Figure_3.tif (1.38 MB)

Quantitative analysis of protein expressions of V-ATPase subunits, ATP6V0A1, ATP6V0A2, ATP6V0A3, ATP6V0C, and ATP6V1E2, as well as autophagosomal marker LC3-II in MEFs after infection with Cre-adenovirus and retroviral transgenic overexpression either with empty control (C), full-length ATP6AP2/PRR (FL) (A), cytosolic domain-deleted ATP6AP2/PRR (ΔCD) (B), the carboxyl-terminal fragment of ATP6AP2/PRR (CTF) (C), or the amino-terminal fragment of ATP6AP2/PRR (NTF) (D).

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posted on 2013-11-04, 03:47 authored by Kenichiro Kinouchi, Atsuhiro Ichihara, Motoaki Sano, Ge-Hong Sun-Wada, Yoh Wada, Hiroki Ochi, Toru Fukuda, Kanako Bokuda, Hideaki Kurosawa, Naohiro Yoshida, Shu Takeda, Keiichi Fukuda, Hiroshi Itoh

The levels of V_O subunits ATP6V0A1, ATP6V0A2, ATP6V0A3, and ATP6V0C were decreased in the floxed MEFs after Ad-Cre infection, but V₁ subunit ATP6V1E2 was unaffected. LC3-II accumulation occurs as a consequence of impaired autophagic degradation. Exogenously expressed FL or ΔCD restored the expression levels of Vo subunits and LC3-II accumulation in ATP6AP2/PRR-ablated MEFs. By contrast, exogenously expressed CTF or NTF failed to rescue the phenotypes. CTF partly restored the expression of ATP6V0A3 and ATP6V0C, and reduced LC3-II accumulation. The data are presented as the ratio of protein expressions in day 10 to those in day 0. Graphed data show the mean ± SD. * P < 0.05 compared with control retrovirus. C, empty control retroviral vector (pMXs-IG); FL, full-length ATP6AP2/PRR; CTF, carboxyl-terminal fragment; CD, cytoplasmic domain; NTF, amino-terminal fragment.