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Altering nuclear pore complex function impacts longevity and mitochondrial function in S. cerevisiae.

Lord CL, Timney BL, Rout MP, Wente SR - J. Cell Biol. (2015)

Bottom Line: Mutants lacking the GLFG domain of Nup116 displayed decreased RLSs, whereas longevity was increased in nup100- mutants.Both Kap121-dependent transport and Nup116 levels decrease in replicatively aged yeast.Together, these studies reveal that specific NPC nuclear transport events directly influence aging.

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Affiliation: Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232.

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Kap121-mediated transport is required for mitochondrial function. (A) Wild-type and mutant strains were serially diluted onto glucose or glycerol and incubated at 25°C until grown as shown. (B) Single representative wild-type or kap121 mutant cells stained with MitoTracker red CMXRos. DIC, differential interference contrast. Bar, 2.5 µm. (C) Quantification of the mean MitoTracker red CMXRos cytoplasmic intensity for strains listed in B. *, P < 0.01 using Tukey’s post-hoc test when compared with wild type after a one-way ANOVA (n ≥ 90 with 30–40 cells quantified in three separate experiments). (D) Immunoblots of lysates derived from nup116Δ5::HIS3 cells transformed with pRS314 vectors containing the constructs listed above each lane. Lysates were immunoblotted with anti-Pgk1 and anti-Nup116 C-terminal (C-term) antibodies. (E) nup116Δ5::HIS3 cells transformed with pRS314 vectors containing the listed constructs were serially diluted onto glucose or glycerol and incubated at 25, 30, or 34°C until grown as shown. (F) nup116Δ5::HIS3 cells transformed with pRS314 vectors containing the listed constructs as well as Spo12NLS-GFP were visualized using fluorescence microscopy. The mean nuclear/cytoplasmic GFP intensity ratios of ≥33 cells from three different experiments were calculated. *, P < 0.01 when compared with NUP116 using Tukey’s post-hoc test following a one-way ANOVA; **, P < 0.05 when compared with nup116ΔGLFG. Error bars represent SEM.
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fig6: Kap121-mediated transport is required for mitochondrial function. (A) Wild-type and mutant strains were serially diluted onto glucose or glycerol and incubated at 25°C until grown as shown. (B) Single representative wild-type or kap121 mutant cells stained with MitoTracker red CMXRos. DIC, differential interference contrast. Bar, 2.5 µm. (C) Quantification of the mean MitoTracker red CMXRos cytoplasmic intensity for strains listed in B. *, P < 0.01 using Tukey’s post-hoc test when compared with wild type after a one-way ANOVA (n ≥ 90 with 30–40 cells quantified in three separate experiments). (D) Immunoblots of lysates derived from nup116Δ5::HIS3 cells transformed with pRS314 vectors containing the constructs listed above each lane. Lysates were immunoblotted with anti-Pgk1 and anti-Nup116 C-terminal (C-term) antibodies. (E) nup116Δ5::HIS3 cells transformed with pRS314 vectors containing the listed constructs were serially diluted onto glucose or glycerol and incubated at 25, 30, or 34°C until grown as shown. (F) nup116Δ5::HIS3 cells transformed with pRS314 vectors containing the listed constructs as well as Spo12NLS-GFP were visualized using fluorescence microscopy. The mean nuclear/cytoplasmic GFP intensity ratios of ≥33 cells from three different experiments were calculated. *, P < 0.01 when compared with NUP116 using Tukey’s post-hoc test following a one-way ANOVA; **, P < 0.05 when compared with nup116ΔGLFG. Error bars represent SEM.

Mentions: Because Kap121 transport was mediated by Nup116’s GLFG domain (Fig. 2), kap121-7 and kap121-21 mutants were analyzed to determine whether they also displayed inhibited mitochondrial activity. This possibility seemed likely as KAP121 was isolated in an overexpression screen for genes that enhance the import of hydrophobic membrane proteins into the mitochondria (Corral-Debrinski et al., 1999). Both kap121-7 and kap121-21 cells were not viable on glycerol at 25°C (Fig. 6 A), indicative of strong defects in mitochondrial function. Quantification of MitoTracker red CMXRos staining showed the ΔΨ of kap121-7 and kap121-21 mutants was significantly decreased compared with wild-type cells (Fig. 6, B and C). When the brightness was adjusted to help visualize MitoTracker signal in kap121 mutants, the mitochondria also often appeared more fragmented compared with wild-type cells (Fig. S5 E).


Altering nuclear pore complex function impacts longevity and mitochondrial function in S. cerevisiae.

Lord CL, Timney BL, Rout MP, Wente SR - J. Cell Biol. (2015)

Kap121-mediated transport is required for mitochondrial function. (A) Wild-type and mutant strains were serially diluted onto glucose or glycerol and incubated at 25°C until grown as shown. (B) Single representative wild-type or kap121 mutant cells stained with MitoTracker red CMXRos. DIC, differential interference contrast. Bar, 2.5 µm. (C) Quantification of the mean MitoTracker red CMXRos cytoplasmic intensity for strains listed in B. *, P < 0.01 using Tukey’s post-hoc test when compared with wild type after a one-way ANOVA (n ≥ 90 with 30–40 cells quantified in three separate experiments). (D) Immunoblots of lysates derived from nup116Δ5::HIS3 cells transformed with pRS314 vectors containing the constructs listed above each lane. Lysates were immunoblotted with anti-Pgk1 and anti-Nup116 C-terminal (C-term) antibodies. (E) nup116Δ5::HIS3 cells transformed with pRS314 vectors containing the listed constructs were serially diluted onto glucose or glycerol and incubated at 25, 30, or 34°C until grown as shown. (F) nup116Δ5::HIS3 cells transformed with pRS314 vectors containing the listed constructs as well as Spo12NLS-GFP were visualized using fluorescence microscopy. The mean nuclear/cytoplasmic GFP intensity ratios of ≥33 cells from three different experiments were calculated. *, P < 0.01 when compared with NUP116 using Tukey’s post-hoc test following a one-way ANOVA; **, P < 0.05 when compared with nup116ΔGLFG. Error bars represent SEM.
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fig6: Kap121-mediated transport is required for mitochondrial function. (A) Wild-type and mutant strains were serially diluted onto glucose or glycerol and incubated at 25°C until grown as shown. (B) Single representative wild-type or kap121 mutant cells stained with MitoTracker red CMXRos. DIC, differential interference contrast. Bar, 2.5 µm. (C) Quantification of the mean MitoTracker red CMXRos cytoplasmic intensity for strains listed in B. *, P < 0.01 using Tukey’s post-hoc test when compared with wild type after a one-way ANOVA (n ≥ 90 with 30–40 cells quantified in three separate experiments). (D) Immunoblots of lysates derived from nup116Δ5::HIS3 cells transformed with pRS314 vectors containing the constructs listed above each lane. Lysates were immunoblotted with anti-Pgk1 and anti-Nup116 C-terminal (C-term) antibodies. (E) nup116Δ5::HIS3 cells transformed with pRS314 vectors containing the listed constructs were serially diluted onto glucose or glycerol and incubated at 25, 30, or 34°C until grown as shown. (F) nup116Δ5::HIS3 cells transformed with pRS314 vectors containing the listed constructs as well as Spo12NLS-GFP were visualized using fluorescence microscopy. The mean nuclear/cytoplasmic GFP intensity ratios of ≥33 cells from three different experiments were calculated. *, P < 0.01 when compared with NUP116 using Tukey’s post-hoc test following a one-way ANOVA; **, P < 0.05 when compared with nup116ΔGLFG. Error bars represent SEM.
Mentions: Because Kap121 transport was mediated by Nup116’s GLFG domain (Fig. 2), kap121-7 and kap121-21 mutants were analyzed to determine whether they also displayed inhibited mitochondrial activity. This possibility seemed likely as KAP121 was isolated in an overexpression screen for genes that enhance the import of hydrophobic membrane proteins into the mitochondria (Corral-Debrinski et al., 1999). Both kap121-7 and kap121-21 cells were not viable on glycerol at 25°C (Fig. 6 A), indicative of strong defects in mitochondrial function. Quantification of MitoTracker red CMXRos staining showed the ΔΨ of kap121-7 and kap121-21 mutants was significantly decreased compared with wild-type cells (Fig. 6, B and C). When the brightness was adjusted to help visualize MitoTracker signal in kap121 mutants, the mitochondria also often appeared more fragmented compared with wild-type cells (Fig. S5 E).

Bottom Line: Mutants lacking the GLFG domain of Nup116 displayed decreased RLSs, whereas longevity was increased in nup100- mutants.Both Kap121-dependent transport and Nup116 levels decrease in replicatively aged yeast.Together, these studies reveal that specific NPC nuclear transport events directly influence aging.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232.

Show MeSH
Related in: MedlinePlus