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Numerous studies have used genome-editing modules such as CRISPR-Cas9 for site-directed mutagenesis; however, evaluation of the efficiency of these modules remains a time-consuming process. Here, we report the development of SKL-mediated Peroxisome Targeting Imaging (SKLPT imaging), an efficient in vivo pre-evaluation method based on the change in subcellular localization of a fluorescent protein. In this method, frameshifts resulting from successful editing cause the fusion of green fluorescent protein to the peroxisome localization signal Serine-Lysine-Leucine (SKL). Using SKLPT imaging, we pre-evaluated three CRISPR-Cas9 modules in vivo at the single-cell level, and then efficiently mutagenized the liverwort (Marchantia polymorpha) genome using a high-efficiency module.

Mammalian gene expression constructs are generally prepared in a plasmid vector, in which a promoter and terminator are located upstream and downstream of a protein-coding sequence, respectively. In this study, we found that front terminator constructs-DNA constructs containing a terminator upstream of a promoter rather than downstream of a coding region-could sufficiently express proteins as a result of end joining of the introduced DNA fragment. By taking advantage of front terminator constructs, FLAG substitutions, and deletions were generated using mutagenesis primers to identify amino acids specifically recognized by commercial FLAG antibodies. A minimal epitope sequence for polyclonal FLAG antibody recognition was also identified. In addition, we analyzed the sequence of a C-terminal Ser-Lys-Leu peroxisome localization signal, and identified the key residues necessary for peroxisome targeting. Moreover, front terminator constructs of hepatitis B surface antigen were used for deletion analysis, leading to the identification of regions required for the particle formation. Collectively, these results indicate that front terminator constructs allow for easy manipulations of C-terminal protein-coding sequences, and suggest that direct gene expression with PCR-amplified DNA is useful for high-throughput protein analysis in mammalian cells.

The biological functions of human acyl-CoA thioesterase III (ACTEIII/PTE-1), initially identified as an HIV-1 Nef binding protein, have remained unclear. We report herein that the stable overexpression of ACTEIII/PTE-1 in human and murine T-cell lines resulted in an increase in both peroxisome number and lipid droplet formation in a manner dependent on the amount of the protein. Peroxisome proliferation was evidenced by immunofluorescence staining for catalase, a peroxisome marker protein, as well as by direct peroxisome enumeration on electron micrographs. Consistently, the amount of catalase was elevated as the amount of ACTEIII/PTE-1 was increased. ACTEIII/PTE-1 mutants with reduced enzymatic activity or with the defect in peroxisome localization did not induce peroxisome proliferation, indicating that peroxisome proliferation was mediated by metabolites generated by ACTEIII/PTE-1 within peroxisomes. Finally, thymocytes isolated from a T-cell-specific ACTEIII/PTE-1 transgenic mouse as well as human and murine cell lines of lymphoid and non-lymphoid origins exhibited a similar proliferation of peroxisomes. Thus, ACTEIII/PTE-1 may be involved in the metabolic regulation of peroxisome proliferation.

The filamentous fungus Alternaria alternata includes seven pathogenic variants (pathotypes) which produce different host-selective toxins and cause diseases on different plants. The Japanese pear pathotype produces the host-selective AK-toxin, an epoxy-decatrienoic acid ester, and causes black spot of Japanese pear. Previously, we identified four genes, AKT1 , AKT2 , AKT3 , and AKTR , involved in AK toxin biosynthesis. AKT1 , AKT2 , and AKT3 encode enzyme proteins with peroxisomal targeting signal type 1 (PTS1)-like tripeptides, SKI, SKL, and PKL, respectively, at the C-terminal ends. In this study, we verified the peroxisome localization of Akt1, Akt2, and Akt3 by using strains expressing N-terminal green fluorescent protein (GFP)-tagged versions of the proteins. To assess the role of peroxisome function in AK-toxin production, we isolated Aa PEX6 , which encodes a peroxin protein essential for peroxisome biogenesis, from the Japanese pear pathotype and made Aa PEX6 disruption-containing transformants from a GFP-Akt1-expressing strain. The ΔAa PEX6 mutant strains did not grow on fatty acid media because of a defect in fatty acid β oxidation. The import of GFP-Akt1 into peroxisomes was impaired in the ΔAa PEX6 mutant strains. These strains completely lost AK toxin production and pathogenicity on susceptible pear leaves. These data show that peroxisomes are essential for AK-toxin biosynthesis. The ΔAa PEX6 mutant strains showed a marked reduction in the ability to cause lesions on leaves of a resistant pear cultivar with defense responses compromised by heat shock. This result suggests that peroxisome function is also required for plant invasion and tissue colonization in A. alternata . We also observed that mutation of Aa PEX6 caused a marked reduction of conidiation.

International audience; Peroxisomes are involved in a variety of metabolic pathways and developmental processes. In the filamentous fungus Podospora anserina, absence of different peroxins implicated in peroxisome matrix protein import leads to different developmental defects. Lack of the RING-finger complex peroxin PEX2 blocks sexual development at the dikaryotic stage, while in absence of both receptors, PEX5 and PEX7, karyogamy and meiosis can proceed and sexual spores are formed. This suggests a complex role for PEX2 that prompted us to study the developmental involvement of the RING-finger complex. We show that, like PEX2, the two other proteins of the complex, PEX10 and PEX12, are equally implicated in peroxisome biogenesis and that absence of each or all these proteins lead to the same developmental defect. Moreover, we demonstrate that peroxisome localization of PEX2 is not drastically affected in the absence of PEX10 and PEX12 and that the upregulation of these latter RING-finger peroxins does not compensate for the lack of a second one, suggesting that the three proteins work together in development but independent of their function in peroxisome biogenesis.

Multimodality imaging using several reporter genes and imaging technologies has become an increasingly important tool in determining the location(s), magnitude, and time variation of reporter gene expression in small animals. We have reported construction and validation of several triple fusion genes composed of a bioluminescent, a fluorescent, and a positron emission tomography (PET) reporter gene in cell culture and in living subjects. However, the bioluminescent and fluorescent components of fusion reporter proteins encoded by these vectors possess lesser activities when compared with the bioluminescent and fluorescent components of the nonfusions. In this study, we first created a mutant (mtfl) of a thermostable firefly luciferase (tfl) bearing the peroxisome localization signal to have greater cytoplasmic localization and improved access for its substrate, d-luciferin. Comparison between the three luciferases [mtfl, tfl, and firefly luciferase (fl)] both in cell culture and in living mice revealed that mtfl possessed 6- to 10-fold (in vitro) and 2-fold (in vivo) higher activity than fl. The improved version of the triple fusion vector carrying mtfl as the bioluminescent reporter component showed significantly (P < 0.05) higher bioluminescence than the previous triple fusion vectors. Of the three different red fluorescent reporter genes (jred, hcred, and mrfp1, isolated from jellyfish chromophore, coral Heteractis crispa, and coral Discosoma, respectively) evaluated, mrfp1 was able to preserve highest expression as a component of the triple fusion reporter gene for in vivo fluorescence imaging. A truncated version of wild-type herpes simplex virus 1 (HSV1) thymidine kinase gene (wttk) retained a higher expression level than the truncated mutant HSV1-sr39 TK (ttk) as the third reporter component of this improved triple fusion vector. Multimodality imaging of tumor-bearing mice using bioluminescence and microPET showed higher luciferase activity [(2.7 ± 0.1 versus 1.9 ± 0.1) × (106 p/s/cm2/sr)] but similar level of fluorine-18–labeled 2′-fluoro-2′-deoxyarabinofuranosyl-5-ethyluracil (18F-FEAU) uptake (1.37 ± 0.15 versus 1.37 ± 0.2) percentage injected dose per gram] by mtfl-mrfp1-wttk–expressing tumors compared with the fl-mrfp1-wttk–expressing tumors. Both tumors showed 4- to 5-fold higher accumulation (P < 0.05) of 18F-FEAU than fluorine-18–labeled 9-(4-fluoro-3-hydroxymethylbutyl)guanine. This improved triple fusion reporter vector will enable high sensitivity detection of lower numbers of cells from living animals using the combined bioluminescence, fluorescence, and microPET imaging techniques. [Cancer Res 2007;67(7):3085–93]

One of the proteins targeted to the peridinin plastid of the dinoflagellate Lingulodinium polyedrum is the iron-containing superoxide dismutase (LpSOD). Like dinoflagellate plastid proteins of class II, LpSOD carries a bipartite presequence comprising a signal peptide followed by a transit peptide. Our bioinformatic studies suggest that its signal peptide is atypical, however, and that the entire presequence may function as a mitochondrial targeting signal. It is possible that LpSOD represents a new class of proteins in algae with complex plastids, which are co-targeted to the plastid and mitochondrion. In addition to the ambiguous N-terminal targeting signal, LpSOD contains a potential type-1 peroxisome-targeting signal (PTS1) located at its C-terminus. In accordance with a peroxisome localization of this dismutase, its mRNA has two in-frame AUG codons. Our bioinformatic analyses indicate that the first start codon resides in a much weaker oligonucleotide context than the second one. This suggests that synthesis of the plastid/mitochondrion-targeted and peroxisome-targeted isoforms could proceed through so-called leaky scanning. Moreover, our results show that expression of the two isoforms could be regulated by a 'hairpin' structure located between the first and second start codons.