Characterization of the mitochondrial inner membrane translocase complex: the Tim23p hydrophobic domain interacts with Tim17p but not with other Tim23p molecules

Abstract

Tim23p is a mitochondrial inner membrane protein essential for the import of proteins from the cytosol. Tim23p contains an amino-terminal hydrophilic segment and a carboxyl-terminal hydrophobic domain (Tim23Cp). To study the functions and interactions of the two parts of Tim23p separately, we constructed tim23N, encoding only the hydrophilic region of Tim23p, and tim23C, encoding only the hydrophobic domain of Tim23p. Only the Tim23C protein is imported into mitochondria, indicating that the mitochondrial targeting information in Tim23p resides in its membrane spans or intervening loops. Tim23Cp, however, cannot substitute for full-length Tim23p, suggesting that the hydrophilic portion of Tim23p also performs an essential function in mitochondrial protein import. We found that overexpression of Tim23Cp is toxic to yeast cells that carry the tim23-1 mutation. Excess Tim23Cp causes Tim23-1p to disappear, leaving tim23-1 cells without a full-length version of the Tim23 protein. If Tim17p, another inner membrane import component, is overexpressed along with Tim23Cp, the toxicity of Tim23Cp is largely reversed and the Tim23-1 protein no longer disappears. In coimmunoprecipitations from solubilized mitochondria, Tim17p associates with the Tim23C protein. In addition, we show that Tim23p and Tim17p can be chemically cross-linked to each other in intact mitochondria. We conclude that the hydrophobic domain encoded by tim23C targets Tim23p to the mitochondria and mediates the direct interaction between Tim23p and Tim17p. In contrast, Tim23Cp cannot be coimmunoprecipitated with Tim23p, raising the possibility that the hydrophobic domain of Tim23p does not interact with other Tim23 molecules.

FIG. 1

The carboxyl-terminal domain of Tim23p is imported into mitochondria. (A) [³⁵S]methionine-labeled proteins were synthesized by in vitro transcription and translation and incubated with mitochondria isolated from wild-type strain D273-10b as described in Materials and Methods. After the import reaction, mitochondria were reisolated by centrifugation. Mitochondrial proteins were separated by SDS-PAGE, and the radiolabeled proteins were detected by fluorography. Lanes: 1, 30% of the translation lysate added to each assay; 2, the mitochondrial pellet after the import reaction; 3, the mitochondrial pellet after the import reaction, following treatment with 100 μg of proteinase K per ml. (B) tim23-1 cells carrying pKR16, which expresses the Tim23C-HA protein, were grown at 24°C to an OD₆₀₀ of 2. The homogenate (HOM) was separated into a postmitochondrial supernatant (PMS) and a mitochondrial pellet (MITO) by centrifugation at 9,600 × g for 10 min. Aliquots of homogenate, mitochondria, and postmitochondrial supernatant representing equal numbers of cells were analyzed by SDS-PAGE and immunoblotting with antibodies to hexokinase, the F₁-ATPase β subunit (F₁β), or the HA epitope tag on Tim23C-HA.

FIG. 2

Tim23Cp cannot substitute for Tim23p or Tim17p. (A) tim17::TRP1 leu2 ura3 cells carrying the URA3-TIM17 plasmid pKR7 were transformed with the indicated CEN-LEU2 or 2μm-LEU2 plasmids as described in Materials and Methods. Transformants were patched onto medium lacking leucine (SD-leu) and then replica plated onto medium containing 5-FOA to detect the loss of pKR7. (B) tim23::URA3 leu2 cyh2 cells carrying a CYH2-TIM23 plasmid (pKR1) were transformed with the indicated CEN-LEU2 or 2μm-LEU2 plasmids as described in Materials and Methods. Transformants were patched onto YEPD medium and then replica plated onto YEPD-CYH medium to detect the loss of pKR1.

FIG. 3

High levels of Tim23Cp are toxic to tim23-1 cells. The tim23-1 strain KRR131 and the wild-type (WT) strain RJ500 were transformed with each of the following plasmids: GAL1-TIM23 (13), GAL1-TIM17 (57), and GAL1-tim23C (pKR17). Transformants were patched onto selective medium containing 2% raffinose as the sole carbon source. The patches were then replica plated onto selective medium containing 2% galactose to induce the expression of the plasmid-borne genes. All the cells were grown at 24°C, the permissive temperature for tim23-1.

FIG. 4

Tim23-1p disappears when Tim23Cp is overexpressed. The tim23-1 strain KRR131 and the wild-type (WT) strain RJ500, each carrying the GAL1-tim23C-HA plasmid pKR18, were grown to an OD₆₀₀ of 0.5 in selective medium containing 2% raffinose. Galactose was added to a final concentration of 4%, and aliquots were removed from each culture at the indicated times. Total cell proteins were extracted (78), and equal amounts of proteins were analyzed by SDS-PAGE followed by immunoblotting with antibodies to F₁β, Tim23p, or the HA epitope on Tim23C-HA. Lanes: 1 to 4, protein from strain RJ500; 5 to 8, protein from strain KRR131.

FIG. 5

Overproduction of Tim23p causes a defect in protein import. The tim23-1 strain KRR131 carrying the GAL1-tim23C plasmid pKR17 was grown to an OD₆₀₀ of 0.5 at the permissive temperature, 23°C, in selective medium containing 2% raffinose and then split into two aliquots. Galactose (GAL) was added to one aliquot to a final concentration of 4%, and the other sample remained in raffinose (RAF) medium. After 4 h, an aliquot of each culture was removed, the cells were pulse-labeled for 5 min with [³⁵S]methionine (13), and further labeling was stopped by the addition of 1 mM sodium azide. Total proteins were extracted from cells and immunoprecipitated with antiserum to F₁β, the β subunit of F₁-ATPase (13, 78). Precipitates were analyzed by SDS-PAGE followed by fluorography. The precursor (p) and mature (m) forms of F₁β are indicated.

FIG. 6

Overexpression of TIM17 relieves tim23C toxicity and prevents the loss of Tim23-1p. (A) The tim23-1 strain KRR131 was transformed with either 2μm-TIM17 (pKR7) (57) or a vector control (pRS426) (67). These cells were then cotransformed with one of the following GAL1 plasmids: pRS314GU (GAL1 vector control) (47), GAL1-TIM23 (13), or pKR17 (GAL1-tim23C). The cells were pregrown on medium containing 2% raffinose and then replica plated onto medium containing 2% galactose. All the cells were grown at 24°C, the permissive temperature for tim23-1. (B) The tim23-1 strain KRR131 was transformed with the plasmids listed below as described in Materials and Methods. Transformants were streaked onto selective medium containing either 2% raffinose or 2% galactose. The cells were grown at 24°C for 5 days to obtain single colonies. Cells labeled control contain the empty vectors pRS314GU and pRS316GU (47). Cells labeled tim23C contain plasmids pRS316GU and GAL1-tim23C (pKR17). Cells labeled tim23C + TIM17 contain plasmids GAL1-tim23C (pKR17) and GAL1-TIM17 (57). (C) The tim23-1 strain KRR131 was transformed with the plasmids listed below and grown to an OD₆₀₀ of 0.5 on selective medium containing 2% raffinose. Galactose was added to a final concentration of 2%, and aliquots were removed from each culture at the indicated times. Total cell proteins were extracted, and equal amounts of proteins were analyzed by SDS-PAGE and immunoblotting with antibodies to Tim23p. The left-hand lanes contain empty GAL1 vectors pRS314GU plus pRS316GU; the middle lanes contain pRS316GU plus GAL1-tim23C (pKR17); the right-hand lanes contain GAL1-tim23C (pKR17) plus GAL1-TIM17.

FIG. 7

Tim23Cp physically interacts with Tim17p in the mitochondrial inner membrane. Mitochondria were isolated from the tim23-1 strain KRR131 and the wild-type (WT) strain RJ500, each carrying either pRS314GU or GAL1-tim23C-HA (pKR18). The mitochondria were solubilized in a buffer containing 0.5% digitonin and immunoprecipitated with antibodies against Tim23p (A) or against the HA epitope on Tim23C-HA (B). Immunoprecipitates and supernatants were analyzed by SDS-PAGE and immunoblotting with antibodies to Tim23p, Tim17p, Tim44p, and the HA epitope. Lanes: 1 and 2, mitochondrial protein from wild-type cells; 3 and 4, mitochondrial protein from wild-type cells expressing Tim23C-HA; 5 and 6, mitochondrial protein from tim23-1 cells; 7 and 8, mitochondrial protein from tim23-1 cells expressing Tim23C-HA. Lanes 1, 3, 5, and 7 contain immunoprecipitate from 60 μg of solubilized mitochondrial protein, and lanes 2, 4, 6, and 8 contain supernatant from the immunoprecipitation, containing 60 μg of mitochondrial protein.

FIG. 8

Tim17p can be cross-linked to Tim23p and four other proteins in intact mitochondria. Mitochondria were isolated from a tim17::URA3 strain carrying pKR11, which expresses Tim17p tagged with the HA epitope. Mitochondria were isolated, treated with SMPB, and split into two aliquots. One aliquot was analyzed directly, while the mitochondrial proteins in the other aliquot were solubilized and precipitated with antibodies to the full-length Tim23 protein. All samples were analyzed by SDS-PAGE and immunoblotting with antibodies to the HA epitope to detect cross-linked products containing Tim17-HA. Lane 1, 50 μg of solubilized mitochondrial protein; lane 2, 100 μg of mitochondrial protein treated with SMPB; lane 3, anti-Tim23p immunoprecipitate from 250 μg of SMPB-treated mitochondrial protein.