Urinalysis.

*<p>Ate1-deficient mice had a significantly lower urine pH.</p

Serum analyses.

*<p>Ate1-deficient mice with more severe phenotypes tended to have higher AST levels.</p

Cre-mediated conversion to <i>Ate1</i>-null genotype in different mouse tissues.

<p>(A) PCR-based genotyping of tail DNA to detect the Cre-mediated <i>Ate1^flox</i>→<i>Ate1⁻</i> conversion of the functionally active flox-on (<i>Ate1^flox</i>) allele to the null <i>Ate1⁻</i> allele in a 27-day old <i>Ate1^flox/−</i>;<i>CaggCreER</i> mouse immediately after the fourth (daily) intraperitoneal (IP) injection of tamoxifen (TM+), or in the absence of TM treatment (TM-). Upper panel: the 512 bp DNA fragment characteristic of the flox-on (<i>Ate1^flox</i>) allele and the 472 bp DNA fragment characteristic of either wild-type or the previously constructed <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007757#pone.0007757-Kwon2" target="_blank">[10]</a> unconditionally null <i>Ate1⁻</i> allele, using primers CB156 and CB157 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007757#pone-0007757-t004" target="_blank">Table 4</a>). Lower panel: the 470 bp DNA fragment characteristic of the Cre-produced flox-off (<i>Ate1⁻</i>) allele, with primers CB110 and CB157 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007757#pone-0007757-t004" target="_blank">Table 4</a>); and the 324 bp DNA fragment (control), amplified from the <i>IL-2</i> gene using primers IMR42 and IMR43, in the same PCR reaction. (B) The Cre-mediated <i>Ate1^flox</i>→<i>Ate1⁻</i> conversion, detected by PCR (as described in panel A) in genomic DNA isolated from the indicated tissues immediately after the fourth (daily) IP injection of tamoxifen in a 24-day old <i>Ate1^flox/−</i>;<i>CaggCreER</i> mouse. (C) Relative in vitro arginylation activity (cpm/reaction) in extracts of the indicated tissues from a wild type mouse (<i>Ate1^+/+</i>) (black bar), a heterozygous mouse (<i>Ate1^+/−</i>) (blue bar), and an <i>Ate1^−/−</i> mouse (the latter mouse was initially <i>Ate1^flox/−</i>;<i>CaggCreER</i>) (red bar) from the same litter 76 days after TM treatment. A white bar on the right indicates the relative arginylation activity obtained with purified recombinant mouse Ate1 (denoted as “rAte1”) that had been expressed in S. cerevisiae. Shown here are “cpm/reaction” after subtracting “cpm/reaction” in the null-control (“buffer alone”) sample. The control incorporation was approximately equal to that observed in extracts from spleen and thymus. In other words, the assay configured as described in this panel and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007757#s4" target="_blank">Materials and Methods</a> was not sensitive enough to robustly detect the arginylation activity in extracts from spleen and thymus. (D) Relative in vitro arginylation activity (cpm/reaction) in the whole brain, cerebellum, and hippocampus harvested from wild type mice (<i>Ate1^+/+</i>; n = 3), heterozygous mice (<i>Ate1^+/−</i>; n = 3), and <i>Ate1^−/−</i> mice (specifically, <i>Ate1^flox/−</i>;<i>CaggCreER</i> mice; n = 3) mice 40 days after TM treatment. Standard deviations are indicated. (E) Relative in vitro arginylation activity (cpm/reaction) in testis extracts from <i>Ate1^+/+</i> mice (n = 3) and <i>Ate1^−/−</i> mice (specifically, <i>Ate1^flox/−</i>;<i>CaggCreER</i> mice; n = 3) ∼130 days after TM treatment. Standard deviations are indicated.</p

Loss of white adipose tissue (WAT), resistance to high fat diet-induced obesity, and ectopic Ucp1 in WAT of Ate1-deficient mice.

<p>(A–C) Visceral fat content of Ate1-containing mice. Shown here are representative examples of Ate1-containing (<i>Ate1^flox/+</i>;<i>CaggCreER</i>) (A) and <i>Ate1^flox/−</i> (B)) and Ate1-deficient (<i>Ate1^flox/−</i>;<i>CaggCreER</i> (C)) mice 37 days after TM-treatment. Note the loss of both visceral fat (large white arrow in A and B) and fat surrounding the kidney (small white arrows in A and B) in an Ate1-deficient mouse (C). (D) Hematoxylin/eosin staining of a 10-µm section of white adipose tissue (WAT) harvested from an Ate1-containing mouse (TM-treated <i>Ate1^flox/+</i>;<i>CaggCreER</i>). The bar denotes 100 µm. (E) Same as in D except that WAT was from an Ate1-deficient mouse (TM-treated <i>Ate1^flox/−</i>;<i>CaggCreER</i>). (F) Average weights of TM-treated Ate1-containing (n = 12; black curve) and Ate1-deficient (n = 11; red curve) mice as a function of time after the beginning of <i>ad libitum</i> high-fat diet. Weights were measured at weekly intervals for 10 weeks. Error bars indicate ±SD. (G) Comparisons, by immunoblotting, of Ucp1 protein levels in extracts from brown adipose tissue (BAT) (lanes 1 and 2) and WAT (lanes 3 through 6) from <i>Ate1^+/−</i> and <i>Ate1^−/−</i> mice 46 days (lanes 1 and 2) or ∼1 year (lanes 3–6) after TM treatment. Specific genotypes were as follows (genotypes after TM treatment are indicated in parentheses here, and also on top of the gel): lane 1, <i>Ate1^flox/−</i> (+/−); lane 2, <i>Ate1^flox/−</i>;<i>CaggCreER</i> (−/−); lane 3, <i>Ate1^flox/−</i>;<i>CaggCreER</i> (−/−); lane 4, <i>Ate1^flox/−</i> (+/−); lane 5, <i>Ate1^flox/−</i>;<i>CaggCreER</i> (−/−); lane 6, <i>Ate1^flox/+</i>;<i>CaggCreER</i> (+/−). Note abnormally high expression of Ucp1 in WAT of Ate1-deficient mice (lanes 3 and 5). An asterisk denotes a protein in WAT that cross-reacts with anti-Ucp1 antibody. (H) RT-PCR analyses of leptin and <i>Ucp1</i> mRNA levels in BAT (lanes 1–4) and WAT (lanes 5–8) of Ate1-containing (denoted as “+/−”; lanes 2, 4, 6, and 8) and Ate1-deficient (denoted as “−/−”; lanes 1, 3, 5, and 7) mice ∼1 year after TM treatment. Specific genotypes: lanes 1 and 5, <i>Ate1^flox/flox</i>;<i>CaggCreER</i> (−/−); lanes 2 and 6, <i>Ate1^flox/+</i>;<i>CaggCreER</i> (+/−); lanes 3 and 5, <i>Ate1^flox/flox</i>;<i>CaggCreER</i> (−/−); lanes 4 and 8, <i>Ate1^flox/−</i> (+/−). (I) RT-PCR analyses of <i>Ucp1</i> and <i>Ucp2</i> mRNA levels in BAT, liver, muscle, and WAT of an <i>Ate1^flox/+</i> mouse (denoted as “+/−”) and an <i>Ate1^flox/−</i>;<i>CaggCreER</i> mouse (denoted as “−/−”)∼1 year after TM treatment.</p

Genomic configurations at the <i>Ate1</i> locus of <i>Cre-lox</i>-based mouse strains constructed in the present work.

<p>(A) The 5′ end of the previously produced unconditional <i>Ate1⁻</i> allele <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007757#pone.0007757-Kwon2" target="_blank">[10]</a>, in which the <i>Ate1</i> exons 1b through 3 were replaced by a cassette encoding a promoter-lacking, NLS-containing LacZ (NLS-βgal) (it was expressed from the endogenous P<i>_Ate1</i> promoter) and the Neo selection marker expressed from the phosphoglycerate kinase P<i>_PGK</i> promoter (green rectangles). (B) A diagram of the 5′ end of wild-type (wt) mouse <i>Ate1</i>, indicating approximate locations of exons 1a through 5. (C) The ∼22.5 kb targeting construct containing a ∼6 kb long-arm region of <i>Ate1</i> homology (shown as a shaded rectangle on the left); a single loxP site (red triangle) upstream of <i>Ate1</i> exon 2, a “floxed”-hygromycin-resistance (<i>hph</i>) cassette, expressed from the P<i>_PGK</i> promoter (blue arrow between two red triangles) downstream of <i>Ate1</i> exon 4; a ∼2 kb short-arm region of homology (an inclined shaded rectangle), and the HSV thymidine kinase (tk) negative-selection cassette expressed from the P<i>_HSV</i> promoter (yellow arrow). Wavy line indicates an abutting sequence of the pBR322 plasmid DNA. (D) The tri-lox <i>Ate1</i> allele obtained after a correctly targeted double crossover event. (E) In the notations here and elsewhere in the paper, “flox-on” indicates a configuration depicted in this panel (the functionally active <i>Ate1^flox</i> allele), whereas “flox-off” indicates a configuration depicted in panel F (the null <i>Ate1⁻</i> allele). The functionally active, “flox-on” (<i>Ate1^flox</i>) allele, obtained by the removal of the <i>hph</i> cassette, using the <i>in vivo</i> expression of Cre-recombinase driven by the P<i>_EIIA</i> promoter, which is active only in pre-implantation blastocysts. (F) The null “flox-off” (<i>Ate1⁻</i>) allele obtained by the inducible expression of CreER recombinase from the P<i>_Cagg</i> promoter and posttranslationally induced by tamoxifen (TM) treatment (see the main text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007757#s4" target="_blank">Materials and Methods</a>). H, approximate locations of HindIII sites used in Southern analyses with DNA probe <b>A</b> (see panel G); E, approximate locations of EcoRI sites used in Southern analyses with DNA probe <b>D</b> (see panel H); black boxes marked “A” and “D” indicate the regions specific for DNA probes <b>A</b> and <b>D</b>, respectively. (G) Southern hybridization analysis using DNA probe <b>A</b> and HindIII-digested genomic DNA. The wt <i>Ate1</i> allele (panel B) yields the 11.8 kb HindIII fragment. The previously constructed <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007757#pone.0007757-Kwon2" target="_blank">[10]</a> unconditionally null <i>Ate1⁻</i> allele (panel A), denoted as “null” on this panel, yields the 9.8 kb HindIII fragment. The functionally active flox-on (<i>Ate1^flox</i>) allele (panel E) yields the 6.3 kb HindIII fragment. Lane 1, <i>Ate1^+/+</i>; lane 2, <i>Ate1^+/−</i>; lane 3, <i>Ate1^+/−</i>; lane 4, <i>Ate1^flox/−</i>. (H) Southern hybridization analysis using DNA probe <b>D</b> (external to targeting vector) and EcoRI-digested genomic DNA. The previously constructed <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007757#pone.0007757-Kwon2" target="_blank">[10]</a> unconditionally null <i>Ate1⁻</i> allele (denoted as “null”) yields the 5.8 kb fragment. Both the wild-type <i>Ate1</i> allele and the flox-on (<i>Ate1^flox</i>) allele yield the 9.7 kB fragment, whereas the null flox-off (<i>Ate1⁻</i>) allele yields the characteristic 3.8 kb fragment. The use of DNA probe <b>D</b> and EcoRI-digested DNA from specific tissues of tamoxifen (TM)-treated <i>Ate1^flox/−</i>;<i>CaggCreER</i> mice allowed approximate estimates of the levels of Cre-mediated recombination that produced the flox-off (<i>Ate1⁻</i>) allele. For example, whereas no flox-on (<i>Ate1^flox</i>) allele could be detected in the kidney and brain of <i>Ate1^flox/−</i>;<i>CaggCreER</i> mice after TM treatment (lanes 5, 6), approximately equal amounts of flox-on (<i>Ate1^flox</i>) and flox-off (<i>Ate1⁻</i>) alleles were present in the heart of TM-treated <i>Ate1^flox/−</i>; <i>CaggCreER</i> mice. Lanes 1–3, 1,000, 250, and 25 ng of EcoRI-digested wt mouse genomic DNA (from a tail biopsy), respectively. Lane 4, EcoRI-digested genomic DNA from the tail of a previously constructed <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007757#pone.0007757-Kwon2" target="_blank">[10] </a><i>Ate1^+/−</i> mouse. Lanes 5–7, EcoRI-digested genomic DNA from the indicated tissues of TM-treated <i>Ate1^flox/−</i>;<i>CaggCreER</i> mice. Lane 8, same as lane 7, but from a TM-treated <i>Ate1^flox/−</i> mouse (lacking the <i>CaggCreER</i> transgene).</p

Brain, behavioral, and testis abnormalities of Ate1-deficient mice.

<p>(A) Enlarged brains of Ate1-deficient mice. Upper panel: comparison of representative brains harvested from an <i>Ate1^+/+</i> and an <i>Ate1^−/−</i> mouse, respectively, 134 days after tamoxifen (TM) treatment. Lower panel: brain weights expressed as percentages of total body weights in <i>Ate1^+/+</i> (n = 41) and <i>Ate1^−/−</i> (n = 40) mice. Horizontal bars and numbers indicate mean values. (B) Wet (0.4053 g versus 0.4608 g) and dry (0.1022 g versus 0.1119 g) weight components of the total mean brain weights (±SD) in <i>Ate1^+/+</i> and <i>Ate1^−/−</i> mice. (C) Total distance traveled (in meters), over 15 min, in an open field test among mice of different genotypes belonging to the same litter, 44 days after TM-treatment. Bar 1, <i>Ate1^flox/+</i>;<i>CaggCreER</i> mouse. Bar 2, <i>Ate1^+/+</i>;<i>CaggCreER</i> mouse. Bar 3, <i>Ate1^+/+</i> mouse. Bar 4, <i>Ate1^flox/−</i>;<i>CaggCreER</i> mouse that was converted to <i>Ate1^−/−</i> by TM treatment. Blue and red bars denote Ate1-containing and Ate1-deficient mice, respectively. (D) Same as in C but maximum lengths of single movements (in centimeters). (E) Same as in C but mean velocities (in cm/second) over 15 min. (F) Paraffin sections (4 µm) of testis showing cross-sections of seminiferous tubules in <i>Ate1^+/+</i> testis stained with hematoxylin and eosin (150× magnification). (G) Same as in F but <i>Ate1^−/−</i> testis. Note that sperm tails in the lumens of <i>Ate1^−/−</i> tubules are sparse in comparison to those in <i>Ate1^+/+</i> testis. (H) Same as in F but at 600× magnification. (I) Same as in G but at 600× magnification. (J) XGal staining for βgal activity in a 10-µm section of <i>Ate1^+/−</i> testis in which one copy of <i>Ate1</i> was replaced by an ORF encoding NLS-β-galactosidase and expressed from the P<i>_Ate1</i> promoter (100× magnification). (K) Immunoblotting analysis, using antibody to poly (ADP-ribose) polymerase (PARP), of testis extracts from an Ate1-containing (<i>Ate1^flox/−</i> (+/−)) and an Ate1-deficient (<i>Ate1^flox/−</i>;<i>CaggCreER</i> (−/−)) mouse 16 days after TM treatment. Note the loss of the full-length length 116 kDa PARP and the presence of the 85 kDa PARP fragment (lane2). An asterisk denotes a protein crossreacting with anti-PARP antibody.</p

Energy balance and metabolic rate in Ate1-deficient mice.

<p>(A) Glucose tolerance test. Glucose concentration (mg/dL) in whole blood of Ate1-containing mice (n = 15; black curve) and Ate1-deficient mice (n = 11; red curve), at different times after a bolus of glucose by gavage, following a 16-hr fast. Glucose was administered at time zero. Error bars indicate ±SD. (B) Fasting blood glucose levels. Average blood glucose levels (mg/dL) in Ate1-containing mice (n = 15; black bar) and Ate1-deficient mice (n = 11; red bar), with measurements shortly before glucose gavage (after a 16-hr fast) and 6 hr after the gavage in A. Standard deviations are indicated. Statistical analysis was performed using an unpaired t-test (p<0.04). (C) Average daily energy consumption (kcal/gm of body weight) for Ate1-containing mice (n = 5; black curve) and Ate1-deficient mice (n = 3; red curve), with measurements from 1 week prior to tamoxifen (TM) treatment. Vertical arrow indicates the beginning of a 5-day TM treatment. Error bars indicate ±SD. (D) Relative efficiencies of the import of ¹⁴C-amino acids and ¹⁴C-peptides from gastrointestinal tract in Ate1-containing mice (black bars) versus Ate1-deficient mice (red bars). Shown here are representative comparisons of the retention of ¹⁴C (in cpm/gm) in the brains, livers, spleens, kidneys, and hearts of indicated mice 16 days after gavage with a single bolus of ¹⁴C-labeled proteins (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007757#s4" target="_blank">Materials and Methods</a>). Mice were gavaged 26 days after TM treatment. (E) Comparison of resting metabolic rate (RMR) (measured in O₂ (ml) consumed per kg of body weight per min) for Ate1-containing mice (n = 6; black bar) versus Ate1-deficient mice (n = 6; red bar). Standard deviations are indicated in E and F. Statistical analysis was performed using an unpaired t-test (p<0.008). (F) Comparison of the respiratory exchange ratio (RER), measured as CO₂ (in ml) per ml of O₂, for Ate1-containing mice (n = 6; black bar) and Ate1-deficient mice (n = 6; red bar) mice. No statistically significant difference in RER was observed. (G) RT-PCR analyses of <i>AgRP, MCH, HPY,</i> and <i>POMC</i> mRNA levels in the hypothalami of TM-treated Ate1-containing mice (Sets 1 and 4) versus Ate1-deficient mice (Sets 2 and 3). Set 1, <i>Ate1^flox/flox</i> (+/+); Set 2, <i>Ate1^flox/flox</i>;<i>CaggCreER</i> (−/−); Set 3, <i>Ate1^flox/flox</i>;<i>CaggCreER</i> (−/−); Set 4, <i>Ate1^flox/+</i>;<i>CaggCreER</i> (+/−). In sets 1 and 2, hypothalami were isolated 93 days after TM treatment. In sets 3 and 4 hypothalami were isolated ∼1 year after TM treatment. Sloping triangles indicate decreasing inputs (by 2-fold) of total RNA.</p

PCR primers used in the present study.

<p>PCR primers used in the present study.</p

Brain abnormalities and behavioral phenotypes of Ate1-deficient mice.

<p>(A) Ate1-deficient mice become hyperactive as a function of time after TM-mediated ablation of <i>Ate1</i>. Total distance (in cm) traveled over 15 min in the open field test box (2500 cm²). This test was repeated every ∼2 weeks after the end of TM treatment. The data for Ate1-containing mice (n = 5; their genotypes were <i>Ate1^flox/+</i>, <i>Ate1^flox/−</i>, and <i>Ate1^flox/+</i>;<i>CaggCreER</i>) and Ate1-deficient mice (n = 3; <i>Ate1^flox/−</i>;<i>CaggCreER</i>) are indicated by black diamonds and red circles, respectively. The horizontal bars indicate mean values. The average total distance traveled over 15 min for all Ate1-containing mice (n = 37) was 4,870 cm. (B) Representative magnetic resonance images showing equivalent horizontal planes of Ate1-containing (<i>Ate1^flox/+</i>;<i>CaggCreER</i> on the left, <i>Ate1^flox/+</i> on the right) brains ∼3 months after TM treatment. The indicated average width of the skull (measured at the widest point from left to right in the same plane) of four Ate1-containing mice was 10.6 mm (±0.89 mm). (C) Same as in B except with brains from Ate1-deficient (<i>Ate1^flox/−</i>;<i>CaggCreER</i>) mice ∼3 months after TM-treatment. The average width of the skull (measured as in B) of four Ate1-deficient mice was 10.8 mm (±0.38 mm). (D) Comparison of the response latency (T_max; recorded in msec) between Ate1-containing (n = 3; black bars) and Ate1-deficient mice (n = 3; red bars) to a 40-msec pulse of 120 dB (p120; p<0.3), a 40-msec pulse of 120 dB preceded by a pre-pulse of 5 dB (pp5; p<0.09), or a 40-msec pulse of 120 dB preceded by a pre-pulse of 15 dB (pp15; p<0.01). Statistical analysis was performed using an unpaired t-test.</p

Postnatal ablation of the mouse Ate1 R-transferase, a component of the N-end rule pathway.

<p>(A) The mammalian N-end rule pathway. N-terminal residues are indicated by single-letter abbreviations for amino acids. Yellow ovals denote the rest of a protein substrate. “Primary”, “secondary” and “tertiary” denote mechanistically distinct subsets of destabilizing N-terminal residues (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007757#s1" target="_blank">Introduction</a>). C* denotes oxidized Cys, either Cys-sulfinate or Cys-sulfonate. MetAPs, Met-aminopeptidases. (B) Bidirectional promoter between the mouse <i>Ate1</i> exons 1A and 1B <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007757#pone.0007757-Hu3" target="_blank">[14]</a>. Green arrows indicate transcriptional units, including a previously uncharacterized gene, termed <i>Dfa</i> (“<u>d</u>ivergent <u>o</u>f <u>A</u>te1), that is transcribed from the bidirectional promoter. (C) Immunoblotting-based comparisons of Ate1 levels in the indicated mouse tissues from <i>Ate1^+/+</i> and <i>Ate1^flox/−</i>;<i>CaggCreER</i> mice 76 days after the tamoxifen (TM)-induced, Cre-mediated <i>Ate1^flox</i>→<i>Ate1⁻</i> conversion that yielded Ate1-deficient mice. The band of 60-kDa Ate1, detected by antibody to mouse Ate1, is indicated on the right. Total (Ponceau-stained) protein patterns are shown below, with positions of molecular-mass markers on the left. (D) IB assays for the levels of Ate1 and Rgs4 (25 kDa) in brain extracts from <i>Ate1^+/+</i> and Ate1-deficient mice (<i>Ate1^flox/−</i>;<i>CaggCreER</i> mice 30 days after TM treatment).</p