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. 2010 Mar 5;285(10):7358-65.
doi: 10.1074/jbc.M109.056960. Epub 2009 Dec 30.

StarD7 mediates the intracellular trafficking of phosphatidylcholine to mitochondria

Yasuhiro Horibata  1 , Hiroyuki Sugimoto
Affiliations

StarD7 mediates the intracellular trafficking of phosphatidylcholine to mitochondria

Yasuhiro Horibata et al. J Biol Chem. .

Abstract

Steroidogenic acute regulatory protein-related lipid transfer (START) domains, found in 15 mammalian proteins termed StarD1-StarD15, are lipid-binding domains implicated in the intracellular lipid transport systems. In the present study, we analyzed the lipid ligand and function of StarD7. We found two variable forms of mammalian StarD7, termed StarD7-I and StarD7-II. Unlike StarD7-II, StarD7-I contained a mitochondrial-targeting sequence in its N terminus. Overexpressed StarD7-I tagged with V5/His in HEPA-1 cells was mainly observed in the mitochondria of cells prepared at low cellular density, but it was distributed in the cytoplasm of high density cells. StarD7-II was constantly distributed in the cytoplasm at any cellular density. Endogenous StarD7 in HEPA-1 cells and rat liver was also distributed in both the cytoplasm and the mitochondria. A protease K protection assay indicated that the mitochondrial StarD7 was associated with the outer mitochondrial membrane. The purified recombinant StarD7 specifically catalyzed the transfer of PC between lipid vesicles in vitro. Furthermore, the intracellular transport of fluorescent PC that was exogenously incorporated into the mitochondria was increased in cells that overexpressed StarD7-I. These results suggest that StarD7 facilitates the delivery of PC to mitochondria in non-vesicular system.

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Figures

FIGURE 1.
FIGURE 1.
Phylogenetic analysis of StarD families and mitochondrial-targeting sequences of StarD7-I. A, phylogenetic analysis and lipid ligands of StarD families. B, amino acid sequences of StarD7-I. The putative mitochondrial-targeting signal is indicated by boldface. The first Met of StarD7-II corresponds to Met76. C, mitochondria-targeting sequences at the N terminus of mammalian StarD7-I are aligned by using ClustalW (27). Identical and chemically similar amino acids are indicated by asterisks and dots, respectively. Gaps inserted into the sequences are indicated by dashed lines.
FIGURE 2.
FIGURE 2.
Proteolytic processing of StarD7-I. A, V5/His-tagged StarD7-I and -II were expressed in HEPA-1 cells (60–70% confluent), and cell lysates were analyzed by Western blotting with anti-V5 antibody. B, pulse-chase experiment. StarD7-I without tag was overexpressed in HEPA-1 cells, and proteins were pulse-labeled with 30 μCi/ml of [35S]Met and [35S]Cys for 20 min with or without CCCP. Then, cells were cultured in normal medium for 3 h. Proteins were immunoprecipitated with anti-StarD7 antibody, and precipitated proteins were separated by SDS-PAGE. C, molecular mass of endogenous StarD7. HEPA-1 cells were transfected with the StarD7-specific siRNA or the expression vector for StarD7-I or StarD7-II, and cell lysates were analyzed by Western blotting with anti-StarD7 antibody.
FIGURE 3.
FIGURE 3.
Subcellular fractionation and protease K protection assay. Localization of the overexpressed V5/His-tagged StarD7-I (A) and StarD7-II (B) in HEPA-1 cells (60–70% confluent). Mitochondrial and cytoplasmic fractions were analyzed by Western blotting with anti-V5 antibody. The purity of the mitochondrial or cytosolic fraction was verified with anti-porin or anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody. C, localization of endogenous StarD7 in HEPA-1 cells (60–70% confluent). Mitochondrial and cytoplasmic fractions were analyzed by Western blotting with anti-StarD7 antibody. D, localization of endogenous StarD7 in rat liver. The purity of microsomal fraction was verified with anti-GM130 antibody. E, mitochondrial fractions from HEPA-1 cells were analyzed by protease K protection assay. StarD7 and porin were sensitive to protease K treatment, whereas complex Vα and core I were resistant. All proteins were digested by protease K in the presence of Triton X-100 (1% w/v).
FIGURE 4.
FIGURE 4.
Lipid extraction and transfer activities of StarD7. A, purified StarD7-I and -II from E. coli. B, lipid extraction activity of StarD7. Purified StarD7-II (100 μg) was incubated with vesicles prepared from total lipids extracted from HEPA-1 cells labeled with [14C]palmitic acid (300 nmol of total phospholipid). StarD7-lipid complexes were separated from the remaining lipid vesicles with 100-kDa cutoff filters. Radioactive lipids in the filtrates were extracted and analyzed by TLC with chloroform, methanol, and water (65:25:4, v/v). 1: total lipids; 2: non-boiled StarD7-II; 3: boiled StarD7-II. C, PC transfer activity of StarD7-I. The intermembrane transfer activities for fluorescent PC analog, C12-NBD-PC, from donor vesicles to acceptor vesicles were analyzed by a fluorescence resonance energy transfer-based assay. StarD7-I prepared by an in vitro translation system in wheat germ lysates was used. Dihydrofolate reductase (DHFR) is a control protein for the in vitro translation. D, specificity of the phospholipid transfer activities of StarD7-I. Phospholipid transfer activities from donor vesicles containing one of the fluorescent phospholipid analogs, C12-NBD-PC, C12-NBD-PE, C12-NBD-PS, or C12-NBD-SM, to acceptor vesicles were assessed with purified StarD7-I. The results are representative of several independent experiments. E, ligand specificities of StarD7-I and -II for several fluorescent phospholipids. Phospholipid transfer activities of both constructs were analyzed with a fluorescence resonance energy transfer-based assay. Values are the means ± S.D. of three independent experiments.
FIGURE 5.
FIGURE 5.
Intracellular transport of fluorescent PC analog in cells that overexpress StarD7-I. A, intracellular localization of fluorescent PC exogenously incorporated into HEPA-1 cells. Cells (60–70% confluent) transfected with empty vector or the expression vector for StarD7-I were incubated with lipid vesicles containing C6-NBD-PC (green) and then with MitoTracker Red (red). Cells were fixed and analyzed by confocal microscopy. Yellow indicates the co-localization of the green and red signals. B, quantification of cells showing the co-localization of NBD-PC and MitoTracker Red. Cells (60–70% confluent) transfected with empty vector, the expression vector for StarD7-I, or StarD7-specific siRNA were incubated with NBD-PC and MitoTracker. Values are means ± S.D. from four independent culture dishes. *, p < 0.01 as compared with the vector control.
FIGURE 6.
FIGURE 6.
Intracellular localization of StarD7-I and -II. HEPA-1 cells plated at low density (20–30% confluent) (A) or high density (100% confluent) (B) were transfected with the expression vector for V5/His-tagged StarD7-I or -II, and then immunostained with anti-V5 antibody followed by anti-mouse IgG Alexa488 (green) and MitoTracker Red (red). Yellow indicates the co-localization of their signals. C, subcellular fractionations of HEPA-1 cells cultured at different cellular densities. Mitochondrial and cytoplasmic fractions were prepared from cells cultured at low density (20–30% confluent) or high density (100% confluent) and analyzed by Western blotting with anti-StarD7 antibody. The purity of the mitochondrial or cytosolic fraction was verified by anti-porin or anti-GAPDH antibody.
FIGURE 7.
FIGURE 7.
Intracellular localization of GFP fused with Met1–Leu75 at the N terminus. GFP (green) and chimeric GFP fused with Met1–Leu75 of StarD7-I at the N terminus were expressed in HEPA-1 cells cultured at low density (20–30% confluent) or high density (100% confluent) followed by treatment with MitoTracker Red (red) and analysis by confocal microscopy. Yellow indicates the co-localization of their signals.
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