Synthetic lethal interaction of the mitochondrial phosphatidylethanolamine biosynthetic machinery with the prohibitin complex of Saccharomyces cerevisiae

Abstract

The majority of mitochondrial phosphatidylethanolamine (PtdEtn), a phospholipid essential for aerobic growth of yeast cells, is synthesized by phosphatidylserine decarboxylase 1 (Psd1p) in the inner mitochondrial membrane (IMM). To identify components that become essential when the level of mitochondrial PtdEtn is decreased, we screened for mutants that are synthetically lethal with a temperature-sensitive (ts) allele of PSD1. This screen unveiled mutations in PHB1 and PHB2 encoding the two subunits of the prohibitin complex, which is located to the IMM and required for the stability of mitochondrially encoded proteins. Deletion of PHB1 and PHB2 resulted in an increase of mitochondrial PtdEtn at 30 degrees C. On glucose media, phb1Delta psd1Delta and phb2Delta psd1Delta double mutants were rescued only for a limited number of generations by exogenous ethanolamine, indicating that a decrease of the PtdEtn level is detrimental for prohibitin mutants. Similar to phb mutants, deletion of PSD1 destabilizes polypeptides encoded by the mitochondrial genome. In a phb1Delta phb2Delta psd1(ts) strain the destabilizing effect is dramatically enhanced. In addition, the mitochondrial genome is lost in this triple mutant, and nuclear-encoded proteins of the IMM are assembled at a very low rate. At the nonpermissive temperature mitochondria of phb1Delta phb2Delta psd1(ts) were fragmented and aggregated. In conclusion, destabilizing effects triggered by low levels of mitochondrial PtdEtn seem to account for synthetic lethality of psd1Delta with phb mutants.

Figure 1

Biosynthesis of phosphatidylethanolamine in yeast. Biosynthesis of PtdEtn in yeast is accomplished by decarboxylation of PtdSer by either Psd1p in the IMM or by Psd2p in the Golgi/vacuole. Alternatively, exogenous Etn can be incorporated into PtdEtn via the CDP-Etn branch of the Kennedy pathway. PtdCho is formed by either methylation of PtdEtn or from exogenous Cho through the CDP-Cho branch of the Kennedy pathway.

Figure 2

psd1Δ phb1Δ and psd1Δ phb2Δ mutants are rescued by Etn on glucose for a limited number of generations, but not on lactate or at elevated temperature. Dilutions (1, 1/10, 1/100, and 1/1000) of cell suspensions were spotted on rich glucose media (YPD) or forced to lose pRB1 on synthetic media containing 2% glucose and FOA (FOA), supplemented with Cho (FOA + Cho), Ser (FOA + Ser), or Etn (FOA + Etn). Strains were transferred from FOA + Etn to a fresh plate (FOA + Etn 2). Additionally, strains were forced to lose pRB1 on synthetic media containing 2% lactate and FOA supplemented with Etn (FOALac + Etn). Plates were incubated at 30°C unless stated otherwise.

Figure 3

Submitochondrial distribution of Psd1-GFP, Phb1-GFP, and Phb2p. Mitochondria (MIT) and subfractions of mitochondria, OMM, contact sites (CS), and IMM, were subjected to Western blot analysis. Psd1-GFP and Phb1-GFP were detected in wild type (A) bearing the respective hybrid proteins by using anti-GFP antibodies. Phb2p was detected in submitochondrial fractions of wild-type and psd1Δ strains (B) by using anti-Phb2p antiserum. Porin and Aac1p (ATP/ADP carrier) were used as marker proteins for the OMM and IMM, respectively.

Figure 4

Viability of a phb1Δ phb2Δ psd1^ts mutant on YPD after a temperature shift to 37°C. A phb1Δ phb2Δ psd1^ts mutant grown on YPD was temperature shifted to 37°C. At time points indicated the number of viable cells was determined by dilution plating on YPD plates and counting of the colony-forming units after incubation at 30°C.

Figure 5

The phb1Δ phb2Δ psd1^ts strain does not grow at elevated temperature or on nonfermentable carbon sources. Growth of phb1Δ psd1^ts (○), phb1Δ phb2Δ psd1^ts (×), psd1^ts (▵), phb1Δ phb2Δ (□), and wild-type (⋄) strains on YPD at 30°C (A) or 37°C (C) and on YPLac at 30°C (B) was monitored by measurement of the optical density at 600 nm at time points indicated.

Figure 6

The prohibitin complex is not involved in import of PtdSer into mitochondria. Strains were labeled for 0, 30, and 60 min with [³H]serine at the indicated temperature. Incorporation of label into PtdSer (□), PtdEtn (▵), and PtdCho (×) was determined by liquid scintillation counting of individual phospholipid spots scraped off a TLC plate (see MATERIALS AND METHODS).

Figure 7

Mitochondrial morphology of phb and psd1 mutants. The OMM protein porin was detected in fixed cells by indirect immunofluorescence (see MATERIALS AND METHODS). Nuclear and mtDNA was stained with DAPI. Arrows indicate mtDNA.

Figure 8

Synthetic slow growth phenotype of psd1Δ and matrix-AAA protease mutants. Growth of yta10Δ (×), yta10Δ psd1Δ (*), yta12Δ (○), yta12Δ psd1Δ (+), yme1Δ (□), yme1Δ psd1Δ (▵), and wild-type (⋄) strains on YPD at 30°C was monitored by measurement of the optical density at 600 nm at time points indicated.

Figure 9

Instability of mitochondrial encoded proteins of phb and psd1 mutant strains. Mitochondrial encoded proteins were pulse labeled and analyzed by autoradiography after 0, 30, 60 and 90 min of chase (see MATERIALS AND METHODS).

Figure 10

Proteins of the IMM are not efficiently assembled in psd1Δ and phb1Δ phb2Δ psd1^ts strains. Homogenates of strains grown at 30°C on YPD were analyzed after a 0-, 1-, 2-, and 4-h temperature shift to 37°C by Western blotting with primary antibodies against the OMM protein porin and the IMM proteins Aac1p and Cox4p.