Figure 6.

Highly reduced levels of Sam35 contribute to phenotypes of Δsam37 cells. (A) Serial fivefold dilutions of wild-type (W303) cells transformed with YEplac195, Δsam37 cells transformed with YEplac195, and Δsam37 cells transformed with YEpSam35 were spotted onto synthetic complete medium plates lacking uracil and incubated at the indicated temperatures for 2–4 d. (B) Cells from the three strains described in A were grown to mid-logarithmic phase in synthetic complete medium with lactate at 30°C and stained with Mitotracker Red; cells were analyzed by confocal microscopy. One hundred cells from each strain were randomly selected, and their mitochondrial morphology was recorded. “Normal” (filled) represents mitochondria that exist as reticulated network of tubules, “Abberant” (open) represents mitochondria that exist as condensed organelles, aggregated organelles, or both. (C) Mitochondria were isolated from the three strains described in A in lactate medium at 25°C and the indicated amount (micrograms of total protein) analyzed by SDS-PAGE, blotted on to nitrocellulose membranes, and immunodecorated using antisera against the indicated proteins. (D) Mitochondria (100 μg of protein) from the indicated strains were solubilized in 1% digitonin buffer. Protein complexes were analyzed by BN-PAGE and blotted onto PVDF membranes, followed by immunodecoration by using anti-Tom40 antisera. (E) ³⁵S-labeled Su9-DHFR and porin precursors were incubated with the indicated strains (described in A) for the indicated time at 25°C. Precursors not imported were removed by treatment with 50 μg/ml proteinase K followed by analysis by SDS-PAGE and digital autoradiography. Quantification of the results is shown on the right. Filled boxes, W303; open boxes, Δsam37; and open triangles, Δsam37-overexpressing Sam35.