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

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.

Coprinopsis cinerea dioxygenase is an oxygenase forming 10(S)-hydroperoxide of linoleic acid, essential for mushroom alcohol, 1-octen-3-ol, synthesis.

1-Octen-3-ol is a volatile oxylipin found ubiquitously in Basidiomycota and Ascomycota. The biosynthetic pathway forming 1-octen-3-ol from linoleic acid via the linoleic acid 10(S)-hydroperoxide was characterized 40 years ago in mushrooms, yet the enzymes involved are not identified. The dioxygenase 1 and 2 genes (Ccdox1 and Ccdox2) in the mushroom Coprinopsis cinerea contain an N-terminal cyclooxygenase-like heme peroxidase domain and a C-terminal cytochrome P450-related domain. Herein, we show that recombinant CcDOX1 is responsible for dioxygenation of linoleic acid to form the 10(S)-hydroperoxide, the first step in 1-octen-3-ol synthesis, whereas CcDOX2 conceivably forms linoleic acid 8-hydroperoxide. We demonstrate that KO of the Ccdox1 gene suppressed 1-octen-3-ol synthesis, although added linoleic acid 10(S)-hydroperoxide was still efficiently converted. The P450-related domain of CcDOX1 lacks the characteristic Cys heme ligand and the evidence indicates that a second uncharacterized enzyme converts the 10(S)-hydroperoxide to 1-octen-3-ol. Additionally, we determined the gene KO strain (ΔCcdox1) was less attractive to fruit fly larvae, while the feeding behavior of fungus gnats on ΔCcdox1 mycelia showed little difference from that on the mycelia of the WT strain. The proliferation of fungivorous nematodes on ΔCcdox1 mycelia was similar to or slightly worse than that on WT mycelia. Thus, 1-octen-3-ol seems to be an attractive compound involved in emitter-receiver ecological communication in mushrooms.

ANKRD22 is an N-myristoylated hairpin-like monotopic membrane protein specifically localized to lipid droplets.

The membrane topology and intracellular localization of ANKRD22, a novel human N-myristoylated protein with a predicted single-pass transmembrane domain that was recently reported to be overexpressed in cancer, were examined. Immunofluorescence staining of COS-1 cells transfected with cDNA encoding ANKRD22 coupled with organelle markers revealed that ANKRD22 localized specifically to lipid droplets (LD). Analysis of the intracellular localization of ANKRD22 mutants C-terminally fused to glycosylatable tumor necrosis factor (GLCTNF) and assessment of their susceptibility to protein N-glycosylation revealed that ANKRD22 is synthesized on the endoplasmic reticulum (ER) membrane as an N-myristoylated hairpin-like monotopic membrane protein with the amino- and carboxyl termini facing the cytoplasm and then sorted to LD. Pro98 located at the center of the predicted membrane domain was found to be essential for the formation of the hairpin-like monotopic topology of ANKRD22. Moreover, the hairpin-like monotopic topology, and positively charged residues located near the C-terminus were demonstrated to be required for the sorting of ANKRD22 from ER to LD. Protein N-myristoylation was found to positively affect the LD localization. Thus, multiple factors, including hairpin-like monotopic membrane topology, C-terminal positively charged residues, and protein N-myristoylation cooperatively affected the intracellular targeting of ANKRD22 to LD.

Protein-N-myristoylation-dependent phosphorylation of serine 13 of tyrosine kinase Lyn by casein kinase 1γ at the Golgi during intracellular protein traffic.

Protein N-myristoylation of Src-family kinases (SFKs) is a critical co-translational modification to anchor the enzymes in the plasma membrane. Phosphorylation of SFKs is also an essential modification for regulating their enzymatic activities. In this study, we used Phos-tag SDS-PAGE to investigate N-myristoylation-dependent phosphorylation of SFKs and their non-N-myristoylated G2A mutants. The serine-13 residue of Lyn (Lyn-S13) was shown to be N-myristoylation-dependently phosphorylated. Although there have been more than 40 reports of mass spectrometric studies on phosphorylation at Lyn-S13, the kinase responsible remained unclear. We succeeded in identifying casein kinase 1γ (CK1γ) as the kinase responsible for phosphorylation of Lyn-S13. In HEK293 cells co-expressing Lyn and CK1γ, the phosphorylation level of Lyn-S13 increased significantly. CK1γ is unique among the CK1 family (α, γ, δ, and ε) in carrying an S-palmitoylation site for membrane binding. Co-expression with the non-S-palmitoylated CK1γ mutant, which localized in the cytosol, gave no increase in the phosphorylation level at Lyn-S13. In HEK293 cells expressing the non-S-palmitoylated Lyn-C3A mutant, on the other hand, the Lyn-C3A mutant was phosphorylated at Lyn-S13, and the mutant remained at the Golgi. These results showed that S-palmitoylated CK1γ can phosphorylate S13 of N-myristoylated Lyn at the Golgi during intracellular protein traffic.

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 ß1 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.

Identification and characterization of protein N-myristoylation occurring on four human mitochondrial proteins, SAMM50, TOMM40, MIC19, and MIC25.

Previously, we showed that SAMM50, a mitochondrial outer membrane protein, is N-myristoylated, and this lipid modification is required for the proper targeting of SAMM50 to mitochondria. In this study, we characterized protein N-myristoylation occurring on four human mitochondrial proteins, SAMM50, TOMM40, MIC19, and MIC25, three of which are components of the mitochondrial intermembrane space bridging (MIB) complex, which plays a critical role in the structure and function of mitochondria. In vitro and in vivo metabolic labeling experiments revealed that all four of these proteins were N-myristoylated. Analysis of intracellular localization of wild-type and non-myristoylated G2A mutants of these proteins by immunofluorescence microscopic analysis and subcellular fractionation analysis indicated that protein N-myristoylation plays a critical role in mitochondrial targeting and membrane binding of two MIB components, SAMM50 and MIC19, but not those of TOMM40 and MIC25. Immunoprecipitation experiments using specific antibodies revealed that MIC19, but not MIC25, was a major N-myristoylated binding partner of SAMM50. Immunoprecipitation experiments using a stable transformant of MIC19 confirmed that protein N-myristoylation of MIC19 is required for the interaction between MIC19 and SAMM50, as reported previously. Thus, protein N-myristoylation occurring on two mitochondrial MIB components, SAMM50 and MIC19, plays a critical role in the mitochondrial targeting and protein-protein interaction between these two MIB components.

N-myristoylation and S-acylation are common modifications of Ca2+ -regulated Arabidopsis kinases and are required for activation of the SLAC1 anion channel.

N-myristoylation and S-acylation promote protein membrane association, allowing regulation of membrane proteins. However, how widespread this targeting mechanism is in plant signaling processes remains unknown. Through bioinformatics analyses, we determined that among plant protein kinase families, the occurrence of motifs indicative for dual lipidation by N-myristoylation and S-acylation is restricted to only five kinase families, including the Ca2+ -regulated CDPK-SnRK and CBL protein families. We demonstrated N-myristoylation of CDPK-SnRKs and CBLs by incorporation of radiolabeled myristic acid. We focused on CPK6 and CBL5 as model cases and examined the impact of dual lipidation on their function by fluorescence microscopy, electrophysiology and functional complementation of Arabidopsis mutants. We found that both lipid modifications were required for proper targeting of CBL5 and CPK6 to the plasma membrane. Moreover, we identified CBL5-CIPK11 complexes as phosphorylating and activating the guard cell anion channel SLAC1. SLAC1 activation by CPK6 or CBL5-CIPK11 was strictly dependent on dual lipid modification, and loss of CPK6 lipid modification prevented functional complementation of cpk3 cpk6 guard cell mutant phenotypes. Our findings establish the general importance of dual lipid modification for Ca2+ signaling processes, and demonstrate their requirement for guard cell anion channel regulation.

Protective role of testis-specific peroxiredoxin 4 against cellular oxidative stress.

Peroxiredoxin (PRDX), a newly discovered antioxidant enzyme, has an important role in hydrogen peroxide reduction. Among six PRDX genes (PRDX1-6) in mammals, PRDX4 gene is alternatively spliced to produce the somatic cell form (PRDX4) and the testis specific form (PRDX4t). In our previous study, PRDX4 knockout mice displayed testicular atrophy with an increase in cell death due to oxidative stress. However, the antioxidant function of PRDX4t is unknown. In this study, we demonstrate that PRDX4t plays a protective role against oxidative stress in the mammalian cell line HEK293T. The PRDX4t-EGFP plasmid was transferred into HEK293T cells; protein expression was confirmed in the cytoplasm. To determine the protective role of PRDX4t in cells, we performed image-based analysis of PRDX4t-EGFP expressed cells exposed to UV irradiation and hydrogen peroxide using fluorescent probe CellROX. Our results suggested that PRDX4t-EGFP expressed cells had reduced levels of oxidative stress compared with cells that express only EGFP. This study highlights that PRDX4t plays an important role in cellular antioxidant defense.

Identification of dually acylated proteins from complementary DNA resources by cell-free and cellular metabolic labeling.

To establish a strategy to identify dually fatty acylated proteins from cDNA resources, seven N-myristoylated proteins with cysteine (Cys) residues within the 10 N-terminal residues were selected as potential candidates among 27 N-myristoylated proteins identified from a model human cDNA resource. Seven proteins C-terminally tagged with FLAG tag or EGFP were generated and their susceptibility to protein N-myristoylation and S-palmitoylation were evaluated by metabolic labeling with [(3)H]myristic acid or [(3)H]palmitic acid either in an insect cell-free protein synthesis system or in transfected mammalian cells. As a result, EEPD1, one of five proteins (RFTN1, EEPD1, GNAI1, PDE2A, RNF11) found to be dually acylated, was shown to be a novel dually fatty acylated protein. Metabolic labeling experiments using G2A and C7S mutants of EEPD1-EGFP revealed that the palmitoylation site of EEPD1 is Cys at position 7. Analysis of the intracellular localization of EEPD1 C-terminally tagged with FLAG tag or EGFP and its G2A and C7S mutants revealed that the dual acylation directs EEPD1 to localize to the plasma membrane. Thus, dually fatty acylated proteins can be identified from cDNA resources by cell-free and cellular metabolic labeling of N-myristoylated proteins with Cys residue(s) close to the N-myristoylated N-terminus.

Phytoestrogen Suppresses Efflux of the Diagnostic Marker Protoporphyrin IX in Lung Carcinoma.

One promising method to visualize cancer cells is based on the detection of the fluorescent photosensitizer protoporphyrin IX (PpIX) synthesized from 5-aminolevulinic acid (ALA), but this method cannot be used in cancers that exhibit poor PpIX accumulation. PpIX appears to be pumped out of cancer cells by the ABC transporter G2 (ABCG2), which is associated with multidrug resistance. Genistein is a phytoestrogen that appears to competitively inhibit ABCG2 activity. Therefore, we investigated whether genistein can promote PpIX accumulation in human lung carcinoma cells. Here we report that treatment of A549 lung carcinoma cells with genistein or a specific ABCG2 inhibitor promoted ALA-mediated accumulation of PpIX by approximately 2-fold. ABCG2 depletion and overexpression studies further revealed that genistein promoted PpIX accumulation via functional repression of ABCG2. After an extended period of genistein treatment, a significant increase in PpIX accumulation was observed in A549 cells (3.7-fold) and in other cell lines. Systemic preconditioning with genistein in a mouse xenograft model of lung carcinoma resulted in a 1.8-fold increase in accumulated PpIX. Long-term genistein treatment stimulated the expression of genes encoding enzymes involved in PpIX synthesis, such as porphobilinogen deaminase, uroporphyrinogen decarboxylase, and protoporphyrinogen oxidase. Accordingly, the rate of PpIX synthesis was also accelerated by genistein pretreatment. Thus, our results suggest that genistein treatment effectively enhances ALA-induced PpIX accumulation by preventing the ABCG2-mediated efflux of PpIX from lung cancer cells and may represent a promising strategy to improve ALA-based diagnostic approaches in a broader set of malignancies. Cancer Res; 76(7); 1837-46. ©2016 AACR.

Identification of Human N-Myristoylated Proteins from Human Complementary DNA Resources by Cell-Free and Cellular Metabolic Labeling Analyses.

To identify physiologically important human N-myristoylated proteins, 90 cDNA clones predicted to encode human N-myristoylated proteins were selected from a human cDNA resource (4,369 Kazusa ORFeome project human cDNA clones) by two bioinformatic N-myristoylation prediction systems, NMT-The MYR Predictor and Myristoylator. After database searches to exclude known human N-myristoylated proteins, 37 cDNA clones were selected as potential human N-myristoylated proteins. The susceptibility of these cDNA clones to protein N-myristoylation was first evaluated using fusion proteins in which the N-terminal ten amino acid residues were fused to an epitope-tagged model protein. Then, protein N-myristoylation of the gene products of full-length cDNAs was evaluated by metabolic labeling experiments both in an insect cell-free protein synthesis system and in transfected human cells. As a result, the products of 13 cDNA clones (FBXL7, PPM1B, SAMM50, PLEKHN, AIFM3, C22orf42, STK32A, FAM131C, DRICH1, MCC1, HID1, P2RX5, STK32B) were found to be human N-myristoylated proteins. Analysis of the role of protein N-myristoylation on the intracellular localization of SAMM50, a mitochondrial outer membrane protein, revealed that protein N-myristoylation was required for proper targeting of SAMM50 to mitochondria. Thus, the strategy used in this study is useful for the identification of physiologically important human N-myristoylated proteins from human cDNA resources.

Homozygous mutation of STXBP5L explains an autosomal recessive infantile-onset neurodegenerative disorder.

We report siblings of consanguineous parents with an infantile-onset neurodegenerative disorder manifesting a predominant sensorimotor axonal neuropathy, optic atrophy and cognitive deficit. We used homozygosity mapping to identify an ∼12-Mbp interval identical by descent (IBD) between the affected individuals on chromosome 3q13.13-21.1 with an LOD score of 2.31. We combined family-based whole-exome and whole-genome sequencing of parents and affected siblings and, after filtering of likely non-pathogenic variants, identified a unique missense variant in syntaxin-binding protein 5-like (STXBP5L c.3127G>A, p.Val1043Ile [CCDS43137.1]) in the IBD interval. Considering other modes of inheritance, we also found compound heterozygous variants in FMNL3 (c.114G>C, p.Phe38Leu and c.1372T>G, p.Ile458Leu [CCDS44874.1]) located on chromosome 12. STXBP5L (or Tomosyn-2) is expressed in the central and peripheral nervous system and is known to inhibit neurotransmitter release through inhibition of the formation of the SNARE complexes between synaptic vesicles and the plasma membrane. FMNL3 is expressed more widely and is a formin family protein that is involved in the regulation of cell morphology and cytoskeletal organization. The STXBP5L p.Val1043Ile variant enhanced inhibition of exocytosis in comparison with wild-type (WT) STXBP5L. Furthermore, WT STXBP5L, but not variant STXBP5L, promoted axonal outgrowth in manipulated mouse primary hippocampal neurons. However, the FMNL3 p.Phe38Leu and p.Ile458Leu variants showed minimal effects in these cells. Collectively, our clinical, genetic and molecular data suggest that the IBD variant in STXBP5L is the likely cause of the disorder.

A cell-free translocation system using extracts of cultured insect cells to yield functional membrane proteins.

Cell-free protein synthesis is a powerful method to explore the structure and function of membrane proteins and to analyze the targeting and translocation of proteins across the ER membrane. Developing a cell-free system based on cultured cells for the synthesis of membrane proteins could provide a highly reproducible alternative to the use of tissues from living animals. We isolated Sf21 microsomes from cultured insect cells by a simplified isolation procedure and evaluated the performance of the translocation system in combination with a cell-free translation system originating from the same source. The isolated microsomes contained the basic translocation machinery for polytopic membrane proteins including SRP-dependent targeting components, translocation channel (translocon)-dependent translocation, and the apparatus for signal peptide cleavage and N-linked glycosylation. A transporter protein synthesized with the cell-free system could be functionally reconstituted into a lipid bilayer. In addition, single and double labeling with non-natural amino acids could be achieved at both the lumen side and the cytosolic side in this system. Moreover, tail-anchored proteins, which are post-translationally integrated by the guided entry of tail-anchored proteins (GET) machinery, were inserted correctly into the microsomes. These results showed that the newly developed cell-free translocation system derived from cultured insect cells is a practical tool for the biogenesis of properly folded polytopic membrane proteins as well as tail-anchored proteins.

Cell-free identification of novel N-myristoylated proteins from complementary DNA resources using bioorthogonal myristic acid analogues.

To establish a non-radioactive, cell-free detection system for protein N-myristoylation, metabolic labeling in a cell-free protein synthesis system using bioorthogonal myristic acid analogues was performed. After Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) with a biotin tag, the tagged proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted on a polyvinylidene fluoride (PVDF) membrane, and then protein N-myristoylation was detected by enhanced chemiluminescence (ECL) using horseradish peroxidase (HRP)-conjugated streptavidin. The results showed that metabolic labeling in an insect cell-free protein synthesis system using an azide analogue of myristic acid followed by CuAAC with alkynyl biotin was the most effective strategy for cell-free detection of protein N-myristoylation. To determine whether the newly developed detection method can be applied for the detection of novel N-myristoylated proteins from complementary DNA (cDNA) resources, four candidate cDNA clones were selected from a human cDNA resource and their susceptibility to protein N-myristoylation was evaluated using the newly developed strategy. As a result, the products of three cDNA clones were found to be novel N-myristoylated protein, and myristoylation-dependent specific intracellular localization was observed for two novel N-myristoylated proteins. Thus, the metabolic labeling in an insect cell-free protein synthesis system using bioorthogonal azide analogue of myristic acid was an effective strategy to identify novel N-myristoylated proteins from cDNA resources.

Cellular localization of CoPK12, a Ca(2+)/calmodulin-dependent protein kinase in mushroom Coprinopsis cinerea, is regulated by N-myristoylation.

Multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs) have been extensively studied in mammals, whereas fungus CaMKs still remain largely uncharacterized. We previously obtained CaMK homolog in Coprinopsis cinerea, designated CoPK12, and revealed its unique catalytic properties in comparison with the mammalian CaMKs. To further clarify the regulatory mechanisms of CoPK12, we investigated post-translational modification and subcellular localization of CoPK12 in this study. In C. cinerea, full-length CoPK12 (65 kDa) was fractionated in the membrane fraction, while the catalytically active fragment (46 kDa) of CoPK12 was solely detected in the soluble fraction by differential centrifugation. Expressed CoPK12-GFP was localized on the cytoplasmic and vacuolar membranes as visualized by green fluorescence in yeast cells. In vitro N-myristoylation assay revealed that CoPK12 is N-myristoylated at Gly-2 in the N-terminal position. Furthermore, calmodulin could bind not only to CaM-binding domain but also to the N-terminal myristoyl moiety of CoPK12. These results, taken together, suggest that the cellular localization and function of CoPK12 are regulated by protein N-myristoylation and limited proteolysis.

Protein N-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.

N-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 N-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 N-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 N-myristoylation by means of replacing Gly2 with Ala. These results indicated that protein N-myristoylation plays a critical role in the ER morphological change induced by overexpression of protein Lunapark.

Acetyl-L-carnitine suppresses thyroid hormone-induced and spontaneous anuran tadpole tail shortening.

Mitochondrial membrane permeability transition (MPT) plays a crucial role in apoptotic tail shortening during anuran metamorphosis. L-carnitine is known to shuttle free fatty acids (FFAs) from the cytosol into mitochondria matrix for ß-oxidation and energy production, and in a previous study we found that treatment with L-carnitine suppresses 3, 3', 5-triiodothyronine (T3 ) and FFA-induced MPT by reducing the level of FFAs. In the present study we focus on acetyl-L-carnitine, which is also involved in fatty acid oxidation, to determine its effect on T3 -induced tail regression in Rana rugosa tadpoles and spontaneous tail regression in Xenopus laevis tadpoles. The ladder-like DNA profile and increases in caspase-3 and caspase-9 indicative of apoptosis in the tails of T3 -treated tadpoles were found to be suppressed by the addition of acetyl-L-carnitine. Likewise, acetyl-L-carnitine was found to inhibit thyroid hormone regulated spontaneous metamorphosis in X. laevis tadpoles, accompanied by decreases in caspase and phospholipase A2 activity, as well as non-ladder-like DNA profiles. These findings support our previous conclusion that elevated levels of FFAs initiate MPT and activate the signaling pathway controlling apoptotic cell death in tadpole tails during anuran metamorphosis.

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 is required for cellular morphological changes induced by two formin family proteins, FMNL2 and FMNL3.

The subcellular localization of 13 recently identified N-myristoylated proteins and the effects of overexpression of these proteins on cellular morphology were examined with the aim of understanding the physiological roles of the protein N-myristoylation that occurs on these proteins. Immunofluorescence staining of HEK293T cells transfected with cDNAs coding for the proteins revealed that most of them were associated with the plasma membrane or the membranes of intracellular compartments, and did not affect cellular morphology. However, two proteins, formin-like2 (FMNL2) and formin-like3 (FMNL3), both of them are members of the formin family of proteins, were associated mainly with the plasma membrane and induced significant cellular morphological changes. Inhibition of protein N-myristoylation by replacement of Gly2 with Ala or by the use of N-myristoylation inhibitor significantly inhibited membrane localization and the induction of cellular morphological changes, indicating that protein N-myristoylation plays critical roles in the cellular morphological changes induced by FMNL2 and FMNL3.

Serum-dependent export of protoporphyrin IX by ATP-binding cassette transporter G2 in T24 cells.

Accumulation of protoporphyrin IX (PpIX) in cancer cells is a basis of 5-aminolevulinic acid (ALA)-induced photodymanic therapy. We studied factors that affect PpIX accumulation in human urothelial carcinoma cell line T24, with particular emphasis on ATP-binding cassette transporter G2 (ABCG2) and serum in the medium. When the medium had no fetal bovine serum (FBS), ALA induced PpIX accumulation in a time- and ALA concentration-dependent manner. Inhibition of heme-synthesizing enzyme, ferrochelatase, by nitric oxide donor (Noc18) or deferoxamine resulted in a substantial increase in the cellular PpIX accumulation, whereas ABCG2 inhibition by fumitremorgin C or verapamil induced a slight PpIX increase. When the medium was added with FBS, cellular accumulation of PpIX stopped at a lower level with an increase of PpIX in the medium, which suggested PpIX efflux. ABCG2 inhibitors restored the cellular PpIX level to that of FBS(-) samples, whereas ferrochelatase inhibitors had little effects. Bovine serum albumin showed similar effects to FBS. Fluorescence microscopic observation revealed that inhibitors of ABC transporter affected the intracellular distribution of PpIX. These results indicated that ABCG2-mediated PpIX efflux was a major factor that prevented PpIX accumulation in cancer cells in the presence of serum. Inhibition of ABCG2 transporter system could be a new target for the improvement of photodynamic therapy.