Figure 3. Starvation leads to large,... | Rockefeller University Press

$Starvation leads to large, rounded mitochondria with increased activity. (A) Wild-type cells were grown to mid-logarithmic phase (growth), washed twice, and cultured in 2% potassium acetate for 2.5 h (starvation). Cells from each condition were labeled with MitoTracker to examine mitochondrial activity and morphology. Cells were visualized by epifluorescence microscopy. DIC, differential interference contrast. Scale bar = 2 μm. (B) Wild-type cells expressing a mitochondrial-targeted DsRed construct were cultured and visualized as in (A). Scale bar = 2 μm. (C) Wild-type cells were grown to mid-logarithmic phase, washed twice, and cultured in 2% potassium acetate for 2.5 h in the presence of low or high 2,4-DNP (100 nM and 1 mM, respectively) and the corresponding low or high amount of DMSO carrier as control (1:2,000 and 1:500, respectively). Cell wall proteins were extracted from equal numbers of cells followed by precipitation with TCA. Lysates and cell wall–extracted proteins were analyzed by Western blot. The loading of the secreted fraction is equivalent to 145× that of the lysate. 2,4-DNP treatment strongly inhibited secretion of all proteins analyzed, with low dose ranging from 15% to 30% reduction, while high-dose 2,4-DNP virtually blocked secretion of the cargo proteins, ranging from 3% to 9% reduction (n = 4). Cof1 is a nonsecreted, cytoplasmic protein used to monitor for cell lysis, while Bgl2 is a conventionally secreted cell wall protein.$

Figure 3.

Starvation leads to large, rounded mitochondria with increased activity. (A) Wild-type cells were grown to mid-logarithmic phase (growth), washed twice, and cultured in 2% potassium acetate for 2.5 h (starvation). Cells from each condition were labeled with MitoTracker to examine mitochondrial activity and morphology. Cells were visualized by epifluorescence microscopy. DIC, differential interference contrast. Scale bar = 2 μm. (B) Wild-type cells expressing a mitochondrial-targeted DsRed construct were cultured and visualized as in (A). Scale bar = 2 μm. (C) Wild-type cells were grown to mid-logarithmic phase, washed twice, and cultured in 2% potassium acetate for 2.5 h in the presence of low or high 2,4-DNP (100 nM and 1 mM, respectively) and the corresponding low or high amount of DMSO carrier as control (1:2,000 and 1:500, respectively). Cell wall proteins were extracted from equal numbers of cells followed by precipitation with TCA. Lysates and cell wall–extracted proteins were analyzed by Western blot. The loading of the secreted fraction is equivalent to 145× that of the lysate. 2,4-DNP treatment strongly inhibited secretion of all proteins analyzed, with low dose ranging from 15% to 30% reduction, while high-dose 2,4-DNP virtually blocked secretion of the cargo proteins, ranging from 3% to 9% reduction (n = 4). Cof1 is a nonsecreted, cytoplasmic protein used to monitor for cell lysis, while Bgl2 is a conventionally secreted cell wall protein.

This Feature Is Available To Subscribers Only