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The yeast gene, MDM20, is necessary for mitochondrial inheritance and organization of the actin cytoskeleton.

Hermann GJ, King EJ, Shaw JM - J. Cell Biol. (1997)

Bottom Line: Together, these results provide compelling evidence that mitochondrial inheritance in yeast is an actin-mediated process.Although MDM20 and TPM1 are both required for the formation and/or stabilization of actin cables, mutations in these genes disrupt mitochondrial inheritance and nuclear segregation to different extents.Thus, Mdm20p and Tpm1p may act in vivo to establish molecular and functional heterogeneity of the actin cytoskeleton.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Utah, Salt Lake City 84112, USA.

ABSTRACT
In Saccharomyces cerevisiae, the growing bud inherits a portion of the mitochondrial network from the mother cell soon after it emerges. Although this polarized transport of mitochondria is thought to require functions of the cytoskeleton, there are conflicting reports concerning the nature of the cytoskeletal element involved. Here we report the isolation of a yeast mutant, mdm20, in which both mitochondrial inheritance and actin cables (bundles of actin filaments) are disrupted. The MDM20 gene encodes a 93-kD polypeptide with no homology to other characterized proteins. Extra copies of TPM1, a gene encoding the actin filament-binding protein tropomyosin, suppress mitochondrial inheritance defects and partially restore actin cables in mdm20 delta cells. Synthetic lethality is also observed between mdm20 and tpm1 mutant strains. Overexpression of a second yeast tropomyosin, Tpm2p, rescues mutant phenotypes in the mdm20 strain to a lesser extent. Together, these results provide compelling evidence that mitochondrial inheritance in yeast is an actin-mediated process. MDM20 and TPM1 also exhibit the same pattern of genetic interactions; mutations in MDM20 are synthetically lethal with mutations in BEM2 and MYO2 but not SAC6. Although MDM20 and TPM1 are both required for the formation and/or stabilization of actin cables, mutations in these genes disrupt mitochondrial inheritance and nuclear segregation to different extents. Thus, Mdm20p and Tpm1p may act in vivo to establish molecular and functional heterogeneity of the actin cytoskeleton.

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Expression of Mdm20p-C-HA in wild-type and tpm1Δ  cells. Extracts were prepared from wild-type cells (JSY999) containing pRS416 (lane 1), pRS426 (lane 2), pRS416-MDM20-CHA (lane 3), and pRS426-MDM20-C-HA (lane 4), or tpm1Δ cells  (JSY707) containing pRS416 (lane 5), pRS426 (lane 6), pRS416- MDM20-C-HA (lane 7), and pRS426-MDM20-C-HA (lane 8).  The cells were harvested after growth to mid log phase at 25°C,  and equal amounts (equivalent OD units) of the cell extracts  were subjected to SDS-PAGE and transferred to nitrocellulose.  The nitrocellulose was probed with antibodies to the HA epitope.  The arrowhead identifies the Mdm20-C-HA protein, and the asterisk identifies a background band that was used to normalize  the amount of protein present in each lane.
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Figure 7: Expression of Mdm20p-C-HA in wild-type and tpm1Δ cells. Extracts were prepared from wild-type cells (JSY999) containing pRS416 (lane 1), pRS426 (lane 2), pRS416-MDM20-CHA (lane 3), and pRS426-MDM20-C-HA (lane 4), or tpm1Δ cells (JSY707) containing pRS416 (lane 5), pRS426 (lane 6), pRS416- MDM20-C-HA (lane 7), and pRS426-MDM20-C-HA (lane 8). The cells were harvested after growth to mid log phase at 25°C, and equal amounts (equivalent OD units) of the cell extracts were subjected to SDS-PAGE and transferred to nitrocellulose. The nitrocellulose was probed with antibodies to the HA epitope. The arrowhead identifies the Mdm20-C-HA protein, and the asterisk identifies a background band that was used to normalize the amount of protein present in each lane.

Mentions: Although overexpression of TPM1 suppressed mitochondrial inheritance defects and restored actin cables in the mdm20 mutant, the functions of the Tpm1 and Mdm20 proteins were not completely interchangeable. As shown in Fig. 6, C and D, and reported previously, actin cables are absent in tpm1Δ mutant cells (Liu and Bretscher, 1989). However, actin cables could not be restored by introducing extra copies of the MDM20 gene into tpm1Δ cells on a high copy number plasmid (Fig. 6, E and F) or by overexpressing epitope-tagged versions of the Mdm20p (Fig. 6, G and H; data not shown). In the latter experiments, Mdm20p overexpression was monitored using amino- and carboxyterminal HA-tagged Mdm20 proteins (see Materials and Methods). Wild-type cells containing Mdm20p-C-HA on a centromere-based plasmid expressed relatively low levels of the tagged protein (Fig. 7, lane 3). The steady state protein level increased more than eightfold when Mdm20-CHA protein was expressed from a multicopy plasmid (Fig. 7, lane 4). Identical results were obtained when expression of the Mdm20p-N-HA was examined in wild-type cells (data not shown). Fusion of the HA tag to the Mdm20 protein had no obvious effects on its function, as these constructs completely rescued temperature-sensitive growth defects, mitochondrial inheritance defects, and loss of actin cables in mdm20 mutant strains (data not shown). Furthermore, overexpression of HA-tagged Mdm20p constructs did not induce additional phenotypes in wild-type cells (data not shown). We found that tpm1Δ cells overexpressing Mdm20p (Fig. 7, lane 8) did not grow significantly better than tpm1Δ cells alone. In addition, overexpression of Mdm20p did not rescue the lethality observed in tpm1Δ tpm2Δ double mutants. Together, these findings suggest that MDM20 cannot bypass the requirement for Tpm1p and Tpm2p in cells. Furthermore, although expression of Tpm1p in wildtype cells results in thicker actin cables (Liu and Bretscher, 1989), the actin cytoskeleton was not affected by overexpressing Mdm20p in a wild-type strain (data not shown). Thus, Mdm20p and Tpm1p appear to perform some distinct functions in cells.


The yeast gene, MDM20, is necessary for mitochondrial inheritance and organization of the actin cytoskeleton.

Hermann GJ, King EJ, Shaw JM - J. Cell Biol. (1997)

Expression of Mdm20p-C-HA in wild-type and tpm1Δ  cells. Extracts were prepared from wild-type cells (JSY999) containing pRS416 (lane 1), pRS426 (lane 2), pRS416-MDM20-CHA (lane 3), and pRS426-MDM20-C-HA (lane 4), or tpm1Δ cells  (JSY707) containing pRS416 (lane 5), pRS426 (lane 6), pRS416- MDM20-C-HA (lane 7), and pRS426-MDM20-C-HA (lane 8).  The cells were harvested after growth to mid log phase at 25°C,  and equal amounts (equivalent OD units) of the cell extracts  were subjected to SDS-PAGE and transferred to nitrocellulose.  The nitrocellulose was probed with antibodies to the HA epitope.  The arrowhead identifies the Mdm20-C-HA protein, and the asterisk identifies a background band that was used to normalize  the amount of protein present in each lane.
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Figure 7: Expression of Mdm20p-C-HA in wild-type and tpm1Δ cells. Extracts were prepared from wild-type cells (JSY999) containing pRS416 (lane 1), pRS426 (lane 2), pRS416-MDM20-CHA (lane 3), and pRS426-MDM20-C-HA (lane 4), or tpm1Δ cells (JSY707) containing pRS416 (lane 5), pRS426 (lane 6), pRS416- MDM20-C-HA (lane 7), and pRS426-MDM20-C-HA (lane 8). The cells were harvested after growth to mid log phase at 25°C, and equal amounts (equivalent OD units) of the cell extracts were subjected to SDS-PAGE and transferred to nitrocellulose. The nitrocellulose was probed with antibodies to the HA epitope. The arrowhead identifies the Mdm20-C-HA protein, and the asterisk identifies a background band that was used to normalize the amount of protein present in each lane.
Mentions: Although overexpression of TPM1 suppressed mitochondrial inheritance defects and restored actin cables in the mdm20 mutant, the functions of the Tpm1 and Mdm20 proteins were not completely interchangeable. As shown in Fig. 6, C and D, and reported previously, actin cables are absent in tpm1Δ mutant cells (Liu and Bretscher, 1989). However, actin cables could not be restored by introducing extra copies of the MDM20 gene into tpm1Δ cells on a high copy number plasmid (Fig. 6, E and F) or by overexpressing epitope-tagged versions of the Mdm20p (Fig. 6, G and H; data not shown). In the latter experiments, Mdm20p overexpression was monitored using amino- and carboxyterminal HA-tagged Mdm20 proteins (see Materials and Methods). Wild-type cells containing Mdm20p-C-HA on a centromere-based plasmid expressed relatively low levels of the tagged protein (Fig. 7, lane 3). The steady state protein level increased more than eightfold when Mdm20-CHA protein was expressed from a multicopy plasmid (Fig. 7, lane 4). Identical results were obtained when expression of the Mdm20p-N-HA was examined in wild-type cells (data not shown). Fusion of the HA tag to the Mdm20 protein had no obvious effects on its function, as these constructs completely rescued temperature-sensitive growth defects, mitochondrial inheritance defects, and loss of actin cables in mdm20 mutant strains (data not shown). Furthermore, overexpression of HA-tagged Mdm20p constructs did not induce additional phenotypes in wild-type cells (data not shown). We found that tpm1Δ cells overexpressing Mdm20p (Fig. 7, lane 8) did not grow significantly better than tpm1Δ cells alone. In addition, overexpression of Mdm20p did not rescue the lethality observed in tpm1Δ tpm2Δ double mutants. Together, these findings suggest that MDM20 cannot bypass the requirement for Tpm1p and Tpm2p in cells. Furthermore, although expression of Tpm1p in wildtype cells results in thicker actin cables (Liu and Bretscher, 1989), the actin cytoskeleton was not affected by overexpressing Mdm20p in a wild-type strain (data not shown). Thus, Mdm20p and Tpm1p appear to perform some distinct functions in cells.

Bottom Line: Together, these results provide compelling evidence that mitochondrial inheritance in yeast is an actin-mediated process.Although MDM20 and TPM1 are both required for the formation and/or stabilization of actin cables, mutations in these genes disrupt mitochondrial inheritance and nuclear segregation to different extents.Thus, Mdm20p and Tpm1p may act in vivo to establish molecular and functional heterogeneity of the actin cytoskeleton.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Utah, Salt Lake City 84112, USA.

ABSTRACT
In Saccharomyces cerevisiae, the growing bud inherits a portion of the mitochondrial network from the mother cell soon after it emerges. Although this polarized transport of mitochondria is thought to require functions of the cytoskeleton, there are conflicting reports concerning the nature of the cytoskeletal element involved. Here we report the isolation of a yeast mutant, mdm20, in which both mitochondrial inheritance and actin cables (bundles of actin filaments) are disrupted. The MDM20 gene encodes a 93-kD polypeptide with no homology to other characterized proteins. Extra copies of TPM1, a gene encoding the actin filament-binding protein tropomyosin, suppress mitochondrial inheritance defects and partially restore actin cables in mdm20 delta cells. Synthetic lethality is also observed between mdm20 and tpm1 mutant strains. Overexpression of a second yeast tropomyosin, Tpm2p, rescues mutant phenotypes in the mdm20 strain to a lesser extent. Together, these results provide compelling evidence that mitochondrial inheritance in yeast is an actin-mediated process. MDM20 and TPM1 also exhibit the same pattern of genetic interactions; mutations in MDM20 are synthetically lethal with mutations in BEM2 and MYO2 but not SAC6. Although MDM20 and TPM1 are both required for the formation and/or stabilization of actin cables, mutations in these genes disrupt mitochondrial inheritance and nuclear segregation to different extents. Thus, Mdm20p and Tpm1p may act in vivo to establish molecular and functional heterogeneity of the actin cytoskeleton.

Show MeSH
Related in: MedlinePlus