Activation of the pleiotropic drug resistance pathway can promote mitochondrial DNA retention by fusion-defective mitochondria in Saccharomyces cerevisiae

Abstract

Genetic and microscopic approaches using Saccharomyces cerevisiae have identified many proteins that play a role in mitochondrial dynamics, but it is possible that other proteins and pathways that play a role in mitochondrial division and fusion remain to be discovered. Mutants lacking mitochondrial fusion are characterized by rapid loss of mitochondrial DNA. We took advantage of a petite-negative mutant that is unable to survive mitochondrial DNA loss to select for mutations that allow cells with fusion-deficient mitochondria to maintain the mitochondrial genome on fermentable medium. Next-generation sequencing revealed that all identified suppressor mutations not associated with known mitochondrial division components were localized to PDR1 or PDR3, which encode transcription factors promoting drug resistance. Further studies revealed that at least one, if not all, of these suppressor mutations dominantly increases resistance to known substrates of the pleiotropic drug resistance pathway. Interestingly, hyperactivation of this pathway did not significantly affect mitochondrial shape, suggesting that mitochondrial division was not greatly affected. Our results reveal an intriguing genetic connection between pleiotropic drug resistance and mitochondrial dynamics.

Keywords: bulk segregant analysis; drug resistance; mitochondrial genome; mitochondrial shape; petite-negative.

Figure 1

A scheme for identifying new genes playing a role in mitochondrial dynamics. Loss of the ATP/ADP antiporter Aac2p prevents survival after mitochondrial DNA (mtDNA) deletion (Kovacova et al. 1968). Blocking mitochondrial fusion by FZO1 deletion in the presence of continued mitochondrial fission causes mtDNA loss (Bleazard et al. 1999; Sesaki and Jensen 1999). A strain was constructed (CDD71) carrying chromosomal fzo1∆ and aac2∆ mutations, in addition to a plasmid encoding FZO1. The FZO1-carrying plasmid was made counterselectable by a linked CYH2 allele existing in the context of a chromosomal cyh2 mutation that provides recessive cycloheximide (CHX) resistance (Sikorski and Boeke 1991). Strain CDD71 was either allowed to spontaneously acquire an sfa mutation, thereby permitting cells lacking FZO1 to maintain mtDNA and to survive in the presence of the aac2∆ mutation, or treated with ultraviolet light to generate such a mutation. Next, cells were replica-plated to medium containing 10 µg/mL CHX to counterselect for the FZO1-containing plasmid. Surviving colonies were isolated and further characterized as described in the Materials and Methods.

Figure 2

Mutation of PDR1 promotes maintenance of mitochondrial DNA (mtDNA) by cells lacking mitochondrial fusion. (A) The PDR1-249 allele allows fusion-incompetent mitochondria to maintain mtDNA in petite-negative cells lacking the ATP/ADP antiporter Aac2p. Strains CDD696 (cyh2), CDD698 (aac2∆ cyh2), CDD132 (fzo1∆ cyh2), CDD71 (fzo1∆ aac2∆ cyh2), CDD687 (fzo1∆ aac2∆ cyh2 fis1∆), CDD664 (fzo1∆ aac2∆ cyh2 PDR1-249), and CDD658 (PDR1-249 CYH2), all containing counterselectable plasmid b19 (pFZO1-CYH2), were cultured overnight in YEPD medium at 30° then serially diluted and spotted to YEPD medium either lacking (+ pFZO1-CYH2) or containing (− pFZO1-CYH2) 10 µg/mL CHX and incubated for 1 or 3 d, respectively. (B) Suppression of mtDNA loss from mutants that cannot fuse mitochondria is not limited to cells lacking FZO1. Strains CDD717 (mgm1∆ aac2∆), CDD716 (mgm1∆ aac2∆ PDR1-249), CDD71 (fzo1∆ aac2∆), and CDD664 (fzo1∆ aac2∆ PDR1-249), all also containing a chromosomal cyh2 allele and plasmids complementing the deletion of a mitochondrial fusion component, were treated as in (A) to allow for the presence or to select for the absence of plasmid b86 (pMGM1-CYH2) or of plasmid b19 (pFZO1-CYH2), as indicated. (C) Deletion of MDM36 or NUM1 does not permit mtDNA maintenance of mtDNA by cells lacking FZO1. Strains CDD71 (fzo1∆ aac2∆), CDD664 (fzo1∆ aac2∆ PDR1-249), CDD714 (fzo1∆ aac2∆ mdm36∆), and CDD750 (fzo1∆ aac2∆ num1∆), also carrying a chromosomal cyh2 mutation and plasmid b19 (pFZO1-CYH2), were treated as in (A).

Figure 3

The PDR1-249 allele dominantly suppresses mitochondrial DNA (mtDNA) loss from mutants unable to fuse mitochondria and activates the PDR pathway. (A) PDR1-249 dominantly allows mtDNA maintenance in cells lacking FZO1. Strains CDD71 (fzo1∆ aac2∆), CDD664 (fzo1∆ aac2∆ PDR1-249), CDD703 (fzo1∆/fzo1∆ aac2∆/aac2∆ PDR1/PDR1), CDD704 (fzo1∆/fzo1∆ aac2∆/aac2∆ PDR1-249/PDR1), and CDD687 (fzo1∆ aac2∆ fis1∆), all also homozygous for the cyh2 allele and carrying plasmid b19 (pFZO1-CYH2), were treated as in Figure 2A. (B) Deletion of PDR1 does not suppress mtDNA loss from cells lacking mitochondrial fusion. Strains CDD71 (fzo1∆ aac2∆), CDD664 (fzo1∆ aac2∆ PDR1-249), and CDD672 (fzo1∆ aac2∆ pdr1∆), all also mutated at the CYH2 locus and carrying plasmid b19 (pFZO1-CYH2), were treated as in Figure 2A. (C) Plasmid-borne PDR1-249 can suppress mtDNA loss from fzo1∆ cells. fzo1∆ aac2∆ strain CDD67, also harboring a chromosomal cyh2 mutation and plasmid b19 (pFZO1-CYH2), was transformed with empty vector pRS313 (Sikorski and Hieter 1989), plasmid b60 (pPDR1), or plasmid b65 (pPDR1-249). Transformants were cultured overnight in SC-Trp medium, then again overnight in SMM-His medium before an assay for proliferation following FZO1 loss as in Figure 2A. (D) The PDR1-249 allele activates the PDR pathway. Strains CDD642 (WT), CDD658 (PDR1-249), and CDD692 (fis1∆) were cultured overnight in YEPD at 30°, then serially diluted and spotted to YEPD medium alone (no drug) for 1 d at 30°, YEPD containing 0.2 µg/mL CHX for 3 d, or YEPD containing 2 µg/mL ketoconazole for 3 d.

Figure 4

PDR activation by the PDR1-249 allele does not cause gross changes to mitochondrial shape. (A) Increased mitochondrial fenestration is not apparent in PDR1-249 cells after disruption of the actin cytoskeleton. Strains CDD642 (wild type; WT), CDD658 (PDR1-249), and CDD692 (fis1∆) were transformed with plasmid pHS12 encoding Cox4p(1-21)-GFP (Sesaki and Jensen 1999). During the logarithmic phase of culture in SD-Leu medium, cells were treated with 10 µM latrunculin A (+LatA) or treated with an equal volume of dimethyl sulfoxide vehicle (−LatA) for 1 hr at 30° (Hammermeister et al. 2010), followed by fluorescence microscopy. (B) The PDR1-249 does not lead to morphology changes indicative of a mitochondrial division defect upon non-fermentable medium. Strains CDD642 (WT), CDD658 (PDR1-249), and CDD692 (fis1∆) were cultured in YEPGE medium at the logarithmic phase of proliferation, and mitochondrial morphology was visualized following staining with 500 nM MitoTracker Green FM. Bar, 5 µm.

Figure 5

PDR activation by the PDR1-249 allele does not cause gross changes to mitochondrial nucleoid distribution or mitochondrial DNA (mtDNA) abundance. (A) The distribution of mitochondrial nucleoids between mother and bud cells does not appear abnormal in PDR1-249 cells. Strains CDD642 (WT) and CDD658 (PDR1-249) were transformed with plasmid pM390, encoding GFP-tagged Abf2p. Mitochondrial morphology was visualized in SD-Leu medium during the logarithmic phase of culture growth. Bar, 5 µm. (B) Mitochondrial DNA is not amplified by PDR1-249 cells. Equal amounts of genomic DNA from an isolate of strain CDD642 lacking mtDNA after treatment with ethidium bromide (WT ρ⁰), CDD658 (PDR1-249), CDD692 (fis1∆), and strains CDD664 (fzo1∆ aac2∆ PDR1-249) and CDD687 (fzo1∆ aac2∆ fis1∆), both lacking plasmid b19 (pFZO1 -CYH2) following CHX counterselection, were cut with EcoRV and Southern blotting was performed using probes recognizing nuclear gene TDH1 or mitochondrial gene COX3.