PMC

Loss of i-AAA protease activity due to yme1, mgr1, or mgr3 mutations allows weak respiratory growth of cox20Δ cells. Cells grown in rich glucose medium were diluted and spotted onto YPAEG (Materials and Methods) nonfermentable ethanol plus glycerol medium (Eth + Gly), incubated at 30° for 14 days, spotted onto YPAD (Materials and Methods) glucose (Gluc), and incubated at 30° for 4 days. Strains are indicated as follows: WT (DFS188), cox20Δ (LEE83), cox20Δmgr1Δ (LEE99), cox20Δmgr3Δ (LEE98), cox20Δyme1Δ (LEE105), and cox20Δyme1Δimp1Δ (LEE139).

Co-immune precipitation of Cox20–Myc with Cox18–HA is dependent on the presence of Cox2. Mitochondria were prepared from strains LEE80 (COX20::Myc), LEE92 (COX20::Myc COX18::HA), and LEE112 (COX20::Myc COX18::HA [cox2-20]), solubilized in buffer containing 1% digitonin, and subjected to immune precipitation with anti-HA beads (Materials and Methods). Five percent of total protein from each extract (Total), and the immune precipitates (α-HA IP) were subjected to SDS–PAGE on a 12% acrylamide gel and Western blotting. The blots were decorated with anti-Myc antibody, stripped, and then redecorated with anti-HA antibody. The presence or absence of Cox20–Myc, Cox18–HA, and Cox2 proteins in the extracts are indicated by + and −.

Expression of a recoded nuclear COX2 gene bypasses the requirement for Cox18 but not Cox20. Cells containing the plasmid pOXA1-W56R-ADH1 () were grown in CSM–Ura, and untransformed cells were grown in CSM. Cultures were diluted and spotted onto YPAEG nonfermentable (Eth + Gly) plates and incubated at 30° for 4 days or spotted onto YPAD fermentable (Gluc) plates and incubated at 30° for 2 days. Strains are indicated as follows: WT (DFS188), cox2-60 (NB60), cox2-60 N-COX2 (LEE121), pet111Δ (NB151-9B), pet111Δ N-COX2 (LEE126), cox18Δ (CAB116), cox18Δ N-COX2 (LEE122), oxa1Δ (MCC319), oxa1Δ N-COX2 (LEE123), imp1Δ (SCS193), imp1Δ N-COX2 (LEE128), cox20Δ (LEE173), cox20Δ N-COX2 (LEE174).

Pre-Cox2 is processed by Imp1 in the absence of i-AAA protease activity, but inefficiently. (A) Crude mitochondrial protein samples () in the amounts indicated were separated on 16% SDS–PAGE and Western blotted. The blot was decorated with both antibody specific to the C-tail of Cox2 and antibody specific against Cit1, as a loading control. Strains are indicated as follows: WT (DFS188), cox20Δ (LEE83), cox2Δ (NB40-3C), cox20Δ mgr1Δ (LEE99), cox20Δ mgr3Δ (LEE98), cox20Δ yme1Δ (LEE105), and cox20Δ yme1Δ imp1Δ (LEE139). pCox2 indicates pre-Cox2; mCox2 indicates mature Cox2; * indicates a novel Cox2 cross-reacting species. (B) Cells were labeled in vivo with [³⁵S]methionine in the presence of cycloheximide for 20 min at 30° (Materials and Methods). Mitochondria were analyzed by SDS–PAGE (16% acrylamide gel) and autoradiography. Strains are indicated as follows: WT (DFS188), cox20Δ (LEE83), cox2Δ (NB40-3C), cox20Δ mgr1Δ (LEE99), cox20Δ mgr3Δ (LEE98), cox20Δ yme1Δ (LEE105), and cox20Δ yme1Δ imp1Δ (LEE139). The identities of major mitochondrial translation products are indicated.

Export of the C terminus of pre-Cox2 is dependent on Cox20, but not on N-terminal processing or cytochrome c oxidase assembly. Mitoplasts (MP) from strains expressing Cox2 with a C-terminal HA tag were either mock treated or treated with 20μg/ml of proteinase K (pK) (Materials and Methods) as indicated. Samples were solubilized with 1% octyl glucoside (OG) prior to protease treatment where indicated. For wild type, 5 μg of proteins were loaded per lane, and for the mutants, 50 μg of protein were loaded per lane. The resulting blots were probed with α-HA, α-Cit1, and α-Yme1 antibodies and developed with the ECL detection system (Invitrogen). Strains are indicated as follows: WT (SCS101), cox2-N15I (SCS114), imp1Δ (SCS184), cox20Δ (SCS182), cox1Δ (SCS218), and cox3Δ (SCS192).