Mitochondrial Localization of ABC Transporter ABCG2 and Its Function in 5-Aminolevulinic Acid-Mediated Protoporphyrin IX Accumulation

Accumulation of protoporphyrin IX (PpIX) in malignant cells is the basis of 5-aminolevulinic acid (ALA)-mediated photodynamic therapy. We studied the expression of proteins that possibly affect ALA-mediated PpIX accumulation, namely oligopeptide transporter-1 and -2, ferrochelatase and ATP-binding cassette transporter G2 (ABCG2), in several tumor cell lines. Among these proteins, only ABCG2 correlated negatively with ALA-mediated PpIX accumulation. Both a subcellular fractionation study and confocal laser microscopic analysis revealed that ABCG2 was distributed not only in the plasma membrane but also intracellular organelles, including mitochondria. In addition, mitochondrial ABCG2 regulated the content of ALA-mediated PpIX in mitochondria, and Ko143, a specific inhibitor of ABCG2, enhanced mitochondrial PpIX accumulation. To clarify the possible roles of mitochondrial ABCG2, we characterized stably transfected-HEK (ST-HEK) cells overexpressing ABCG2. In these ST-HEK cells, functionally active ABCG2 was detected in mitochondria, and treatment with Ko143 increased ALA-mediated mitochondrial PpIX accumulation. Moreover, the mitochondria isolated from ST-HEK cells exported doxorubicin probably through ABCG2, because the export of doxorubicin was inhibited by Ko143. The susceptibility of ABCG2 distributed in mitochondria to proteinase K, endoglycosidase H and peptide-N-glycosidase F suggested that ABCG2 in mitochondrial fraction is modified by N-glycans and trafficked through the endoplasmic reticulum and Golgi apparatus and finally localizes within the mitochondria. Thus, it was found that ABCG2 distributed in mitochondria is a functional transporter and that the mitochondrial ABCG2 regulates ALA-mediated PpIX level through PpIX export from mitochondria to the cytosol

Protein N-myristoylation plays a critical role in the mitochondrial localization of human mitochondrial complex I accessory subunit NDUFB7

Abstract The present study examined human N-myristoylated proteins that specifically localize to mitochondria among the 1,705 human genes listed in MitoProteome, a mitochondrial protein database. We herein employed a strategy utilizing cellular metabolic labeling with a bioorthogonal myristic acid analog in transfected COS-1 cells established in our previous studies. Four proteins, DMAC1, HCCS, NDUFB7, and PLGRKT, were identified as N-myristoylated proteins that specifically localize to mitochondria. Among these proteins, DMAC1 and NDUFB7 play critical roles in the assembly of complex I of the mitochondrial respiratory chain. DMAC1 functions as an assembly factor, and NDUFB7 is an accessory subunit of complex I. An analysis of the intracellular localization of non-myristoylatable G2A mutants revealed that protein N-myristoylation occurring on NDUFB7 was important for the mitochondrial localization of this protein. Furthermore, an analysis of the role of the CHCH domain in NDUFB7 using Cys to Ser mutants revealed that it was essential for the mitochondrial localization of NDUFB7. Therefore, the present results showed that NDUFB7, a vital component of human mitochondrial complex I, was N-myristoylated, and protein N-myrisotylation and the CHCH domain were both indispensable for the specific targeting and localization of NDUFB7 to mitochondria

A strategy to identify protein-N-myristoylation-dependent phosphorylation reactions of cellular proteins by using Phos-tag SDS-PAGE.

To establish a strategy for identifying protein-N-myristoylation-dependent phosphorylation of cellular proteins, Phos-tag SDS-PAGE was performed on wild-type (WT) and nonmyristoylated mutant (G2A-mutant) FMNL2 and FMNL3, phosphorylated N-myristoylated model proteins expressed in HEK293 cells. The difference in the banding pattern in Phos-tag SDS-PAGE between the WT and G2A-mutant FMNL2 indicated the presence of N-myristoylation-dependent phosphorylation sites in FMNL2. Phos-tag SDS-PAGE of FMNL2 mutants in which the putative phosphorylation sites listed in PhosphoSitePlus (an online database of phosphorylation sites) were changed to Ala revealed that Ser-171 and Ser-1072 are N-myristoylation-dependent phosphorylation sites in FMNL2. Similar experiments with FMNL3 demonstrated that N-myristoylation-dependent phosphorylation occurs at a single Ser residue at position 174, which is a Ser residue conserved between FMNL2 and FMNL3, corresponding to Ser-171 in FMNL2. The facts that phosphorylation of Ser-1072 in FMNL2 has been shown to play a critical role in integrin β1 internalization mediated by FMNL2 and that Ser-171 in FMNL2 and Ser-174 in FMNL3 are novel putative phosphorylation sites conserved between FMNL2 and FMNL3 indicate that the strategy used in this study is a useful tool for identifying and characterizing physiologically important phosphorylation reactions occurring on N-myristoylated proteins

Protein <i>N</i>-Myristoylation Plays a Critical Role in the Endoplasmic Reticulum Morphological Change Induced by Overexpression of Protein Lunapark, an Integral Membrane Protein of the Endoplasmic Reticulum

<div><p><i>N</i>-myristoylation of eukaryotic cellular proteins has been recognized as a modification that occurs mainly on cytoplasmic proteins. In this study, we examined the membrane localization, membrane integration, and intracellular localization of four recently identified human <i>N</i>-myristoylated proteins with predicted transmembrane domains. As a result, it was found that protein Lunapark, the human ortholog of yeast protein Lnp1p that has recently been found to be involved in network formation of the endoplasmic reticulum (ER), is an <i>N</i>-myristoylated polytopic integral membrane protein. Analysis of tumor necrosis factor-fusion proteins with each of the two putative transmembrane domains and their flanking regions of protein Lunapark revealed that transmembrane domain 1 and 2 functioned as type II signal anchor sequence and stop transfer sequence, respectively, and together generated a double-spanning integral membrane protein with an N-/C-terminal cytoplasmic orientation. Immunofluorescence staining of HEK293T cells transfected with a cDNA encoding protein Lunapark tagged with FLAG-tag at its C-terminus revealed that overexpressed protein Lunapark localized mainly to the peripheral ER and induced the formation of large polygonal tubular structures. Morphological changes in the ER induced by overexpressed protein Lunapark were significantly inhibited by the inhibition of protein <i>N</i>-myristoylation by means of replacing Gly2 with Ala. These results indicated that protein <i>N</i>-myristoylation plays a critical role in the ER morphological change induced by overexpression of protein Lunapark.</p></div

Role of the zinc finger motif of protein Lunapark in the ER morphological change induced by protein Lunapark.

<p>A. Alignment of the zinc finger motif of Lunapark protein family members. Highly conserved cysteine residues are indicated by red arrows. B. Detection of protein <i>N</i>-myristoylation of Lunapark-CtoA-FLAG expressed in HEK293T cells. cDNAs encoding Lunapark-FLAG, Lunapark-G2A-FLAG, and Lunapark-CtoA-FLAG were transfected in to HEK293T cells, and their expression and the <i>N</i>-myristoylation of the products in the total cell lysates were evaluated by Western blotting analysis and [³H]myristic acid ([³H]Myr) labeling, respectively. C. Intracellular localization of Lunapark-FLAG, Lunapark-G2A-FLAG, and Lunapark-CtoA-FLAG was determined by immunofluorescence staining of HEK293T cells transfected with cDNAs encoding these three proteins using an anti-FLAG antibody. Right panel shows a close-up view of the area surrounded by a white box in the immunofluorescence image. D. Quantitative analysis of the ER morphological change in HEK293T cells induced by Lunapark-FLAG (WT), Lunapark-G2A-FLAG (G2A), and Lunapark-CtoA-FLAG (CtoA). cDNAs encoding Lunapark-FLAG, Lunapark-G2A-FLAG, and Lunapark-CtoA-FLAG were transfected in to HEK293T cells and the morphological change of the ER in each cell was determined by immunofluorescence staining and the extent of the ER morphological change was compared. The extent of ER morphological changes is expressed as a percentage of the number of cells with highly tubular, partially tubular, and non-tubular ER against the total number of transfected cells. Data are expressed as mean ± SD for five independent experiments. **<i>P</i><0.001 vs. WT. *<i>P</i><0.01 vs. WT.</p

Protein Lunapark mainly localized to the peripheral ER and induced the ER morphological change in an <i>N</i>-myristoylation-dependent manner.

<p>A. EGFP-Sec61β cDNA alone, Lunapark-FLAG cDNA and EGFP-Sec61β cDNA, or Lunapark-G2A-FLAG cDNA and EGFP-Sec61β cDNA, were transfected in to HEK-293T cells, and distribution of these proteins was evaluated by immunofluorescence analysis or fluorescence microscopic analysis. b′, c′, d′, e′, f′, g′, h′, and i′ show a close-up and over-exposed image of the area surrounded by a white box in b, c, d, e, f, g, h, and i, respectively. B. EGFP-Sec61β cDNA was transfected in to HEK-293T cells and the distribution of the protein was evaluated by fluorescence microscopic analysis. ER was detected with ER-Tracker Red.</p