Prion-dependent switching between respiratory competence and deficiency in the yeast nam9-1 mutant

Abstract

Nam9p is a protein of the mitochondrial ribosome. The respiration-deficient Saccharomyces cerevisiae strain MB43-nam9-1 expresses Nam9-1p containing the point mutation S82L. Respiratory deficiency correlates with a decrease in the steady level of some mitochondrially encoded proteins and the complete lack of mitochondrially encoded cytochrome oxidase subunit 2 (Cox2). De novo synthesis of Cox2 in MB43-nam9-1 is unaffected, indicating that newly synthesized Cox2 is rapidly degraded. Respiratory deficiency of MB43-nam9-1 is overcome by transient overexpression of HSP104, by deletion of HSP104, by transient exposure to guanidine hydrochloride, and by expression of the C-terminal portion of Sup35, indicating an involvement of the yeast prion [PSI(+)]. Respiratory deficiency of MB43-nam9-1 can be reinduced by transfer of cytosol from S. cerevisiae that harbors [PSI(+)]. We conclude that nam9-1 causes respiratory deficiency only in combination with the cytosolic prion [PSI(+)], presenting the first example of a synthetic effect between cytosolic [PSI(+)] and a mutant mitochondrial protein.

FIG. 1

Both Nam9p and Nam9-1p are localized in the mitochondria. (A) MB43-NAM9 and MB43-nam9-1 strains were grown on low-glucose medium, harvested at the point of glucose exhaustion, and fractionated as described in Materials and Methods. Two hundred micrograms of protein from total extract (tot), 200 μg of protein from postmitochondrial supernatant (cyt), and 20 μg of protein from crude mitochondria (mit) were analyzed by SDS-PAGE followed by immunodecoration with antibodies specific for Nam9p, Hsp104, mitochondrial malate dehydrogenase (Mdh1), and cytosolic hexokinase. (B) Nam9p and Nam9-1p were synthesized in a yeast translation system in the presence of [³⁵S]methionine (for details, see Materials and Methods). In vitro import reactions into mitochondria were performed for the indicated times with either radiolabeled Nam9p or Nam9-1p in the presence (+ΔΨ) or absence (−ΔΨ) of a membrane potential. Precursor that had not crossed the mitochondrial membranes after the import reaction was removed by treatment with proteinase K. std 5% and std 10% correspond to 5 and 10% of the import reaction, respectively. (C) Kinetics of the import reactions shown in panel B. The amount of imported Nam9p or Nam9-1p is given as a percentage of the total amount of Nam9p or Nam9-1p added to the import reaction. ○, Nam9p; ●, Nam9-1p.

FIG. 2

Nam9p and Nam9-1p localize to the small subunit of mitochondrial ribosome. (A) Extracts of crude mitochondria (100 μg) from MB43-NAM9 or MB43-nam9-1, respectively, were either precipitated with TCA (T) or separated into ribosomal pellet (P) and supernatant (S) by centrifugation through a sucrose cushion. After SDS-PAGE and Western blotting, samples were analyzed by immunodecoration with antibodies specific for Nam9p, Hsp60 (mitochondrial matrix), or Mrp49 (mitochondrial large ribosomal subunit). (B) Association of Nam9p with the mitochondrial ribosome was assessed by coimmunoprecipitation of the ribosomal components with antibodies specific for Nam9p (αNam9). Preimmune serum was used in the control reaction (control). Coimmunoprecipitation reactions were performed with 100 μg of total mitochondrial protein (T) under conditions that either stabilize (+Mg) or destabilize (−Mg) the interaction between the two ribosomal subunits. B, material bound to αNam9; U, material unbound to αNam9. T, B, and U were analyzed by Western blotting followed by immunodecoration with antibodies against Nam9p, Hsp78 (mitochondrial matrix), Mrp20 (mitochondrial large ribosomal subunit), and Mrp13 (mitochondrial small ribosomal subunit).

FIG. 3

The absence of Cox2 in MB43-nam9-1 is due to an event downstream of translation. (A) The steady-state levels of mitochondrially encoded proteins were assessed in strains MB43-NAM9 and MB43-nam9-1, respectively. Crude mitochondria (50 μg of protein) were analyzed by immunoblotting with antibodies directed against Nam9p, Cox2, Cox3, Cox4, Atp1, Atp4, Atp6, and mitochondrial malate dehydrogenase (Mdh1). In order to determine the relative amounts of the various proteins in the nam9-1 and NAM9 strains, respectively, the amount of each protein present in the NAM9 strain was set to 1. Numbers to the right give the ratios between the nam9-1 and the NAM9 strains. (B) In vivo synthesis of mitochondrially encoded proteins in MB43-NAM9 and MB43-nam9-1, respectively. Mitochondrial translation was performed in the presence of [³⁵S]methionine and cycloheximide as described in Materials and Methods and analyzed by SDS–12% PAGE. To ascertain the position of Cox2 and Cob in SDS-PAGE, the first two lanes show the mitochondrial translation products of the [mit⁻] strains M3041 (Δcob) and V25 (Δcox2), containing stop mutations in COB and COX2, respectively. As a result, the full-length translation products of the respective genes are absent. Var1, ribosomal protein. The Atp8 and Atp9 bands and the Cox3 and Atp6 bands are not separated from each other.

FIG. 4

Deletion as well as overexpression of Hsp104 stabilizes Cox2 in the nam9-1 strain. (A) Stability of newly synthesized Cox2 in MB43-NAM9, MB43-nam9-1, MB43-nam9-1Δhsp104, and MB43-nam9-1[HSP104↑]. Cells were pulse-labeled with [³⁵S]methionine in the presence of cycloheximide, and a chase reaction was performed for the times indicated (see Materials and Methods). The mitochondrial translation products from cells, corresponding to an OD₆₀₀ of 0.5, were analyzed by SDS–16% PAGE. M3041 (Δcob) and V25 (Δcox2) were used as references for the migration of Cox2 and Cob, respectively. Note that Cox2 and Cob migrate in the reverse order in SDS–16% PAGE compared to SDS–12% PAGE (compare to Fig. 6). (B) Steady-state levels of Cox2 in strains MB43-NAM9, MB43-nam9-1, MB43-nam9-1 hsp104, and MB43-nam9-1[HSP104↑]. The respective strains were grown on low-glucose medium and harvested either at the point of glucose exhaustion (glucose +/−) or after an additional incubation of 12 h (glucose −) as described in Materials and Methods. Total protein was extracted from cells corresponding to an OD₆₀₀ of 0.5. Western blots were decorated with antibodies directed against the mitochondrial matrix protein aconitase (Aco) and cytochrome c oxidase subunit 2 (Cox2).

FIG. 5

Respiratory deficiency of MB43-nam9-1 is suppressed by the absence of or by overproduction of Hsp104. The respective derivatives of MB43 (compare to Table 1) were transferred to YPGly plates and incubated for 3 days at 30°C. In order to determine the content of Hsp104 and Nam9 or Nam9-1p, the strains were grown on low-glucose medium and harvested at the point of glucose exhaustion (see Materials and Methods). Total protein was extracted by alkaline lysis and analyzed by Western blotting using antibodies specific for Hsp104 and Nam9p.

FIG. 6

The nam9-1 mutation leads to respiratory deficiency only in the presence of a prion-like element. In order to test the respiratory competence of the MB43-nam9-1 derivatives (Table 1; see also Materials and Methods), the respective strains were transferred to YPGly plates and incubated for 3 days at 30°C. (A) MB43-nam9-1 was grown on YPGly (lane 1), YPGly containing 5 mM GuHCl (lane 2), and YPGly after transient growth on YPD containing 5 mM GuHCl (lane 3). (B) As outlined in the scheme, JC25 was turned [rho⁰] by treatment with EtBr and the resulting strain, JC25[rho⁰], was used as a recipient for the cytoplasm of respiration-deficient MB43-nam9-1. Strain JC25 containing the cytoplasm of respiration-deficient MB43-nam9-1 was termed JC25/1 and was used as the donor strain in a second cytoduction, either directly or after transient exposure to 5 mM GuHCl. The recipient strain for the second cytoduction was MB43-nam9-1[rho⁰], generated as follows: MB43-nam9-1 was transformed with a plasmid overexpressing Hsp104, the plasmid was cured, and finally the strain was treated with EtBr. For details compare Results and Materials and Methods. (C) Diploids generated by crosses of respiration-competent MB43-nam9-1[mit⁻-V25] with the [rho⁰] derivatives of the [PSI⁺] strain OT72, the [psi⁻] strain OT74, JC25, and JC25/1. The presence of a prion-like element in panels B and C is indicated by an asterisk.

FIG. 7

Respiratory deficiency of MB43-nam9-1 can be overcome by expression of the C-terminal portion of Sup35. MB43-nam9-1 derivatives were transferred to YPGly plates and incubated for 3 days at 30°C. MB43-nam9-1 was transformed with empty control vector ([−]), pEMBLyex4-3ATG ([SUP35-C]), pEMBLyex4-SUP35 ([SUP35]), and pA1/4 ([HSP104↑]) (Table 1; see also Materials and Methods).