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The access of metabolites into yeast mitochondria in the presence and absence of the voltage dependent anion selective channel (YVDAC1)

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Kmita H., Stobienia O., Michejda J.

Acta Biochimica Polonica

1999

vol. 46

issue 4

991-1000

Since yeast Saccharomyces cerevisiae mutants depleted of the voltage dependent anion selective channel (YVDAC1) are still able to grow on a non-fermentable carbon source, a functional transport system in the outer mitochondrial membrane must exist to support the access of metabolites into mitochondria. It was assumed that the properties of the system could be inferred from the differences in the results observed between wild type and mutant mitochondria since no crucial differences in this respect between the two types of mitoplasts were observed. YVDAC1-depleted mitochondria displayed a highly reduced permeability of the outer membrane, which was reflected in increased values of K^{NADH}_{0.5} for respiration and K^{ADP}_{0.5} for triggering phosphorylating state as well as in delayed action of carboxyatractylate (CATR) in inhibition of phosphorylating state. The parameters were chosen to express the accessibility of the applied species to the intermembrane space. The passage of the molecules through the outer membrane depleted of YVDAC1 could be partially improved in the presence of bivalent cations (Mg^{2+}, Ca^{2+}), as in their presence lower values of the calculated parameters were obtained. The restrictions imposed on the transport of molecules through the YVDAC1-depleted outer membrane resulted in a competition between them for the access to the intermembrane space as measured by changes in parameters observed for a given species in the presence of another one. The competition was stronger in the absence of Mg^{2+} and depended on charge and size of transported molecules, as the strongest competitor was CATR and the weakest one - {NADH}. Thus, it can be concluded that the transport system functioning in the absence of YVDAC1 is modulated by bivalent cations and charge as well as size of transported molecules. Since an increased level of respiration due to the dissipation of Δψ causes an increase of K^{NADH}_{0.5} in both wild type and YVDAC1-depleted mitochondria it is concluded that a common property of YVDAC1 and the system functioning in YVDAC1-depleted mitochondria seems to be the dependence of the capacity on the level of mitochondrial respiration.

The influence of depletion of voltage dependent anion selective channel on protein import into the yeast Saccharomyces cerevisiae mitochondria.

100%

Szczechowicz A., Hryniewiecka L., Kmita H.

2001

vol. 48

issue 3

719-728

The supply of substrates to the respiratory chain as well as of other metabolites (e.g. ATP) into inner compartments of mitochondria is crucial to preprotein import into these organelles. Transport of the compounds across the outer mitochondrial membrane is enabled by mitochondrial porin, also known as the voltage-dependent anion-selective channel (VDAC). Our previous studies led to the conclusion that the transport of metabolites through the outer membrane of the yeast Saccharomyces cerevisiae mitochondria missing VDAC (now termed YVDAC1) is considerably restricted. Therefore we expected that depletion of YVDAC1 should also hamper protein import into the mutant mitochondria. We report here that YVDAC1-depleted mitochondria are able to import a fusion protein termed pSu9-DHFR in the amount comparable to that of wild type mitochondria, although over a considerably longer time. The rate of import of the fusion protein into YVDAC1-depleted mitochondria is distinctly lower than into wild type mitochondria probably due to restricted ATP access to the intermembrane space and is additionally influenced by the way the supporting respiratory substrates are transported through the outer membrane. In the presence of ethanol, diffusing freely through lipid membranes, YVDAC1-depleted mitochondria are able to import the fusion protein at a higher rate than in the presence of external NADH which is, like ATP, transported through the outer membrane by facilitated diffusion. It has been shown that transport of external NADH across the outer membrane of YVDAC1-depleted mitochondria is supported by the protein import machinery, i.e. the TOM complex (Kmita & Budzińska, 2000, Biochim. Biophys. Acta 1509, 86-94.). Since the TOM complex might also contribute to the permeability of the membrane to ATP, it seems possible that external NADH and ATP as well as the imported preprotein could compete with one another for the passage through the outer membrane in YVDAC1-depleted mitochondria.

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2 Acta Biochimica Polonica

1 2001

1 1999

2 Kmita H.

1 Hryniewiecka L.

1 Michejda J.

1 Stobienia O.

1 Szczechowicz A.

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The access of metabolites into yeast mitochondria in the presence and absence of the voltage dependent anion selective channel (YVDAC1)

The influence of depletion of voltage dependent anion selective channel on protein import into the yeast Saccharomyces cerevisiae mitochondria.