Mutagenesis of the NLS and NES of human PanK2(82–570).

<p>(A) Schematic diagram of human PanK2 without the MTS and the mutant derivatives named: hPanK2-noNLS-mCherry and hPanK2-noNES-mCherry, each containing disrupted NLS or NES motifs, respectively. Arginine (R) or leucine (L) residues were replaced by the alanine (A) as indicated. (B) HeLa cells were transfected with expression plasmids encoding hPanK2(82–570)-mCherry (a, b), hPank2(82-570-noNLS)-mCherry (c, d), hPanK2(82-570-noNES)-mCherry (e, f) or mCherry alone (g,h). After 48 hours cells were stained with Hoetsch 33342 to visualize nuclei (green, b, d, f, h) and analyzed by live-cell confocal microscopy. Results are representative of at least 2 independent experiments. Scale bar, 10 µm.</p

Identification of the NLS of human PanK2.

<p>(A) Schematic diagram of the full length hPanK2(1–570) and derivatives named hPanK2(1–210), hPanK2(1–150), hPanK2(1–52), hPanK2(82–570), hPanK2(95–570), hPanK2(82–210), and hPanK2(82–94) fused with ZsGreen1. The indicated numbers are the amino acid residues encoded by the constructs. (B) HEK293 cells were transfected with the indicated hPanK2 sequences fused with ZsGreen1 and the fusion proteins (green) were visualized by live cell confocal microscopy. Cells were counterstained with MitoTracker Red CMXRos (mito, red), WGA-Alexa 647 (white) and Hoetsch 33342 (blue) to visualize mitochondria, plasma membrane and nuclei, respectively. Merged images (b, d, f, h, j, l, n, p) show the co-localization indicated by yellow pixels containing both red and green pseudocolored contributions. Results are representative of at least 2 experiments. Scale bar, 10 µm.</p

Cytosolic PanK1β partially associates with clathrin-coated and recycling endosomes.

<p>HEK293 cells were transfected with expression plasmids encoding mPanK1β fused to ZsGreen1 (green, a, c, d, f, g, i, j, l, m, o), together with different plasmids encoding the fluorescent protein mCherry fused to distinct markers for different subcellular vesicles: human clathrin LCB (magenta, b, c), human Rab5 (magenta, e, f), human Rab11 (magenta, h, i), firefly luciferase peroxisomal targeting signal (magenta, k, l) and rat Lamp1 (magenta, n, o). Rab5 designates early endosomes, Rab11 designates recycling endosomes and Lamp1 designates lysosomes. Cells were visualized using live-cell confocal imaging. Merged images show co-localization as indicated by white pixels resulting from both magenta and green pseudocolored contributions. Insets in merged images show details at higher magnification. Results are representative of 2 or more independent experiments. Scale bar, 10 µm.</p

Alignment of the amino-terminal sequences of human and mouse PanK isoforms.

<p>(A) Identical (black) or conserved (gray) residues between the human and mouse isoforms are indicated. The amino acids corresponding to the beginning of the catalytic domain of each isoform are indicated in red. The mitochondrial targeting sequence (MTS) of hPanK2 is indicated in yellow, and the nuclear localization signals (NLS) are highlighted in cyan. (B) Schematic diagram of human and mouse full-length PanK proteins. The numbers indicate the total length of the PanK proteins. The results that follow demonstrate the functionality of nuclear localization signals (NLS) and nuclear export signals (NES), which are highlighted in cyan and orange, respectively.</p

Subcellular distribution of PanK isoforms.

<p>PanK1α is sequestered in the nucleus, with preferential association within the nucleolus. PanK1β associates with clathrin-coated vesicles and recycling endosomes. Human PanK2 (hPanK2) is distributed in both the nucleus and the mitochondria. Nuclear hPanK2 translocates from nucleus to the intermembrane space of mitochondria. Mouse PanK2 (mPanK2) is cytosolic. PanK3 is distributed throughout the cytoplasm.</p

Mutagenesis of NLS in the human and mouse PanK1α isoforms.

<p>(A) Schematic diagram of human and mouse PanK1α proteins and their mutant derivatives named: hPanK1α-noNLS-His and mPanK1α-noNLS-His, containing the disrupted NLS motifs as indicated. (B) HEK293 cells were transfected with expression plasmids encoding wild type hPanK1α-His (a, b), mPanK1α-His (g, h), hPanK1α-noNLS-His (c, d), mPanK1α-noNLS-His (i, j). His-tagged PanK proteins are green, cell nuclei were stained with DAPI (blue, b, d, f, h, j, l). Cotransfection with an expression plasmid encoding Lamin A/C-mCherry (LmnA/C, magenta, d, b, d, f, h, j, l) designated nuclear membrane. The PanK1α constructs were visualized by immunocytochemistry in fixed cells using an anti-His tag antibody (a, c, g, i). Results are representative of at least 2 independent experiments. Scale bar, 10 µm.</p

Compartmentalization of Mammalian Pantothenate Kinases

<div><p>The pantothenate kinases (PanK) catalyze the first and the rate-limiting step in coenzyme A (CoA) biosynthesis and regulate the amount of CoA in tissues by differential isoform expression and allosteric interaction with metabolic ligands. The four human and mouse PanK proteins share a homologous carboxy-terminal catalytic domain, but differ in their amino-termini. These unique termini direct the isoforms to different subcellular compartments. PanK1α isoforms were exclusively nuclear, with preferential association with the granular component of the nucleolus during interphase. PanK1α also associated with the perichromosomal region in condensing chromosomes during mitosis. The PanK1β and PanK3 isoforms were cytosolic, with a portion of PanK1β associated with clathrin-associated vesicles and recycling endosomes. Human PanK2, known to associate with mitochondria, was specifically localized to the intermembrane space. Human PanK2 was also detected in the nucleus, and functional nuclear localization and export signals were identified and experimentally confirmed. Nuclear PanK2 trafficked from the nucleus to the mitochondria, but not in the other direction, and was absent from the nucleus during G2 phase of the cell cycle. The localization of human PanK2 in these two compartments was in sharp contrast to mouse PanK2, which was exclusively cytosolic. These data demonstrate that PanK isoforms are differentially compartmentalized allowing them to sense CoA homeostasis in different cellular compartments and enable interaction with regulatory ligands produced in these same locations.</p> </div

Distribution of human and mouse PanK isoforms.

<p>HEK293 cells were transfected with expression plasmids encoding hPanK1α-His (a, b), mPanK1α-His (c, d), hPanK1β-ZsGreen1 (e, f), mPanK1β-ZsGreen1 (g, h), hPanK2-ZsGreen1 (i, j), mPanK2-ZsGreen1 (k, l), hPanK3 (m, n), mPanK3 (o, p) and ZsGreen1 (q-t). PanK proteins are green, cell nuclei were stained with DAPI (blue, b, d) or Hoechst 33342 (blue, f, h, j, l, n, p, r, t). Cotransfection with an expression plasmid encoding Lamin A/C-mCherry (LmnA/C, magenta, f, h, j, l, n, p, r, t) designated nuclear membrane and staining with wheat germ agglutinin (WGA)-Alexa647 designated plasma membrane (white, f, h, j, l, n, p, r, t). The PanK1α constructs were visualized by immunocytochemistry in fixed cells using an anti-His tag antibody (a, b, c, d). The PanK1β, PanK2, PanK3 fluorescent fusion proteins as well as ZsGreen1 protein alone were visualized by live-cell confocal fluorescent microscopy. The results are representative of 2 or more independent experiments. The results for PanK2 were confirmed in a mouse cell line, NIH3T3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049509#pone.0049509-Leonardi3" target="_blank">[5]</a>. Scale bar, 10 µm.</p

Identification of NLS in the human and mouse PanK1α isoforms.

<p>HEK293 cells were transfected with expression plasmids encoding PanK1α partial sequences fused to ZsGreen1. (A) Schematic diagram of human and mouse PanK1α proteins and their derivatives named: hPanK1α(1–235), hPanK1α(1–217), hPanK1α(218–233), mPanK1α(1–185), mPanK1α(9–185), mPanK1α(61–185), mPanK1α(168–185), mPanK1α(9–150), mPanK1α(61–150), mPanK1α(1–60) and mPanK1α(1–8). The numbers indicate amino acid positions included in the fusion proteins. Dashed lines represent internal deletions. The NLS are indicated in dark gray. (B and C) Functional analysis of predicted NLS of PanK1α. hPanK1α expression constructs (panel B, a–f, green) or mPanK1α constructs (panel C, a-p, green) were co-transfected with an expression plasmid encoding lamin A/C fused to mCherry (LmnA/C, magenta), which designates the nuclear membrane, and before imaging, cells were counterstained with wheat germ agglutinin (WGA)-Alexa 647 (white) and Hoetsch 33342 (blue) to visualize the plasma membrane and the nucleus, respectively. The results are representative of at least 2 independent experiments. Scale bar, 10 µm.</p

Localization of PanK1α within the nucleolus and with the perichromosomal region during mitosis.

<p>HEK293 cells were transfected with expression plasmids encoding mPanK1α(1–185) fused to ZsGreen1 (panels A and B, a, c, d, f, green). (A) Cells were co-transfected with plasmid pAA076 encoding human fibrillarin fused to mCherry (Fibrillarin, magenta, b, c) or plasmid pAA079 encoding human B23 fused to mCherry (B23, magenta, e, f). Fibrillarin designates the dense fibrillar component and B23 designates the granular component of nucleoli. Cell nuclei were visualized by staining with Hoechst 33342 (blue). Cells were visualized using live-cell confocal imaging. Co-localization in merged images are indicated by white pixels containing both magenta and green pseudocolored contributions. Insets in merged images show details at higher magnification. (B) HEK293 cells were co-transfected with with a plasmid encoding human heterochromatin protein 1α fused to mCherry (HP1a, b, c, e, f, magenta). Cells were visualized using live-cell confocal imaging. During interphase, mPanK1α associated with nucleoli and HP1a associated with relaxed heterochromatin, thus designating the nuclear region. In mitotic cells, the nuclear envelope is absent and mPanK1α associated with condensed chromosomes, while HP1a was dissipated throughout the cytoplasm. Dashed lines delimit cell borders (note that mitotic cells are spherical and detached from the substratum). Results are representative of at least 2 independent experiments. Results obtained using hPanK1α were the same. Scale bar, 10 µm.</p