(A) Multiple copies of TIM18 suppress the growth defect of tim54-1 mutants but not tim23-1 mutants. tim54-1 his3 strain 735 was transformed with one of the following high-copy plasmids: 2μ-TIM18 plasmid pOK66, 2μ-TIM54 plasmid pOK59, 2μ-TIM22 plasmid pJH203, or the empty vector pRS423 (Sikorski and Hieter, 1989). tim23-1 leu2 ura3 strain 574 was transformed with 2μ-TIM18 plasmid pOK66, 2μ-TIM23 plasmid pKR21 (Ryan et al., 1998), 2μ-TIM17 plasmid pKR7 (Ryan et al., 1994), or the empty vector pRS426 (Sikorski and Hieter, 1989). Transformants were streaked onto YEPmedium containing 2% glycerol and ethanol and incubated at 24 or 34°C for 5 d. (B) Multiple copies of TIM18 do not suppress the tim54::KAN or tim22::TRP1 disruptions. tim22::TRP1 strain 935, which carries the TIM22-URA3 plasmid pJH202 (Kerscher et al., 1997), was transformed with 2μ-TIM18 plasmid pOK66, 2μ-TIM22 plasmid pJH203, or the empty vector pRS423 (Sikorski and Hieter, 1989). tim54::KAN strain 1078, which carries the TIM54-URA3 plasmid pOK30 (Kerscher et al., 1997), was transformed with 2μ-TIM18 plasmid pOK66, CEN-TIM54 plasmid pOK22 (Kerscher et al., 1997), or the empty vector pRS424 (Sikorski and Hieter, 1989). Transformants were patched onto YEPD medium and then replica plated onto medium containing 5-FOA to select for loss of the TIM22-URA3 or TIM54-URA3 plasmids.

TIM18 is required for normal cell growth. (A) tim18::HIS3 disruptions are cold sensitive for growth. The tim18::HIS3 disruption in diploid strain 605 was sporulated, and meiotic progeny were allowed to grow on YEP medium containing 2% glucose for 5 d at 34, 30, or 24°C. Lanes 1–9 show different tetrads, and individual spores are labeled A–D. (B) The cold-sensitive growth defect of tim18::HIS3 is limited to glucose medium. tim18::HIS3 strain 994 and TIM18 strain 993 were pregrown at 34°C on YEP medium containing 2% glucose (YEPD), YEP medium containing 2% raffinose (YEPraf), or YEP medium containing 2% glycerol and ethanol (YEPgly/eth). Two OD₆₀₀ units of cells were diluted in 10-fold increments, and 10 μl of each dilution (starting with the 1:100 dilution) was spotted onto YEPD, YEPraf, or YEPgly/eth medium and incubated at 24, 30, or 34°C for 3–8 d. (C) tim18::HIS3 cells cannot tolerate loss of mitochondrial DNA. TIM18 and tim18::HIS3 cells were grown at 30°C on YEPD medium in the presence (YEPD+EtBr) or absence (YEPD) of 25 μg/ml ethidium bromide, streaked onto YEPD or YEPgly/eth medium lacking ethidium bromide, and incubated at 30°C for 5 d. WT, wild type.

TIM18 genetically interacts with both TIM54 and TIM22. (A) tim18::HIS3 is lethal in combination with tim54-1. tim54-1/tim18::HIS3 diploid strain 1009 was sporulated, and meiotic progeny were allowed to grow for 8 d at 24°C on YEP medium containing 2% glycerol and ethanol (YEPgly/eth). Lanes 1–9 show different tetrads, and individual spores are labeled A–D. (B) Multiple copies of TIM22 suppress the growth defect of tim18::HIS3. tim18::HIS3 strain 992 (tim18Δ) was transformed with the following high-copy plasmids: 2μ-TIM22 plasmid pJH202 (Kerscher et al., 1997), 2μ-TIM54 plasmid pOK31 (Kerscher et al., 1997), 2μ-TIM18 plasmid pOK94, or the empty vector pRS426. Wild-type TIM18 ura3 strain 993 was transformed with the empty vector pRS426. Cells were grown at 34°C in YEPD, diluted in 10-fold steps, spotted onto YEPD plates at 24°C, and incubated for 5 d.

The Tim18 protein is homologous to two other yeast proteins. (A) DNA sequence of TIM18 and its predicted protein product. The underlined amino acids represent potential membrane-spanning domains predicted from hydropathy analysis. The triangle indicates a potential mitochondrial processing protease cleavage site. (B) Increased levels of Sdh4p and YLR164w, proteins homologous to Tim18p, do not suppress the tim54-1 growth defect. tim54-1 strain 809 was transformed with one of the following high-copy plasmids: 2μ-SDH4 plasmid pOK91, 2μ-YLR164w plasmid pOK90, 2μ-TIM18 plasmid pOK66, 2μ-TIM18-HA plasmid pOK1030, or the empty vector pRS423. Transformants were streaked onto YEP medium containing 2% glycerol and ethanol and incubated at 24°C for 5 d or at 34°C for 3 d.

TIM18 encodes a mitochondrial inner membrane protein with its carboxyl terminus facing the mitochondrial intermembrane space. (A) Tim18p is a mitochondrial protein. tim18::HIS3 strain 992 containing plasmid pOK1032, which expresses the Tim18p-HA fusion protein, was grown in YEP medium containing 2% glycerol and ethanol. Cells were homogenized (H) and separated into a mitochondrial pellet fraction (M) and crude cytosol (C) by centrifugation. Aliquots of homogenate, mitochondria, and cytosol representing cell equivalents were subjected to SDS-PAGE. Immune blots were decorated with antibodies to the HA epitope (Tim18p-HA),antiserum to the β-subunit of the F₁-ATPase (F1β), or antiserum to hexokinase (Hex) and detected by chemiluminescence. (B) Tim18p is imported into mitochondria and processed to a mature form. Mitochondria were isolated from wild-type strain D273-10b and incubated for 30 min at 30°C with the ³⁵S-labeled Tim18 protein. After all manipulations, mitochondria were reisolated by centrifugation and analyzed by SDS-PAGE and phosphorimaging. Lane 1, 20% of the Tim18p precursor added to each import reaction. Lane 2, translation product of Tim18p(44–192), a truncated version of Tim18p lacking its first 43 amino acids. Lane 3, import in the presence of 25 μM CCCP and 0.5 μM valinomycin (−ΔΨ). Lane 4, import. Lane 5, import followed by digestion with 100 μg/ml trypsin for 30 min. The precursor (p) and mature (m) forms of Tim18p are indicated. (C) Tim18p-HA is an integral membrane protein. Tim18p-HA mitochondria were treated with either 0.1 M sodium carbonate or 1.5 M sodium chloride or left untreated (BB) and then separated into supernatant (S) and pellet (P) fractions by centrifugation. After SDS-PAGE, proteins were analyzed by immune blotting with antibodies to the HA epitope (Tim18p-HA), F1β, a peripheral membrane protein, and PiC, an integral membrane protein. (D) Tim18p-HA is located in the mitochondrial IM. Tim18p-HA mitochondria were sonicated, and membrane vesicles were loaded on sucrose step gradients. After centrifugation, fractions were collected and analyzed by immune blotting with antibodies to the HA epitope (Tim18p-HA), the IM protein F1β, or the OM protein OM45. Fraction 1 represents the top of the gradient. (E) The carboxyl terminus of Tim18p-HA faces the IMS. Tim18p-HA mitochondria were digested with 50 μg/ml proteinase K (PK) for 30 min on ice and analyzed by immune blotting with antibodies to the HA epitope (Tim18p-HA), F1β, Tim23p, or the OM protein Tom70p. To expose proteins located in the IMS, the mitochondrial OM was ruptured by osmotic shock (OS) and then treated with protease.

The Tim18 protein is part of the Tim22p-Tim54p complex. (A) Tim54p and Tim22p coprecipitate with Tim18p-HA. Tim18p-HA mitochondria were solubilized in 0.5% digitonin buffer. Mitochondrial extracts (M) were immune precipitated with antibodies against the HA epitope. Immunoprecipitates (P) and supernatants (S) were analyzed by SDS-PAGE, and duplicate samples were immune blotted with antibodies to the HA epitope, Tim22p, Tim54p, Tim23p, Tim17p, and PiC and then detected by chemiluminescence. (B) Tim18p-HA, Tim54p-HA, and Tim22p-HA are part of an ∼300-kDa complex. Mitochondria were isolated from cells expressing Tim18p-HA, Tim54p-HA, or Tim22p-HA, solubilized in 1% digitonin buffer, and separated by blue native electrophoresis (6–16.5% polyacrylamide). Immune blots were decorated with antibodies to HA or with antiserum to Tim23p. The protein complexes at ∼300 kDa (dark arrowhead) and ∼90 kDa (white arrowhead) are indicated. The molecular masses of standards run in adjacent lanes are indicated. (C) Tim18p is required for formation of the ∼300-kDa complex containing Tim54p. Strain 494, which expresses Tim54p-HA, TIM18 (wild-type) strain 993, tim18::HIS3 strain 992, and strain 992 carrying TIM54-HA plasmid pJH301 (Kerscher et al., 1997) were grown at 30°C on either galactose- or glycerol/ethanol-containing medium. Isolated mitochondria were solubilized in 1% digitonin buffer and separated by blue native electrophoresis (BN-PAGE). Mitochondria were also subjected to SDS-PAGE. Immune blots were decorated with antibodies to Tim54p, the HA epitope of Tim54p (HA), Tim23p, or PiC. The dark arrowheads indicate an ∼300-kDa complex in wild-type (WT) mitochondria, and the white arrowheads mark an ∼200-kDa complex in tim18::HIS3 (Δ) mitochondria. (D) tim18::HIS3 mitochondria contain an ∼200-kDa complex containing Tim22p and Tim12p. Mitochondria from TIM18 (WT) strain 993 and tim18::HIS3 (Δ) strain 992 were first subjected to blue native electrophoresis (BN-PAGE) and then to SDS-PAGE. Immune blots were decorated with antibodies to Tim22p, Tim12p, or Tom40p. Films were quantitated by scanning densitometry, and the results are shown below each pair of immune blots. (●) WT; (○) Δ.

tim18::HIS3 cells contain reduced steady-state levels of several IM proteins. TIM18 (WT) strain 995 and tim18::HIS3 (Δ) strain 994 were grown at 34°C in YEPD medium, diluted, and then grown at 24, 30, or 34°C to an OD₆₀₀ of ∼1.5. Total protein was isolated and separated by SDS-PAGE. Immune blots (75 μg of protein) were decorated with antibodies to PiC, Tim22p, Cox4p, Tim54p, Tim23p, or the α-subunit of the F₁-ATPase (F1α) and detected by chemiluminescence.