Figure 3.

Mmm2p is a mitochondrial outer membrane protein. (A) Mmm2p-HA is a mitochondrial protein. Strain RJ892, expressing Mmm2p-HA, was grown and homogenized (H) and then separated into a mitochondrial pellet (M) and a post-mitochondrial supernatant (PMS) by centrifugation. Aliquots of the fractions were analyzed by SDS-PAGE and Western blotting, using antibodies to the HA epitope (Mmm2p-HA), or antiserum to the mitochondrial protein, Tim23p, or to the cytosolic hexokinase protein. (B) Mmm2p is an integral membrane protein. Mitochondria from cells expressing Mmm2p-HA were incubated with buffer, 1.5 M sodium chloride, or 0.1 M sodium carbonate. After centrifugation, membrane pellets were analyzed by SDS-PAGE and Western blotting with HA antibodies (Mmm2p-HA), or antiserum to Tim23p or the β subunit of the F₁-ATPase (F₁β). (C) The COOH terminus of Mmm2p faces the cytosol. Mmm2p-HA mitochondria were incubated in the presence or absence of 100 μg/ml trypsin. In one aliquot of mitochondria, the outer membrane was disrupted by osmotic shock (OS). After centrifugation, mitochondrial proteins were analyzed by Western blotting using HA antibodies (Mmm2p-HA), or antiserum to Tim23p or cytochrome b₂ (Cyt. b₂). (D) Mmm2p is located in the outer membrane. Mmm2p-HA–containing mitochondria were sonicated, and membrane vesicles were loaded onto a sucrose density gradient. Fractions were collected and analyzed by Western blotting, using HA antibodies (Mmm2p-HA) or antiserum to the OM45 and F1β proteins. Fraction 1 corresponds to the top of the gradient.