of wild-type and
+/
embryos developed on the feeder cells into colonies, attached to
the plastic surface and formed monolayers. Three of the blastocysts
were
-adaptin-deficient. The trophoblast cells and cells of the
pluripotent inner cell mass enlarged significantly, indicating
differentiation or cell death (not shown). The
/
cells failed to
spread onto the plastic surface, divide, and establish colonies.
-Adaptin and AP-1 in
-Adaptin +/
Cells and
Tissues--
Analysis of expression levels of the
-adaptin protein
in cultured day 12.5 pc embryonic fibroblasts and in liver, brain, and
thymus of adult animals by Western blotting revealed a decrease of 50%
in +/
cells compared with the +/+ cells (see Fig.
3).
-Adaptin mRNA levels were
reduced to 30% in cultured embryonic fibroblasts, liver, kidney, and
brain. In all tissues, a 2.8-kb
-adaptin transcript was seen. Fig.
3B shows mRNA levels of adaptins in embryonic
fibroblasts. µ1 and
1 adaptin mRNAs were not altered in
fibroblasts.
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Fig. 3.
A, Western blot analysis of -adaptin
protein levels in crude cell extracts of liver and thymus prepared from
2 +/+ and 2 +/
adult mice. Numbers identify the individuals. LAMP 1 protein was used as internal control. B, Northern blot
analysis of adaptin mRNA levels in embryonic fibroblasts. 10 µg
of total RNA were loaded per lane. Numbers give the relative signal
intensities between the cell lines in percent. Membranes were stripped
for successive hybridization experiments with 32P-labeled
cDNAs. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
was used as internal control. Signals were visualized and quantified by
PhosphoImager analysis.
embryonic fibroblasts were
separated by gel filtration to analyze whether the excess of
1,
µ1, and
1 adaptins of the AP-1 complex form smaller or larger complexes or accumulate as free subunits. Column fractions were analyzed by Western blotting (see Fig.
4). The wild-type AP-1 complex was
detected by anti-
mouse monoclonal antibody, a mouse monoclonal
antibody recognizing
1 and
2 or an antiserum specific for µ1.
The AP-1 complex was found in fractions 7-10
(Mr of 400,000). No adaptor complexes of a
smaller or larger size or free
and µ1 adaptins were found in the
other fractions of the gradient. This suggests that the excess of
1
and µ1 are not stable.
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Fig. 4.
AP-1 adaptins in cultured +/ embryonic
fibroblasts. Elution profile at 280 nm and molecular mass
calibration for the gel filtration (see Experimental Procedures).
Fractions containing adaptins are indicated by a bar. Western blot
analysis of these fractions is shown. Others did not contain antigenic
material. a,
-adaptin; b,
-adaptin;
c, µ1-adaptin. Membranes were stripped and reprobed with
the respective anti-adaptin antibody.
cell lines (not shown).
littermates. A cohort of 9 +/
male mice and 6 +/+ males of the outbred (C57/Bl6) colony were weighed
from day 6.5 postnatal to 100 postnatal, and a second group of 27 +/
and 11 +/+ mice were weighed between 3 and 10 months of age. Data of
the periods between day 6-21 and day 100-320 were analyzed by least
square linear regression (Fig.
5A). The weight difference
developed within the first 3 weeks after birth. Between day 6-21
postnatal the body weight of +/
male mice increased by 0.23 g/d
compared with 0.40 g/d in +/+ males. At day 21 postnatal, litters were
separated from their mothers, and weaning caused a growth arrest for 2 to 4 days. Thereafter +/
mice grew as fast as +/+ mice, thus
maintaining their 2.5 g lighter body weight for at least up to 10 months of age. This growth difference was independent of the genetic
background, although animals of the inbred colony grew slower then
outbred animals, and females grew slower then males. Histological
analysis of liver, thymus, spleen, and kidney did not reveal
morphological abnormalities in heterozygous animals.
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Fig. 5.
Phenotypes of +/ mice. A,
growth of heterozygous mice. Data are from C57/Bl6 males, 17 +/+ and 36 +/
. Analysis by least square linear regression gave confidence values
of 80-95%.
, +/+;
, +/
. B, T cell development in
the thymus of 4-7-week-old animals analyzed by flow cytometry.
animals, the number of
CD4+ single-positive T cells was reduced by 20%, whereas
the number of CD8+ single-positive T cells was hardly
affected (see Fig. 5B). Cell surface expression of CD4 and
CD8 on CD3+ cells was not altered. No abnormalities were
found for splenocytes or the tissue architecture of thymus and spleen.
DISCUSSION
-adaptin gene of AP-1 nonfunctional in mice.
-Adaptin
/
embryos were found at day 3.5 pc, but they died
within the following 24 h before nidation. At day 2.5 pc of mouse
development, translation of maternal mRNA stops and zygotic mRNA appears (31). Thus protein levels of
-adaptin will begin to
decline in
/
blastocysts at day 2.5 pc. Apparently development of
/
blastocysts ceases, when
-adaptin declines below a threshold because of the lack of embryonically encoded
-adaptin.
-adaptin subunit, but by the absence of the entire AP-1 complex. In
+/
cells the amount of
-adaptin is reduced to 50%. We tested whether
1, µ1, and
1 are in excess in these cells. mRNA
levels of these subunits are not altered, but we failed to detect
1 or µ1 as free subunits or as partial complexes missing the
-adaptin. We conclude from this that
-adaptin governs AP-1
complex formation, probably by acting as a scaffold, and that other
adaptins are unstable in the absence of
.
/
cell lines by cultivating cells of the
inner cell mass from day 3.5 pc blastocysts. The inner cell mass of +/+
and +/
blastocysts grew in vitro, but cells from
/
blastocysts did not proliferate. The severity of the
-adaptin
/
phenotype in mice is surprising given the absence of any phenotype of
the yeast AP-1 mutants. It is suggestive to ascribe the lethality to
the missorting of some functionally important proteins caused by the
absence of AP-1. Receptors that normally interact with AP-1 are
expected to become transferred in the absence of AP-1 to the plasma
membrane rather than to endosomes. This occurs for example when, in
these transmembrane proteins, domains interacting with AP-1 are deleted
(33, 34). Lethality could thus be due to the abnormal presence of these
transmembrane proteins at the cell surface and/or from their deficiency
at the normal site of their action. The MPRs are responsible for the
recruitment of the majority of AP-1 complexes to the TGN (35), and they
serve as receptors for soluble lysosomal enzymes and insulin-like
growth factor II. Mice lacking both MPRs are viable albeit at a reduced rate (36, 37), and embryonic development is normal in I-cell disease
(38), in which M6P-containing ligands are missorted because of the
absence of the M6P recognition marker. AP-1 could be important for
prohormone processing and secretion of proteins critical for embryonic
development. AP-1 is found on maturing secretory granula where it binds
to the prohormone-processing protease furin (5). The presently known
growth factors controlling embryonic development during the prenidation
period are provided to the embryo by the mother. The failure of proper
preimplantation development of
/
embryos is therefore unlikely to
result from defects in growth factor processing and secretion (39, 40). It could, however, also result from a failure of growth factor trafficking. Blastocyst development depends on maternal growth factors,
which stimulate blastocyst growth and maintain the pluripotent state of
the inner cell mass. These factors are transcytosed through the
trophectoderm (41). At the day 3.5 to 4.5 of mouse development, blastocysts hatch out of the zona pellucida. Cells of the mural trophectoderm produce a trypsin-like protease, which generates an
opening in the zona pellucida through which the blastocyst hatches,
leaving an empty zona pellucida behind (42).
-Adaptin-deficient blastocysts are not able to proceed through these developmental steps.
That AP-1 is dispensable for the unicellular yeast in fact argues for
an essential role of AP-1 during the development of a multicellular organism.
-adaptin +/
heterozygotes, in which AP-1 is reduced to half the normal level. The growth deficiency in heterozygotes as well as the decrease of CD4+/CD8
T-cells in the thymus points to a
rate-limiting function of AP-1 in some tissues, which cannot be
explained by the known AP-1 transport functions. However, the transport
of lysosomal enzymes in
-adaptin +/
embryonic fibroblasts was
normal. This indicates that AP-1 is not rate limiting for trafficking
of MPRs, despite the fact that MPRs constitute the major cargo proteins
recruiting AP-1 to the TGN (35). Related adaptor complexes exist, which
may compensate the loss of AP-1. The sequences of a human and mouse
-adaptin homologue, named
2-adaptin, have recently been
published,4 which are 60%
homologous to
-adaptin. Obviously this protein cannot compensate for
the loss of
-adaptin and must therefore fullfill a different function.
-adaptin has a particular
role is demonstrated by a mouse knock-out of the µ1-adaptin subunit.
These mice develop until midorganogenesis. In cell lines established
from the embryos, a trimeric
µAP-1 complex is formed, indicating
that this complex is able to fulfill some AP-1 function in
µ1
cells.5
-adaptin +/
heterozygotes may be helpful to achieve a graded reduction in the
-adaptin levels so that the critical threshold of AP-1 for viability
could be determined, and the consequences of AP-1 deficiency could be analyzed.
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ACKNOWLEDGEMENTS |
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We thank A. Berns (Amsterdam) for the
generous gift of a DNA library, M. Robinson (Cambridge) for the
-adaptin cDNA, A. Wobus (Gatersleben) for technical advice for
in vitro blastocyst cultivation, P. L. Chiodera
(Brescia) for histological analyses, and R. Dressel (Göttingen)
for technical assistance. Special thanks go to M. Pauly-Evers for the
introduction into handling the blastocysts and M. Horst for critical
reading of the manuscript. Image processing was done using Adobe
Photoshop 3.0 and Deneba Canvas 5.0.
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FOOTNOTES |
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* This work was supported by Grant SFB 523/A6 (to P. S.) from the Deutsche Forschungsgemeinschaft.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 49-551-395932;
Fax: 49-551-395979; E-mail: schu{at}uni-bc2.gwdg.de.
2 G. Payne, personal communication.
3 Generous gift of A. Berns, Amsterdam.
4 Lewin, D. A., Sheff, D., Ooi, C. E., Whitney, J. A., Yornamoto, E., Chicione, L., Webster, P., Bonifacino, J. S., and Mellman, I. (1998) GenBankTM accession numbers AF068707 and AF068706.
5 C. Meyer and P. Schu, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: TGN, trans-Golgi network; AP, adaptor protein; AP-1, AP complex 1; CD, cluster of determination; MPR, mannose-6-phosphate receptor; neoR, neomycin resistance; kb, kilobase(s); bp, base pair(s); PBS, phosphate-buffered saline; MES, 4-morpholineethanesulfonic acid; FITC, fluorescein isothiocyanate; pc, post coitus.
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REFERENCES |
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