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Conditional knockdown of hMRS2 results in loss of mitochondrial Mg(2+) uptake and cell death.

Piskacek M, Zotova L, Zsurka G, Schweyen RJ - J. Cell. Mol. Med. (2008)

Bottom Line: Constitutive shRNA-mediated knockdown of hMRS2 in human HEK-293 cell line was found here to cause death.Long-term expression of shRNAs resulted in loss of mitochondrial respiratory complex I, decreased mitochondrial membrane potential and cell death.We conclude that hMrs2 is the major transport protein for Mg (+) uptake into mitochondria and that expression of hMrs2 is essential for the maintenance of respiratory complex I and cell viability.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Vienna University, Austria.

ABSTRACT
The human gene MRS2L encodes a mitochondrial protein distantly related to CorA Mg(2+) transport proteins. Constitutive shRNA-mediated knockdown of hMRS2 in human HEK-293 cell line was found here to cause death. To further study its role in Mg(2+) transport, we have established stable cell lines with conditionally expressing shRNAs directed against hMRS2L. The cells expressing shRNA for several generations exhibited lower steady-state levels of free mitochondrial Mg(2+) ([Mg(2+)](m)) and reduced capacity of mitochondrial Mg(2+) uptake than control cells. Long-term expression of shRNAs resulted in loss of mitochondrial respiratory complex I, decreased mitochondrial membrane potential and cell death. We conclude that hMrs2 is the major transport protein for Mg (+) uptake into mitochondria and that expression of hMrs2 is essential for the maintenance of respiratory complex I and cell viability.

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Determination of free Mg2+ concentrations [Mg2+]m in isolated mitochondria dependent on [Mg2+]e. Mitochondria isolated from clones MPL-O (with empty vector; red curve) and MPL-E1 (expressing shRNA-E; blue curve) after 10 days of doxycy-cline treatment were loaded with the Mg2+-sensitive dye mag-fura 2. Changes in free Mg2+ concentrations [Mg2+]m over time were determined upon raising extramitochondrial Mg2+ concentrations [Mg2+]e from nominally 0 mM to 1 mM. (For details of methods cf. legend to Supplementary Fig. 3 and [5]).
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fig03: Determination of free Mg2+ concentrations [Mg2+]m in isolated mitochondria dependent on [Mg2+]e. Mitochondria isolated from clones MPL-O (with empty vector; red curve) and MPL-E1 (expressing shRNA-E; blue curve) after 10 days of doxycy-cline treatment were loaded with the Mg2+-sensitive dye mag-fura 2. Changes in free Mg2+ concentrations [Mg2+]m over time were determined upon raising extramitochondrial Mg2+ concentrations [Mg2+]e from nominally 0 mM to 1 mM. (For details of methods cf. legend to Supplementary Fig. 3 and [5]).

Mentions: Figure 3 presents continuous recordings of [Mg2+]m obtained with mitochondria isolated from clones MPL-E1, expressing shRNA-E and mitochondria from control cells MPL-O, all grown for 10 days in the presence of doxycycline. In MPL-O mitochondria [Mg2+]m was calculated to be about 1 mM at resting conditions and to rapidly rise to 2 mM with [Mg2+]e of 1 mM (Fig. 3). A further increase of [Mg2+]e to 5 mM led to a minor increase in [Mg2+]m only (not shown). The response to raising [Mg2+]e above 1 mM was somewhat variable from experiment to experiment, but generally much less pronounced than the increase observed with [Mg2+]e of 1 mM. This indicated that the mitochondrial Mg2+ uptake system was already saturated at about 2 mM [Mg2+]m. Plateau levels remained constant over extended periods of time, confirming that neither mag-fura 2 leaked in nor magnesium leaked out from the mitochondria. The rapid increase in [Mg2+]m within a few seconds was comparable to that observed with yeast mitochondria and reflected a high capacity influx driven by the (inside negative) membrane potential ΔΨm of mitochondria.


Conditional knockdown of hMRS2 results in loss of mitochondrial Mg(2+) uptake and cell death.

Piskacek M, Zotova L, Zsurka G, Schweyen RJ - J. Cell. Mol. Med. (2008)

Determination of free Mg2+ concentrations [Mg2+]m in isolated mitochondria dependent on [Mg2+]e. Mitochondria isolated from clones MPL-O (with empty vector; red curve) and MPL-E1 (expressing shRNA-E; blue curve) after 10 days of doxycy-cline treatment were loaded with the Mg2+-sensitive dye mag-fura 2. Changes in free Mg2+ concentrations [Mg2+]m over time were determined upon raising extramitochondrial Mg2+ concentrations [Mg2+]e from nominally 0 mM to 1 mM. (For details of methods cf. legend to Supplementary Fig. 3 and [5]).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3822876&req=5

fig03: Determination of free Mg2+ concentrations [Mg2+]m in isolated mitochondria dependent on [Mg2+]e. Mitochondria isolated from clones MPL-O (with empty vector; red curve) and MPL-E1 (expressing shRNA-E; blue curve) after 10 days of doxycy-cline treatment were loaded with the Mg2+-sensitive dye mag-fura 2. Changes in free Mg2+ concentrations [Mg2+]m over time were determined upon raising extramitochondrial Mg2+ concentrations [Mg2+]e from nominally 0 mM to 1 mM. (For details of methods cf. legend to Supplementary Fig. 3 and [5]).
Mentions: Figure 3 presents continuous recordings of [Mg2+]m obtained with mitochondria isolated from clones MPL-E1, expressing shRNA-E and mitochondria from control cells MPL-O, all grown for 10 days in the presence of doxycycline. In MPL-O mitochondria [Mg2+]m was calculated to be about 1 mM at resting conditions and to rapidly rise to 2 mM with [Mg2+]e of 1 mM (Fig. 3). A further increase of [Mg2+]e to 5 mM led to a minor increase in [Mg2+]m only (not shown). The response to raising [Mg2+]e above 1 mM was somewhat variable from experiment to experiment, but generally much less pronounced than the increase observed with [Mg2+]e of 1 mM. This indicated that the mitochondrial Mg2+ uptake system was already saturated at about 2 mM [Mg2+]m. Plateau levels remained constant over extended periods of time, confirming that neither mag-fura 2 leaked in nor magnesium leaked out from the mitochondria. The rapid increase in [Mg2+]m within a few seconds was comparable to that observed with yeast mitochondria and reflected a high capacity influx driven by the (inside negative) membrane potential ΔΨm of mitochondria.

Bottom Line: Constitutive shRNA-mediated knockdown of hMRS2 in human HEK-293 cell line was found here to cause death.Long-term expression of shRNAs resulted in loss of mitochondrial respiratory complex I, decreased mitochondrial membrane potential and cell death.We conclude that hMrs2 is the major transport protein for Mg (+) uptake into mitochondria and that expression of hMrs2 is essential for the maintenance of respiratory complex I and cell viability.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Vienna University, Austria.

ABSTRACT
The human gene MRS2L encodes a mitochondrial protein distantly related to CorA Mg(2+) transport proteins. Constitutive shRNA-mediated knockdown of hMRS2 in human HEK-293 cell line was found here to cause death. To further study its role in Mg(2+) transport, we have established stable cell lines with conditionally expressing shRNAs directed against hMRS2L. The cells expressing shRNA for several generations exhibited lower steady-state levels of free mitochondrial Mg(2+) ([Mg(2+)](m)) and reduced capacity of mitochondrial Mg(2+) uptake than control cells. Long-term expression of shRNAs resulted in loss of mitochondrial respiratory complex I, decreased mitochondrial membrane potential and cell death. We conclude that hMrs2 is the major transport protein for Mg (+) uptake into mitochondria and that expression of hMrs2 is essential for the maintenance of respiratory complex I and cell viability.

Show MeSH