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Distribution, cleavage and lipidation of Atg8 fusion proteins in Spodoptera litura Sl-HP cells.

Zhang X, Lu H, Ai H, Peng R, Yang Y, Li A, Hong H, Peng J, Liu K - PLoS ONE (2014)

Bottom Line: Our results showed that endogenous Spodoptera litura Atg8 and HA tagged Atg8 driven by the baculovirus ie2 promoter were enriched in cytoplasm.Both the mutant mCherry-Atg8F77/79A resulting in non-cleavage of the Atg8 and the mutant mCherry-Atg8G exposing its glycine residue at the end of C-terminus were also localized in cytoplasm and nucleoplasm.The increase of autophagosomes decreased the abundance of mCherry-Atg8 in nucleoplasm.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Central China Normal University, Wuhan, China.

ABSTRACT
Atg8 proteins fused with tags are commonly used to detect autophagy. The expression patterns of Lepidopteran insect Atg8 are relatively well documented. However, the influence of protein tags on characterization of Atg8 is still not very clear. Our results showed that endogenous Spodoptera litura Atg8 and HA tagged Atg8 driven by the baculovirus ie2 promoter were enriched in cytoplasm. The recombinant plasmid pEGFP-Atg8(EGFP) in which Atg8 contained a stop codon was constructed and expressed. Green fluorescence was accumulated in cytoplasm. However, red fluorescence was located in both cytoplasm and nucleoplasm in most cells transfected with the recombinant plasmid pmCherry-Atg8(EGFP). In contrast to pEGFP-Atg8(EGFP), green fluorescence was also located in both cytoplasm and nucleoplasm in most cells transfected with the recombinant plasmid pie2/EGFP-Atg8 driven by the baculovirus ie2 promoter in which the CMV promoter and EGFP nucleotide sequences were removed, and the high level of the EGFP-Atg8 expression significantly increased its abundance in nucleoplasm. HA-Atg8 expressed at high level through baculovirus under the control of polyherin promoter was also localized in cytoplasm and nucleoplasm. The cleavage of mCherry-Atg8 was different from that of EGFP-Atg8. Both the mutant mCherry-Atg8F77/79A resulting in non-cleavage of the Atg8 and the mutant mCherry-Atg8G exposing its glycine residue at the end of C-terminus were also localized in cytoplasm and nucleoplasm. The increase of autophagosomes decreased the abundance of mCherry-Atg8 in nucleoplasm. In addition, the ratio of HA-Atg8-PE/HA-Atg8 was less than that of endogenous Atg8-PE/Atg8. These results demonstrated that the Atg8 is located in both nucleus and cytoplasm when expressed at high level and exported to the cytoplasm when autophagy is activated, and the fusion tags of Atg8 might have influence on the processing of Atg8 fusion proteins.

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A multiple alignment of amino acid sequences of Atg8 from different insect species.S. litura SlAtg8 (GenBank accession number: JX183217), Spodoptera frugiperda SfAtg8 (Spodobase: Sf2M09420-5-1), Trichonplusia ni TnAtg8 (GenBank accession number: JX183216), Helicoverpa armigera HaAtg8 (GenBank accession number: JQ739159), Manduca sexta MsAtg8 (Manduca Base: CUFF.28656.1), Bombyx mori BmAtg8 (GenBank accession number: FJ416330.1), Drosophila melanogaster DmAtg8 (GenBank accession number: NM167245.2), Aedes aegypti gaba(a) receptor (GenBank accession number: AY736002.1).
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pone-0096059-g002: A multiple alignment of amino acid sequences of Atg8 from different insect species.S. litura SlAtg8 (GenBank accession number: JX183217), Spodoptera frugiperda SfAtg8 (Spodobase: Sf2M09420-5-1), Trichonplusia ni TnAtg8 (GenBank accession number: JX183216), Helicoverpa armigera HaAtg8 (GenBank accession number: JQ739159), Manduca sexta MsAtg8 (Manduca Base: CUFF.28656.1), Bombyx mori BmAtg8 (GenBank accession number: FJ416330.1), Drosophila melanogaster DmAtg8 (GenBank accession number: NM167245.2), Aedes aegypti gaba(a) receptor (GenBank accession number: AY736002.1).

Mentions: The amino acid sequences of Atg8 among insects are highly conserved, and the amino acid sequences of the Atg8 of the S. Litura and the H. armigera are identical although they are from different genera of Lepidopteran insect (Figure 2). Thus, a series of plasmids expressing Atg8 fusion proteins were constructed using H. armigera Atg8 gene in the present study.


Distribution, cleavage and lipidation of Atg8 fusion proteins in Spodoptera litura Sl-HP cells.

Zhang X, Lu H, Ai H, Peng R, Yang Y, Li A, Hong H, Peng J, Liu K - PLoS ONE (2014)

A multiple alignment of amino acid sequences of Atg8 from different insect species.S. litura SlAtg8 (GenBank accession number: JX183217), Spodoptera frugiperda SfAtg8 (Spodobase: Sf2M09420-5-1), Trichonplusia ni TnAtg8 (GenBank accession number: JX183216), Helicoverpa armigera HaAtg8 (GenBank accession number: JQ739159), Manduca sexta MsAtg8 (Manduca Base: CUFF.28656.1), Bombyx mori BmAtg8 (GenBank accession number: FJ416330.1), Drosophila melanogaster DmAtg8 (GenBank accession number: NM167245.2), Aedes aegypti gaba(a) receptor (GenBank accession number: AY736002.1).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4008565&req=5

pone-0096059-g002: A multiple alignment of amino acid sequences of Atg8 from different insect species.S. litura SlAtg8 (GenBank accession number: JX183217), Spodoptera frugiperda SfAtg8 (Spodobase: Sf2M09420-5-1), Trichonplusia ni TnAtg8 (GenBank accession number: JX183216), Helicoverpa armigera HaAtg8 (GenBank accession number: JQ739159), Manduca sexta MsAtg8 (Manduca Base: CUFF.28656.1), Bombyx mori BmAtg8 (GenBank accession number: FJ416330.1), Drosophila melanogaster DmAtg8 (GenBank accession number: NM167245.2), Aedes aegypti gaba(a) receptor (GenBank accession number: AY736002.1).
Mentions: The amino acid sequences of Atg8 among insects are highly conserved, and the amino acid sequences of the Atg8 of the S. Litura and the H. armigera are identical although they are from different genera of Lepidopteran insect (Figure 2). Thus, a series of plasmids expressing Atg8 fusion proteins were constructed using H. armigera Atg8 gene in the present study.

Bottom Line: Our results showed that endogenous Spodoptera litura Atg8 and HA tagged Atg8 driven by the baculovirus ie2 promoter were enriched in cytoplasm.Both the mutant mCherry-Atg8F77/79A resulting in non-cleavage of the Atg8 and the mutant mCherry-Atg8G exposing its glycine residue at the end of C-terminus were also localized in cytoplasm and nucleoplasm.The increase of autophagosomes decreased the abundance of mCherry-Atg8 in nucleoplasm.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Central China Normal University, Wuhan, China.

ABSTRACT
Atg8 proteins fused with tags are commonly used to detect autophagy. The expression patterns of Lepidopteran insect Atg8 are relatively well documented. However, the influence of protein tags on characterization of Atg8 is still not very clear. Our results showed that endogenous Spodoptera litura Atg8 and HA tagged Atg8 driven by the baculovirus ie2 promoter were enriched in cytoplasm. The recombinant plasmid pEGFP-Atg8(EGFP) in which Atg8 contained a stop codon was constructed and expressed. Green fluorescence was accumulated in cytoplasm. However, red fluorescence was located in both cytoplasm and nucleoplasm in most cells transfected with the recombinant plasmid pmCherry-Atg8(EGFP). In contrast to pEGFP-Atg8(EGFP), green fluorescence was also located in both cytoplasm and nucleoplasm in most cells transfected with the recombinant plasmid pie2/EGFP-Atg8 driven by the baculovirus ie2 promoter in which the CMV promoter and EGFP nucleotide sequences were removed, and the high level of the EGFP-Atg8 expression significantly increased its abundance in nucleoplasm. HA-Atg8 expressed at high level through baculovirus under the control of polyherin promoter was also localized in cytoplasm and nucleoplasm. The cleavage of mCherry-Atg8 was different from that of EGFP-Atg8. Both the mutant mCherry-Atg8F77/79A resulting in non-cleavage of the Atg8 and the mutant mCherry-Atg8G exposing its glycine residue at the end of C-terminus were also localized in cytoplasm and nucleoplasm. The increase of autophagosomes decreased the abundance of mCherry-Atg8 in nucleoplasm. In addition, the ratio of HA-Atg8-PE/HA-Atg8 was less than that of endogenous Atg8-PE/Atg8. These results demonstrated that the Atg8 is located in both nucleus and cytoplasm when expressed at high level and exported to the cytoplasm when autophagy is activated, and the fusion tags of Atg8 might have influence on the processing of Atg8 fusion proteins.

Show MeSH
Related in: MedlinePlus