Figure 4(A) Predicted protein domains for Mcp3. Mcp3 is predicted to contain a canonical N-terminal mitochondrial targeting signal (black bar) that contains a hydrophobic stretch (hatched part). The predicted MPP and the Imp1/2 cleavage sites are indicated by a black or grey arrow head, respectively. The two predicted transmembrane domains are highlighted as grey bars. (B) Internally tagged HA-Mcp3 is functional. mdm10∆ cells were transformed with the empty plasmid pYX142 () or pYX142 encoding Mdm10, Mcp3, or an internally HA-tagged Mcp3 (MDM10, MCP3, HA-MCP3). Cells were grown to an OD600 of 1.0 and spotted on YPG plates in a 1:5 dilution series. Plates were incubated for growth at 37°C for 4 days. (C) HA-Mcp3 is processed in vivo. Mitochondria isolated from mdm10∆ (∆) cells containing the empty plasmid or expressing HA-Mcp3 were analysed by SDS-PAGE and immunodecoration with antibodies against the indicated proteins. Two different bands are detectable in case of HA-Mcp3 (p, precursor; m, mature protein). The MOM protein Tom40 serves as loading control. (D) Mcp3 is a mitochondrial protein. Whole cell lysate (WCL) and fractions corresponding to cytosol (Cyt.), light microsomal fraction (ER) and mitochondria (Mito.) of mcp3∆ cells either containing the empty plasmid () or expressing HA-Mcp3 were analysed by SDS-PAGE and immunodecoration with antibodies against the HA-tag, the mitochondrial protein Tom70, a marker protein for the cytosol (Bmh1) and an ER marker protein (Erv2). m, mature protein. (E) Mcp3 co-localizes with the mitochondrial marker protein AAC. Cells over-expressing HA-Mcp3 were fixed and analysed by fluorescence microscopy using antibodies against the HA-epitope (αHA) and the mitochondrial ADP/ATP carrier (αAAC). (scale bar = 5 µm). (F) C-terminally tagged Mcp3-HA is located to mitochondria. A mitochondria enriched fraction (Mito.) and the post mitochondrial supernatant (PMS) of wild-type cells carrying the empty plasmid () or over-expressing the C-terminally HA-tagged Mcp3 (Mcp3-HA) were analysed by SDS-PAGE and immunodecortion with antibodies against the HA-Tag, the mitochondrial protein Tom40 and a marker protein for the cytosol (Hxk1). m, mature protein. (G) Mcp3 is membrane-embedded. Mitochondria isolated from mcp3Δ cells expressing HA-Mcp3 were left untreated (I, input) or subjected to alkaline extraction. The supernatant (SN) and pellet (P) fractions were analysed by SDS-PAGE and immunodecoration with antibodies against the indicated proteins. Tom20 an integral MOM proteins; Aco1, soluble matrix protein. m, mature protein. (H) Mcp3 is a MOM protein. Mitochondria isolated from cells expressing HA-Mcp3 were treated with proteinase K (PK, 100 µg/ml) in the absence or presence of the detergent Triton X-100 (TX). Samples were precipitated with TCA and analysed by SDS-PAGE and immunodecoration with antibodies against the HA-tag or the indicated mitochondrial proteins. Tom20, a MOM protein exposed to the cytosol; Dld1, a protein located in the IMS. (I) Mcp3 behaves like an outer membrane protein in density gradient centrifugation. Mitochondrial vesicles isolated from cells over-expressing HA-Mcp3 were subjected to sucrose density gradient centrifugation. Fractions of the gradient were collected and analysed by SDS-PAGE and immunodecoration with antibodies against the indicated proteins. The processed form of HA-Mcp3 is shown. Right panel: The intensities of the various bands were quantified and depicted. The sum of all intensities for each protein was set to 100%. (J) Mcp3 lacking either transmembrane domain fails to rescue loss of Mdm10. Wild-type cells carrying the empty plasmid (wt+) or mdm10∆ cells transformed with the empty plasmid () or plasmid encoding Mdm10, Mcp3, or Mcp3 lacking TMD1 or TMD2 (MDM10, MCP3, ΔTMD1, ΔTMD2) were grown to an OD600 of 1.0 and spotted on YPG plates in a 1:5 dilution series. Plates were incubated for growth at 37°C for 4 days. (K) Mcp3 lacking either of the two transmembrane domains is unstable. Crude mitochondria were obtained from wild-type cells containing an empty plasmid (Ø) or mcp3Δ cells (Δ) transformed with plasmid encoding Mcp3 or Mcp3 lacking TMD1 or TMD2 (MCP3, ΔTMD1, ΔTMD2). Samples were analysed by SDS-PAGE and immunodecoration with antibodies against Mcp3 and Tom20 as loading control.