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A protein conjugation system essential for autophagy

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Autophagy is a process for the bulk degradation of proteins, in which cytoplasmic components of the cell are enclosed by double-membrane structures known as autophagosomes for delivery to lysosomes or vacuoles for degradation. This process is crucial for survival during starvation and cell differentiation. No molecules have been identified that are involved in autophagy in higher eukaryotes. We have isolated 14 autophagy-defective (apg) mutants of the yeast Saccharomyces cerevisiae and examined the autophagic process at the molecular level. We show here that a unique covalent-modification system is essential for autophagy to occur. The carboxy-terminal glycine residue of Apg12, a 186-amino-acid protein, is conjugated to a lysine at residue 149 of Apg5, a 294-amino-acid protein. Of the apg mutants, we found that apg7 and apg10 were unable to form an Apg5/Apg12 conjugate. By cloning APG7, we discovered that Apg7 is a ubiquitin-E1-like enzyme. This conjugation can be reconstituted in vitro and depends on ATP. To our knowledge, this is the first report of a protein unrelated to ubiquitin that uses a ubiquitination-like conjugation system. Furthermore, Apg5 and Apg12 have mammalian homologues, suggesting that this new modification system is conserved from yeast to mammalian cells.
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Nature © Macmillan Publishers Ltd 1998
8
Received 21 May; accepted 21 July 1998.
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Acknowledgements. We thank D. Smith and J. Olesker for assistancein writing the manuscript; T. So
¨
llner,
J. E. Rothman, P. Szabo, G. van Meer, D. Nikolov, A. Koff, G. Bacher, and members of the Wiedmann,
Duvoisin and Rothman laboratories for discussions and comments; N. Min and L. Cohen-Gould for
performing the initial immunohistochemistry and electron microscopy experiments. We thank P. Marks
and R. Rifkind for suggesting the eye lens experiments. This work was supported by the Memorial Sloan-
Kettering Cancer Center, a Fellowship by the Deutsche Forschungsgemeinschaft (to K.v.L.), and by the
Samuel and May Rudin Foundation and a Tolly Vinik Pilot Grant Award (to R.M.D.).
Correspondence and requests for materials should be addressed to M.W.
letters to nature
NATURE
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VOL 395
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A protein conjugation system
essential for autophagy
Noboru Mizushima*, Takeshi Noda*, Tamotsu Yoshimori*,
Yae Tanaka
, Tomoko Ishii
, Michael D. George
,
Daniel J. Klionsky
, Mariko Ohsumi
& Yoshinori Ohsumi*
* Department of Cell Biology, National Institute for Basic Biology,
Okazaki 444-8585, Japan
Department of Biosciences, Teikyo University of Science & Technology,
Yamanashi 409-0193, Japan
Section of Microbiology, University of California, Davis, California 95616, USA
.........................................................................................................................
Autophagy is a process for the bulk degradation of proteins, in
which cytoplasmic components of the cell are enclosed by double-
membrane structures known as autophagosomes for delivery to
lysosomes or vacuoles for degradation
1–4
. This process is crucial
for survival during starvation and cell differentiation. No mol-
ecules have been identified that are involved in autophagy in
higher eukaryotes. We have isolated 14 autophagy-defective (apg)
mutants of the yeast Saccharomyces cerevisiae
5
and examined the
autophagic process at the molecular level
6–9
. We show here that a
unique covalent-modification system is essential for autophagy to
occur. The carboxy-terminal glycine residue of Apg12, a 186-
amino-acid protein, is conjugated to a lysine at residue 149 of
Apg5, a 294-amino-acid protein. Of the apg mutants, we found
that apg7 and apg10 were unable to form an Apg5/Apg12 con-
jugate. By cloning APG7, we discovered that Apg7 is a ubiquitin-
E1-like enzyme. This conjugation can be reconstituted in vitro
and depends on ATP. To our knowledge, this is the first report of a
protein unrelated to ubiquitin that uses a ubiquitination-like
conjugation system. Furthermore, Apg5 and Apg12 have mam-
malian homologues, suggesting that this new modification system
is conserved from yeast to mammalian cells.
In yeast, autophagy is induced by various starvation conditions,
and its progression is easily monitored under a light microscope
1
:
when wild-type cells were cultured under nitrogen-starvation con-
ditions in the presence of phenylmethylsulphonyl fluoride (PMSF),
autophagic bodies accumulated in the vacuoles (arrows in Fig. 1a).
The apg12-1 mutant did not accumulate autophagic bodies during
starvation. We cloned the APG12 gene by the method described
previously
7,8
. APG12 encodes a hydrophilic protein of 186 amino
acids with a predicted relative molecular mass (M
r
) of 21K (Fig. 1b).
Figure 1 Cloning of APG12 and phenotype of apg12 disruptant. a, Wild-type,
apg12-1 mutant and Dapg12 cells were cultured in nitrogen-starvation medium
containing 1 mM PMSF. After incubation for 6 h, cells were observed under a
phase-contrast microscope. Arrows indicate autophagic bodies. b, Amino-acid
sequence of Apg12. c, Wild-type (squares) and Dapg12 (circles) were cultured in
nitrogen-starvation medium and their viability was determined by phloxine B
staining
5
. d, Quantification of autophagic activity of wild-type and Dapg12 cells by
alkaline phosphatase (ALP) assay before (black bars) and after (white bars)
nitrogen starvation for 4 h. Error bars indicate s.d. of three independent
experiments. e, Homology between Apg12 and potential human and C. elegans\-
counterparts. C. elegans U32305 is 46% similar and 22% identical to amino acids
67–186 of yeast Apg12. A human cDNA (THC173313) encodes a protein that is
59% similar and 32% identical to amino acids 102186 of Apg12.
Nature © Macmillan Publishers Ltd 1998
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Gene disruption experiments revealed that APG12 is not essential
for growth (data not shown) but is essential for autophagy (Fig. 1a)
and for maintaining viability during starvation (Fig. 1c). We
confirmed this in an assay system for measuring autophagic activity
(Fig. 1d), in which a truncated form of pro-alkaline phosphatase
expressed in the cytoplasm was delivered to vacuoles in an autop-
hagy-dependent manner and processed to the active enzyme
10
. A
vacuolar enzyme, aminopeptidase I, is delivered from the cytoplasm
to vacuoles constitutively to yield the mature, active enzyme
11
. This
‘Cvt pathway’ is closely linked to the autophagic process
12
, and all
apg mutants
13
, including Dapg12 cells, show defects in this pathway
(see Fig. 3d). The amino-acid sequence of Apg12 did not provide
any insight into its function, but a BLAST search identified a
potential Caenorhabditis elegans homologue whose function is
unknown (Fig. 1e). In addition, a search of the EST (expressed-
sequence tag) database identified several cDNA fragments encoding
parts of a potential human homologue (Fig. 1e).
To detect Apg12, we constructed a 3 × haemagglutinin(HA)-
tagged APG12. On immunoblotting, Apg12 presented as a ladder of
bands between 31K32.5K (Fig. 2a). As phosphatase treatment of
the lysate yielded a single band at 31K representing tagged Apg12
(data not shown), we concluded that Apg12 is phosphorylated in
vivo. Furthermore, we found that about half of the Apg12 was
present as a much larger band of ,70K (asterisked in Fig. 2a, b).
Although the 31K Apg12 was detected in all apg mutant strains, the
Dapg5, apg7-1 and apg10-1 strains did not show the 70K band
(Fig. 2b; Dapg1 is representative of the other mutants). These results
indicate that these three APG products are essential for the genera-
tion of the 70K band.
We have previously shown that the APG5 gene encodes a 294-
amino-acid protein
6
. Immunoblot analysis of 1 × HA-tagged Apg5
indicated that it also generated two bands in nearly equal amounts,
one of the size of tagged Apg5 (32.5K) and the other at about 70K
(Fig. 2c). In the Dapg12 strain, the higher band was not seen,
whereas the 32.5K band of Apg5 was slightly increased (Fig. 2c).
Immunoprecipitation analysis revealed that the 70K band included
both Apg5 and Apg12 (Fig. 2d). We concluded that it was a one-to-
one conjugate of Apg5 and Apg12.
To characterize the 70K band further, we did mutagenic analysis
of Apg12 (Fig. 3a). We found that the carboxy-terminal portion of
Apg12 was important for the conjugation (Fig. 3b: D57 and D121).
83
62
47.5
32.5
25
HA-APG5 + + -
anti-Myc
IP Ab
Blot
Myc-Apg12
IgG
Myc-Apg12/
HA-Apg5
WT
apg5
apg7-1
apg10-1
apg1
*
HA-
Apg12
32.5
47.5
62
83
25
*
HA-
Apg12
apg12
WT
WT
HA-Apg5
*
a
b
cd
anti-Myc
anti-HA
M
r
(K)
HA-APG12
Figure 2 Apg12 is conjugated to Apg5. a, b, Lysates from Dapg12 cells carrying
only vector or 3 × HA-APG12 (a), and Dapg5, apg7-1, apg10-1 and Dapg1 cells
carrying 3 × HA-Apg12 plasmid (b) were immunoblotted using anti-HA antibody.
The positions of 3 × HA-Apg12 and the larger product (asterisks) are indicated. c,
Immunoblot analysis of wild-type and Dapg12 cells harbouring HA-APG5 plasmid.
d, Dapg5 Dapg12 cells were co-transformed with Myc-APG12 and HA-APG5.
Their lysates were immunoprecipitated with anti-Myc or anti-HA antibodies and
detected by immunoblotting using anti-Myc antibody. The position of the
crossreacting IgG heavy chain is indicated.
vector
WT
G
G186A
vector
WT
G
G186A
0
20
40
60
Apg12 WT
Apg12 121
Apg12 57
Apg12 G
Apg12 G186A
3xHA
G186A
Apg5/
Apg12
Apg12
Apg1257
WT
G
57
121
proAPI
API
ALP activity (U)
FG
FA
F
a
d
c
b
Figure 3 The C-terminal Gly residue of Apg12 is essential for interaction with
Apg5 and for autophagy. a, Diagram of Apg12 C-terminal mutants. b, Dapg12 cells
were transformed with the mutant plasmids and their lysates were immunoblotted
with anti-HA antibody. c, Autophagic activity was measured as described for Fig.1d.
d, Transport of pro-API to the vacuole was examined by immunoblotting with anti-
API antiserum. The positions of pro-API and mature API are indicated.
a
b
c
d
K
149
294
R
APG5
1xHA
vector WT K149R
0
20
40
60
80
ALP activity (U)
vector
WT
K149R
proAPI
API
vector
WT
K149R
Apg5/
Apg12
Apg5
Figure 4 Apg5
K149R
is unable to generate Apg5/Apg12 conjugate and is defective
in autophagy. a, Position of the putative Apg12-interacting Lys residue. Dapg5
cells were transformed with vector alone, wild-type APG5 or APG5
K149R
, and then
immunoblotted with anti-HA (b) and anti-API (d). Autophagic activity was
determined by alkaline phosphatase assay (c).
Nature © Macmillan Publishers Ltd 1998
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Even a single Gly 186 deletion at the C terminus (Apg12DG) caused
complete loss of the Apg12/Apg5 conjugate, although free Apg12DG
was detected in an amount comparable to that in the wild type
(Fig. 3b: DG). Apg12
G186A
, in which the Gly 186 is replaced by
alanine, was incorporated into the higher band inefficiently, but still
significantly (Fig. 3b: G186A). This indicates that Gly 186 is impor-
tant for Apg5/Apg12 conjugation. We next assessed the functional
activities of these mutants. Apg12DG showed an Apg-negative
phenotype (Fig. 3c), and was also unable to produce mature
aminopeptidase I (Fig. 3d), indicating that the Apg5/Apg12 con-
jugate is required for both autophagy and cytosol-to-vacuole
targeting of this enzyme. The Apg12
G186A
mutant showed an
almost normal phenotype for autophagy and for maturation of
aminopeptidase I (Fig. 3c, d), suggesting that a small amount of
Agp5/Apg12 conjugate is enough for it to function normally.
By analogy with ubiquitin
1416
, conjugation of Apg5 and Apg12
probably occurs through formation of an isopeptide bond between
the C-terminal Gly 186 of Apg12 and an e-amino group of one of the
19 lysine residues in Apg5. To test this, we systematically replaced
each lysine residue of Apg5 with arginine. Both free Apg5 and the
Apg5/Apg12 conjugate were detected in 18 mutants (data not
shown). The Apg5
K149R
variant had no conjugate at all, but a
higher amount of free Apg5
K149R
(Fig. 4a, b), indicating that the
Lys 149 residue of Apg5 is the acceptor site for Apg12 conjugation.
As expected, Apg5
K149R
was defective in both autophagy and in
generating mature aminopeptidase I (Fig. 4c, d), whereas the other
18 mutants were normal (data not shown). Starvation did not alter
the relative amounts of free Apg5, free Apg12 or of the Apg5/Apg12
conjugate. We conclude that the conjugate functions as a common
machinery in both pathways: for the autophagic pathway during
starvation and for the Cvt pathway in the growing phase.
As shown in Fig. 2, the apg7 and apg10 mutants failed to
conjugate Apg5 and Apg12, suggesting that these two APG products
may function as an enzyme system for conjugation. Cloning of the
APG7 gene revealed that it encodes a 630-amino-acid protein with
predicted M
r
of 71.4K (Fig. 5a). The region containing amino acids
322392 of Apg7 shows significant homology with the correspond-
ing region in E1, the ubiquitin-activating enzyme in S. cerevisiae
(Fig. 5b) and in other species (data not shown). This region
encompasses a putative ATP-binding site (GxGxxG)
17
, suggesting
that Apg7 may be an Apg12-activating enzyme. Although the
sequence around the active-site cysteine is less conserved, align-
ments between Apg7 and other E1-like enzymes indicate that
Cys 507 is a putative active-site cysteine (Fig. 5b). Apg10 might be
an E2 ubiquitin-conjugating enzyme type of protein because its size
is similar to various E2 enzymes and one of its cysteine residues is
essential for its function (T. Shintani et al., unpublished results). We
reconstituted the conjugation reaction in vitro. Lysates of Dapg5
cells and Dapg12 cells were mixed in vitro and incubated with or
without ATP. Figure 5c shows that the 70K band appeared in a time-
dependent and ATP-dependent manner. The conjugation was
sensitive to 1 mM N-ethylmaleimide (data not shown). These
results show that the Apg12 conjugation pathway contains an
ATP-dependent step, which is probably the activation of Apg12 by
Apg7.
Autophagy involves a dynamic membrane rearrangement
24
.
Morphological studies have indicated that all APG products func-
tion at or before the autophagosome formation step (M. Baba and
Y.O., unpublished results). Some Apg proteins are present on
membrane structures
9
. Most of the Apg5 and Apg5/Apg12 con-
jugate, and more than half of the free Apg12, were present in
100,000g pellet fractions (data not shown), suggesting that they
associate with some membrane compartments. We therefore exam-
ined their intracellular localization by sucrose density-gradient
centrifugation analysis and found that free Apg5 and the Apg5/
Apg12 conjugate co-fractionated (Fig. 6); in contrast, most of the
MSSERVLSYAPAFKSFLDTSFFQELSRLKLDVLKLDSTCQPLTVNLDLHN
IPKSADQVPLFLTNRSFEKHNNKRTNEVPLQGSIFNFNVLDEFKNLDKQL
FLHQRALECWEDGIKDINKCVSFVIISFADLKKYRFYYWLGVPCFQRPSS
TVLHVRPEPSLKGLFSKCQKWFDVNYSKWVCILDADDEIVNYDKCIIRKT
KVLAIRDTSTMENVPSALTKNFLSVLQYDVPDLIDFKLLIIRQNEGSFAL
NATFASIDPQSSSSNPDMKVSGWERNVQGKLAPRVVDLSSLLDPLKIADQ
SVDLNLKLMKWRILPDLNLDIIKNTKVLLLGAGTLGCYVSRALIAWGVRK
ITFVDNGTVSYSNPVRQALYNFEDCGKPKAELAAASLKRIFPLMDATGVK
LSIPMIGHKLVNEEAQHKDFDRLRALIKEHDIIFLLVDSRESRWLPSLLS
NIENKTVINAALGFDSYLVMRHGNRDEQSSKQLGCYFCHDVVAPTDSLTD
RTLDQMCTVTRPGVAMMASSLAVELMTSLLQTKYSGSETTVLGDIPHQIR
GFLHNFSILKLETPAYEHCPACSPKVIEAFTDLGWEFVKKALEHPLYLEE
ISGLSVIKQEVERLGNDVFEWEDDESDEIA
50
100
150
200
250
300
350
400
450
500
550
600
630
a
b
c
Apg5/Apg12HA
apg12
apg5
0 2 10 30 30 (min)
+ATP -ATP
1 2 3 4 5 6 7
322 IKNTKVLLLGAGTLGCYVSR--ALIAWGVRK---ITFVDNGTVSYSNPVRQALYNFEDCGKPKAELAAASLKRIFP Apg7
| |:|| |:|:| :|| : : ||: | | || :: || || |: :| || |:| || :: : |
432 IANSKVFLVGSGAIGCEMLKNWALLGLGSGSDGYIVVTDNDSIEKSNLNRQFLFRPKDVGKNKSEVAAEAVCAMNP Uba1
Uba1
Cys
600
Apg7
Cys
507
506 MCTV Apg7
599 LCTL Uba1
176 VCTI Uba2
167 MCTI Uba3
Figure 5 Apg7 is an E1-like protein and Apg12 is conjugated to Apg5 in an ATP-
dependent manner. a, Amino-acid sequence of Apg7. b, Homology between
Apg7 and Uba1, S. cerevisiae E1 enzyme. Black circles indicate a putative ATP-
binding site (GxGxxG). The putative active-site Cys residue of Apg7 is indicated. c,
In vitro conjugation of Apg5 and Apg12. A cell lysate of Dapg12 carrying HA-APG5
(lane 1) was incubated with an equal amount of lysate from Dapg5 carrying HA
APG12 (2 m plasmid) (lane 2) at 30 8C with (lane 46) or without (lane 7) 5 mM ATP.
Samples were mixed with SDS-sample buffer at the times indicated.
15105
0.0
0.1
0.2
0.3
Apg5
Apg12
Apg5/Apg12
Fraction number
TOP
BOTTOM
Recovery
ADH ALP Kex2 Sec12
Figure 6 Apg5/Apg12 conjugate co-fractionates with free Apg5 but not free
Apg12. Spheroplasts were generated from cells expressing either HA-Apg12 or
HA-Apg5. Their lysates were mixed and layered on top of a 10-step (1854 % w/w)
sucrose gradient, and centrifuged at 174,000g for 2.5 h (ref. 29). Fifteen fractions
were collected and the positions of free Apg5, free Apg12 and Apg5/Apg12
conjugate were examined by western blotting. The peak fractions of alcohol
dehydrogenase (ADH)(cytosol), ALP (vacuole), Kex2 (Golgi) and Sec12 (endo-
plasmic reticulum) are indicated by arrows.
Apg7
Apg7
Apg12
G–
C
Apg12
G
(Apg10?)
Apg5
Apg12
G–
K149
C
ATP
AMP
Figure 7 Model of the Apg12-conjugation system.
Nature © Macmillan Publishers Ltd 1998
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Apg12 was in the denser fractions. These results indicate that the
conjugation of Apg5 and Apg12 is associated with a change in the
subcellular localization of Apg12.
We have described a new covalent modification system that is
required for autophagy in yeast. Four of 14 APG products function
in this pathway. Our model is shown in Fig. 7: Apg12 is activated by
binding to Apg7 via a high-energy thioester bond; through transfer
to an E2-like molecule (possibly Apg10), Apg12 is finally conjugated
to Lys 149 of Apg5 via an isopeptide bond. Although the steps in this
conjugation pathway are similar to those that occur in ubiquitina-
tion
1416
and in the modification by other ubiquitin-like proteins
such as SUMO-1 (refs 1821), Smt3 (ref. 22), Rub1 (refs 23, 24) and
Nedd8 (ref. 25), Apg12 has several unique features. It has no
significant homology to ubiquitin and is much larger than ubiquitin
and ubiquitin-related modifiers
1825
. Only a single specific substrate,
Apg5, has been found. Apg12 homologues in human and C. elegans
have a glycine residue at the C terminus (Fig. 1c). We have cloned
human Apg12 and found that it is conjugated to human Apg5
(N.M., H. Sugita, T.Y. and Y.O., manuscript in preparation). Human
Apg5 was recently cloned as ‘apoptosis specific protein by another
group
26
, although its physiological significance is not clear yet. This
conjugation system is conserved from yeast to mammalian cells, and
may be critical for autophagy in every eukaryote.
M
. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Methods
Yeast strains. The Saccharomyces cerevisiae strains used for cloning and
immunochemical analysis were MT3-4-4(MATa apg12-1 ura3), MT87-4-
5(MATa apg7-1 ura3), MT91-4-2(MATa apg10-1 ura3), SKD5-1D(MATa ura3
leu2 trp1 Dapg5::LEU2) and YYK36(MATa ura3 leu2 trp1 his3 Dapg1::LEU2).
Gene disruptions of APG5 and APG12 were performed with YW5-1B(MATa
ura3 leu2 trp1) or KA31(MATa ura3 leu2 trp1 his3).
Alkaline phosphatase assay. The APG12 or APG5 gene was disrupted in
TN125(MATa ura3 leu2 trp1 his3 ade2 lys2 PHO8::pho8D60), and the assay was
done as described
27
.
Immunochemical procedures. Whole-cell extracts were prepared by
suspending cells in 0.2M NaOH, 0.5% b-mercaptoethanol, and precipitated
with acetone. Extracts were separated by SDSPAGE, followed by immuno-
blotting using anti-HA antibody (16B12, BAbCO) or anti-API (aminopepti-
dase I) polyclonal antibody. Immunoprecipitation was done as described
28
using 16B12 or anti-Myc antibody (9E10).
Site-directed mutagenesis. Mutation and deletion constructs were generated
by PCR-based site-directed mutagenesis and confirmed by automated DNA
sequencing.
In vitro Apg12 conjugation assay. Total cell lysates were prepared from
Dapg12 strain expressing HA-Apg5 and Dapg5 strain expressing HA-Apg12
after spheroplasting. Both lysates (30 mg ml
1
) were mixed in 50 mM Tris (pH
7.5), 100 mM NaCl, 10 mM MgCl
2
, 1 mM DTT, 0.3 mM PMSF and 2mg ml
1
pepstatin, and incubated at 30 8C for the indicated times with or without 5 mM
ATP. The reaction was stopped by mixing with SDSPAGE buffer and boiling.
Received 20 May; accepted 29 June 1998.
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Acknowledgements. We thank Y. Wada for the genomic library. N.M. is a research fellow of the Japan
Society for the Promotion of Science.
Correspondence and requests for materials should be addressed to Y.O. (e-mail: yohsumi@nibb.ac.jp).
The sequences of APG12 and APG7 are available from GenBank under accession numbers Z36086 (ORF
YBR217w) and U00027 (ORF YHR171w), respectively.
Retinoid-X receptor signalling
in the developing spinal cord
Ludmila Solomin*, Clas B. Johansson
, Rolf H. Zetterstro
¨
m
,
Reid P. Bissonnette§, Richard A. Heyman§, Lars Olson
,
Urban Lendahl
, Jonas Frise
´
n
& Thomas Perlmann*
* The Ludwig Institute for Cancer Research, Stockholm Branch, PO Box 240,
S-171 77 Stockholm, Sweden
Departments of
Cell and Molecular Biology and
Neuroscience,
Karolinska Institute, S-171 77 Stockholm, Sweden
§ Ligand Pharmaceuticals, 10275 Science Center Drive, San Diego,
California 92121, USA
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Retinoids regulate gene expression through the action of retinoic
acid receptors (RARs) and retinoid-X receptors (RXRs), which
both belong to the family of nuclear hormone receptors
1,2
. Reti-
noids are of fundamental importance during development
2
, but it
has been difficult to assess the distribution of ligand-activated
receptors in vivo. This is particularly the case for RXR, which is a
critical unliganded auxiliary protein for several nuclear receptors,
including RAR
1
, but its ligand-activated role in vivo remains
uncertain. Here we describe an assay in transgenic mice, based
on the expression of an effector fusion protein linking the ligand-
binding domain of either RXR or RAR to the yeast Gal4 DNA-
binding domain, and the in situ detection of ligand-activated
effector proteins by using an inducible transgenic lacZ reporter
gene. We detect receptor activation in the spinal cord in a pattern
that indicates that the receptor functions in the maturation of
limb-innervating motor neurons. Our results reveal a specific
activation pattern of Gal4RXR which indicates that RXR is a
critical bona fide receptor in the developing spinal cord.
Ligands for retinoid receptors are all-trans retinoic acid (RA),
which binds to RAR, and 9-cis RA, which binds both RAR and

Supplementary resources (2)

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The Saccharomyces cerevisiae APE1 gene product, aminopeptidase I (API), is a soluble hydrolase that has been shown to be localized to the vacuole. API lacks a standard signal sequence and contains an unusual amino-terminal propeptide. We have examined the biosynthesis of API in order to elucidate the mechanism of its delivery to the vacuole. API is synthesized as an inactive precursor that is matured in a PEP4-dependent manner. The half-time for processing is approximately 45 min. The API precursor remains in the cytoplasm after synthesis and does not enter the secretory pathway. The precursor does not receive glycosyl modifications, and removal of its propeptide occurs in a sec-independent manner. Neither the precursor nor mature form of API are secreted into the extracellular fraction in vps mutants or upon overproduction, two additional characteristics of soluble vacuolar proteins that transit through the secretory pathway. Overproduction of API results in both an increase in the half-time of processing and the stable accumulation of precursor protein. These results suggest that API enters the vacuole by a posttranslational process not used by most previously studied resident vacuolar proteins and will be a useful model protein to analyze this alternative mechanism of vacuolar localization.
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