Subcellular localization of HA-tagged Mmm1.

<p>A. Cell fractionation by differential and sucrose flotation gradient centrifugation was performed on the indicated strains. Flotation gradient purified mitochondrial, post mitochondrial pellet (PMP) and cytosolic fractions (30 µg) were subjected to SDS-PAGE followed by Western blot analysis using antibodies to the indicated proteins. Kar2, ER marker; Tom40, mitochondrial marker; Arginase, cytosolic marker. B. Outer membrane vesicles (OMVs) were isolated from cells expressing Mmm1-HA protein, subjected to SDS-PAGE and analyzed by Western blot for the presence or absence of the indicated proteins. The leftmost lane contains whole mitochondria C. Cell fractionation and detection of indicated proteins. As in panel A but Tom22 is the mitochondrial marker. D. Flotation gradient purified mitochondria and PMP isolated from the Mmm1-HA strain were treated with 0.1 M sodium carbonate at pH 11.0, 11.5 or 12.0. Membrane sheets were pelleted by ultracentrifugation. Proteins in the supernatant were precipitated with trichloroacetic acid. Pellet (pel) and supernatant (sup) fractions were then subjected to SDS-PAGE, transferred to nitrocellulose and analyzed by Western blot using antibodies to the indicated proteins.