FIG. 4.

F1L displays classical tail-anchored orientation in infected cells. (A and B) F1L membrane orientation determined by confocal microscopy. HeLa cells were infected with VVWR-FLAG-F1Lwt and permeabilized with either NP-40, digitonin, or SLO. F1L was detected using anti-F1L (A) or anti-FLAG (B) antibodies, and mitochondria were detected using anti-cytochrome c antibody. (C) F1L inserts into mitochondria during virus infection. Jurkat cells were infected with the recombinant VV VVWR-FLAG-F1Lwt for 6 h at an MOI of 5. Mitochondrial pellet fractions (P) were purified from supernatants (S) by centrifugation. The presence of F1L in mitochondria was detected using either anti-F1L or anti-Flag antibody. (D) F1L is an integral membrane protein. Mitochondria pellet fractions (P) were purified and separated from supernatants (S) by centrifugation. Pellet fractions were washed with either PBS or 0.1 M Na₂CO₃ (pH 11.5). (E) F1L displays tail-anchored orientation in infected cells. Following purification of mitochondria from virally infected cells, mitochondria were treated with either proteinase K (ProK), trypsin, or enterokinase (EK). Supernatants (S) and mitochondrial pellet (P) fractions were separated by centrifugation and washed with Na₂CO₃ (pH 11.5), and F1L was detected by immunoblotting using anti-FLAG or anti-F1L antibody.