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. 1999 May 31;145(5):973–978. doi: 10.1083/jcb.145.5.973

Figure 1.

Figure 1

Figure 1

Soluble cytosolic domain of hTom20 stimulates insertion of VDAC into the outer membrane of rat heart mitochondria in vitro by a pathway that is independent of the preprotein translocation pore. (A) 35S-labeled transcription-translation products of human VDAC and rat pOCT in reticulocyte lysate were subjected to standard protein import reactions in the presence of purified rat heart mitochondria for the indicated times at 4°C (lanes 2 and 7) or 30°C (lanes 1, 3–6, and 8–11) in the absence (lane 1) or presence (lanes 2–11) of mitochondria. Also included in the reactions were 15 μg purified pODHFR (Sheffield et al., 1990) and 2 mM MTX (lanes 4, 6, 9, and 11), or 15 μg purified cytosolic domain of hTom20, hTom20Δ1-29 (lanes 5, 6, 10, and 11). At the end of the reactions, the mitochondria were either treated with trypsin (pOCT, lanes 7–11; McBride et al., 1992) or extracted with 0.1 M NaCO3, pH 11.5 (alkali, VDAC lanes 8–11; Goping et al., 1998). Reaction products were resolved by 10% SDS-PAGE and visualized by fluorography. Lane 1, 10% of input [35S]VDAC or [35S]pOCT. p, Precursor form of OCT; m, mature form of OCT. The bar graph quantifies the radioactive bands from import reactions of VDAC at 30 min, using a Power MacIntosh 7200/120 and NIH image v1.61 image analysis software. Shown are the averages from four separate experiments with standard deviations. The bar numbers refer to the lane numbers in the VDAC (30 min) fluorogram. (B) Import (30 min) of [35S]VDAC, [35S]pOCT, and [35S]yTom70(1-29)DHFR (previously called pOMD29; McBride et al., 1992) was conducted in the presence or absence of pODHFR + MTX. Alkaline-resistant VDAC and yTom70(1-29)DHFR and processed pOCT were analyzed and quantified as in A. (C) Same as A, except that import of [35S]VDAC or yeast [35S]pCox Va was conducted using mitochondria isolated (Daum et al., 1982) from wild-type Saccharomyces cerevisiae (strain D273-10B) or from a yeast strain (KKY3.3) harboring a temperature sensitive mutation in Tom40 (Kassenbrock et al., 1995), and incubated at the nonpermissive (37°C) or permissive (23°C) temperatures for Tom40 for 60 min before conducting import reactions. Import was for 30 min at 4, 23, or 37°C, as indicated. p, Precursor form of Cox VA; m, mature form of Cox Va.