Optimization of mNeonGreen for Homo sapiens increases its fluorescent intensity in mammalian cells.

Green fluorescent protein (GFP) is tremendously useful for investigating many cellular and intracellular events. The monomeric GFP mNeonGreen is about 3- to 5-times brighter than GFP and monomeric enhanced GFP and shows high photostability. The maturation half-time of mNeonGreen is about 3-fold faster than that of monomeric enhanced GFP. However, the cDNA sequence encoding mNeonGreen contains some codons that are rarely used in Homo sapiens. For better expression of mNeonGreen in human cells, we synthesized a human-optimized cDNA encoding mNeonGreen and generated an expression plasmid for humanized mNeonGreen under the control of the cytomegalovirus promoter. The resultant plasmid was introduced into HEK293 cells. The fluorescent intensity of humanized mNeonGreen was about 1.4-fold higher than that of the original mNeonGreen. The humanized mNeonGreen with a mitochondria-targeting signal showed mitochondrial distribution of mNeonGreen. We further generated an expression vector of humanized mNeonGreen with 3xFLAG tags at its carboxyl terminus as these tags are useful for immunological analyses. The 3xFLAG-tagged mNeonGreen was recognized well with an anti-FLAG-M2 antibody. These plasmids for the expression of humanized mNeonGreen and mNeonGreen-3xFLAG are useful tools for biological studies in mammalian cells using mNeonGreen.

The mouse APG10 homologue, an E2-like enzyme for Apg12p conjugation, facilitates MAP-LC3 modification.

Autophagy is a process for the bulk degradation of cytosolic compartments by lysosomes/vacuoles. The formation of autophagosomes involves a dynamic rearrangement of the membrane for which two ubiquitin-like modifications (the conjugation of Apg12p and the modification of a soluble form of MAP-LC3 to a membrane-bound form) are essential. In yeast, Apg10p is an E2-like enzyme essential for Apg12p conjugation. The isolated mouse APG10 gene product interacts with mammalian Apg12p dependent on mammalian Apg7p (E1-like enzyme), and facilitates Apg12p conjugation. The interaction of Apg10p with Apg12p is dependent on the carboxyl-terminal glycine of Apg12p. Mutational analysis of the predicted active site cysteine (Cys161) within mouse Apg10p shows that mutant Apg10pC161S, which can form a stable intermediate with Apg12p, inhibits Apg12p conjugation even in the presence of Apg7p, while overexpression of Apg7p facilitates formation of an Apg12p-Apg5p conjugate. Furthermore, the coexpression of Apg10p with Apg7p facilitates the modification of a soluble form of MAP-LC3 to a membrane-bound form, a second modification essential for autophagy. Mouse Apg10p interacts with MAP-LC3 in HEK293 cells, while no mutant Apg10pC161S forms any intermediate with MAP-LC3. Direct interaction between Apg10p and MAP-LC3 is also demonstrated by yeast two-hybrid analysis. The inability of mutant Apg10pC161S to form any intermediate with MAP-LC3 has ruled out the possibility that MAP-LC3 interacts with Apg10p as a substrate.

Murine Apg12p has a substrate preference for murine Apg7p over three Apg8p homologs.

Apg7p is a unique E1 enzyme which is essential for both the Apg12p- and Apg8p-modification systems, and plays indispensable roles in yeast autophagy. A cDNA encoding murine Apg7p homolog (mApg7p) was isolated from a mouse brain cDNA library. The predicted amino acid sequence of the clone shows a significant homology to human Apg7p and yeast Apg7p. Murine Apg12p as well as the three mammalian Apg8p homologs co-immunoprecipitate with mApg7p. Site-directed mutagenesis revealed that an active-site cysteine within mApg7p is Cys(567), indicating that mApg7p is an authentic E1 enzyme for murine Apg12p and mammalian Apg8p homologs. The mutagenesis study also revealed that Apg12p has a substrate preference for mApg7p over the three Apg8p homologs, suggesting that the Apg12p conjugation by Apg7p occurs preferentially in mammalian cells compared with the modification of the three Apg8p homologs. We also report here on the ubiquitous expression of human APG7 mRNA in human adult and fetal tissues and of rat Apg7p in adult tissues.

Human Apg3p/Aut1p homologue is an authentic E2 enzyme for multiple substrates, GATE-16, GABARAP, and MAP-LC3, and facilitates the conjugation of hApg12p to hApg5p.

Autophagy is a process of bulk degradation of cytoplasmic components by the lysosome/vacuole and has a significant relationship to several neurodegenerative disorders and myopathies in mammals. One of APG gene products essential for autophagy in yeast, Apg3p, is a protein-conjugating enzyme for Apg8p lipidation (Ichimura, Y., Kirisako, T., Takao, T., Satomi, Y., Shimonishi, Y., Ishihara, N., Mizushima, N., Tanida, I., Kominami, E., Ohsumi, M., Noda, T., and Ohsumi, Y. (2000) Nature 408, 488-492). In this study, the cloning of a human Apg3p homologue (hApg3p) as an E2 enzyme essential for human Apg8p homologues (i.e. GATE-16, GABARAP, and MAP-LC3) is shown, and its unique characteristics are described. The predicted amino acid sequence of the isolated clone shows 34.1% identity and 48.1% similarity to yeast Apg3p. Site-directed mutagenesis revealed that Cys(264) of hApg3p is an authentic active-site cysteine residue essential for the formation of hApg3p small middle dothApg8p homologue intermediates. Overexpression of hApg7p enhances the formation of a stable E2-substrate complex between hApg3p(C264S) and each of the hApg8p homologues, and MAP-LC3 is preferred as the substrate over the other two Apg8p homologues. These results indicate that hApg3p is an E2-like enzyme essential for three human Apg8p homologues. Co-immunoprecipitation of hApg7p with hApg3p indicates that hApg3p forms an E1.E2 complex with hApg7p as in the case of yeast Apg3p and Apg7p. Furthermore, hApg3p coimmunoprecipitates with hApg12p, and the overexpression of hApg3p facilitates the formation of the GFPhApg12p.thApg5p conjugate, suggesting that hApg3p cross-talks with the hApg12p conjugation system.