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. 2000 May 1;191(9):1487–1498. doi: 10.1084/jem.191.9.1487

Figure 2.

Figure 2

Figure 2

M11L localizes to mitochondria in infected cells. (A) BGMK cells infected for 20 h with M11L-expressing myxoma virus (vMyxlac) or the M11L knockout virus (vMyxM11L−) were treated with Mitotracker Red to identify mitochondria, and M11L was detected by indirect immunofluorescence. Cells were visualized by confocal microscopy. As expected, M11L was detected in cells infected with vMyxlac (a) but not in cells infected with the M11L knockout virus (d), and Mitotracker Red produced punctate mitochondrial staining (b and e). Superimposed Mitotracker Red and M11L signals (c) yielded a yellow image, indicating that M11L localizes to mitochondria. This was not observed in cells infected with the knockout virus (f). Bar, 10 nm. (B) The proteinase K (PK) sensitivity of the 18-kD M11L (top) or 17-kD COX IV (bottom) proteins was assessed. Digitonin lysates of HepG2 cells infected with M11L-expressing VVM11L or the control virus VV601 (CNTL) were prepared 12 h after infection. Pellet (lanes 1, 2, 5, and 6) and supernatant (sup; lanes 3, 4, 7, and 8) fractions were isolated. Samples were subjected to proteinase K treatment for 20 min (PK 20 min; lanes 1–4) or left untreated (PK 0 min; lanes 5–8), and M11L or COX IV were detected by SDS-PAGE and immunoblotting. M11L (top) but not COX IV (bottom) in the pellet fraction (lane 1) was sensitive to proteinase K treatment, indicating that although M11L is membrane associated, it is orientated towards the cytosol.