PhoE is sorted to mitochondria in a TOM- and TOB complex-dependent manner. (A) Mitochondrial import of PhoE depends on the import receptor Tom20. Mitochondria isolated from tom20Δ, tom70Δ, and their corresponding wild-type cells transformed with p2μTPI/PhoE were analyzed by SDS/PAGE and immunodecoration with antibodies against PhoE and the indicated mitochondrial proteins. (B) Cells from wild-type strain or from a strain expressing Tob55 under the control of the GAL10 promoter (Gal/His₈-Tob55) were harvested at the indicated time points after a shift to glucose-containing medium (see Fig. S4A). Crude mitochondria were isolated and proteins were analyzed by SDS/PAGE and immunodecoration with antibodies against PhoE and the indicated mitochondrial proteins. Tob55, Tom40, and porin, β-barrel proteins; Tom70, a signal-anchored protein in the outer membrane; Oxa1, an inner membrane protein.

The C-terminal phenylalanine residue of PhoE is dispensable for targeting to mitochondria but not for proper assembly. (A) Mitochondria isolated from PhoEΔF-expressing cells were loaded directly on SDS/PAGE gel (T, Total) or were treated with urea and then centrifuged to separate the nonextractable pellet (P) from the urea-soluble fraction (S). Proteins were analyzed by SDS/PAGE and immunodecoration with antibodies against the indicated proteins. A proteolytic fragment of PhoE is labeled with asterisk. (B) PhoEΔF is present in mitochondria in a protease-sensitive conformation. Mitochondria isolated from cells expressing low amounts of PhoEΔF were treated and analyzed as described in Fig. 3A.

Bacterial β-barrel proteins are targeted to mitochondria in yeast cells. Whole-cell lysate of cells expressing PhoE and fractions corresponding to mitochondria, endoplasmic reticulum (ER), and cytosol were analyzed by SDS/PAGE and immunodecoration with antibodies against the bacterial protein, the mitochondrial protein Tom70, the ER protein Erv2, and a control marker protein for the cytosol (hexokinase). Mitochondria isolated from wild-type, untransformed cells were coanalyzed as a control.

PhoE expressed at low levels is properly integrated into the mitochondrial outer membrane. (A) Mitochondria isolated from cells expressing PhoE under the control of the POR1 promoter were loaded directly on SDS/PAGE gel (input) or were subjected first to alkaline extraction and then centrifuged to discriminate between membrane proteins (pellet) and soluble proteins in the supernatant (sup.). Additional aliquots of mitochondria were left intact or were treated by hypoosmotic swelling (HS) before their incubation with the indicated amounts of proteinase K. Proteins were analyzed by SDS/PAGE and immunodecorated with antibodies against the indicated proteins. Of note, for unknown reason, the recovery of PhoE in the carbonate extraction experiment was only 75%. Porin, protein embedded in the outer membrane; Tom20, outer-membrane protein exposed to the cytosol; Hsp60, soluble matrix protein; OxaI, an inner-membrane protein exposed to IMS. (B) Mitochondria isolated from PhoE-expressing cells were loaded directly on SDS/PAGE gel (T, Total) or were treated with urea and then centrifuged to separate the nonextractable pellet (P) from the urea-soluble fraction (S). Proteins were analyzed by SDS/PAGE and immunodecoration with antibodies against the indicated proteins. A proteolytic fragment of PhoE is labeled with an asterisk. (C Upper) PhoE is sorted to the mitochondrial outer membrane when expressed at low levels. Mitochondria from cells expressing PhoE under the POR1 promoter were analyzed by sucrose density gradient centrifugation. Proteins from fractions were analyzed by SDS/PAGE and immunodecoration with antibodies against PhoE, Tom20, and the inner-membrane protein CoxII. (C Lower) The bands corresponding to the detected proteins were quantified by densitometry. The relative amount in each fraction is presented as the portion from the total amount of this protein.

PhoE can form native-like trimeric structures in mitochondria. (A) Full-length PhoE assembles into a trimeric structure in mitochondrial membranes. Cell envelopes from E. coli or mitochondria isolated from cells transformed with plasmid encoding PhoE under either the GAL1 or POR1 promoter were analyzed by seminative PAGE after heat denaturation or without further treatment. The positions of monomeric and oligomeric forms of PhoE are indicated with M and O, respectively. (B) Deletion of the C-terminal Phe residue abolishes PhoE trimer formation in mitochondrial membranes. Mitochondria isolated from cells transformed with the empty plasmid or expressing the PhoEΔF variant from the GAL1 or POR1 promoter were treated and analyzed as in Fig. 5A. Mitochondria harboring high levels of full-length PhoE are coanalyzed as control. (C) Mitochondria derived from cells expressing PhoE (i), PhoE under control of the POR1 promoter (ii), PhoEΔF under the control of the GAL1 promoter (iii), or no PhoE (empty vector control) (iv) were stained with a monoclonal antibody that specifically recognizes native PhoE trimers.