Ubiquitin-Conjugating Enzymes Ubc1 and Ubc4 Mediate the Turnover of Hap4, a Master Regulator of Mitochondrial Biogenesis in Saccharomyces cerevisiae

Abstract

Mitochondrial biogenesis is tightly regulated in response to extracellular and intracellular signals, thereby adapting yeast cells to changes in their environment. The Hap2/3/4/5 complex is a master transcriptional regulator of mitochondrial biogenesis in yeast. Hap4 is the regulatory subunit of the complex and exhibits increased expression when the Hap2/3/4/5 complex is activated. In cells grown under glucose derepression conditions, both the HAP4 transcript level and Hap4 protein level are increased. As part of an inter-organellar signaling mechanism coordinating gene expression between the mitochondrial and nuclear genomes, the activity of the Hap2/3/4/5 complex is reduced in respiratory-deficient cells, such as ρ⁰ cells lacking mitochondrial DNA, as a result of reduced Hap4 protein levels. However, the underlying mechanism is unclear. Here, we show that reduced HAP4 expression in ρ⁰ cells is mediated through both transcriptional and post-transcriptional mechanisms. We show that loss of mitochondrial DNA increases the turnover of Hap4, which requires the 26S proteasome and ubiquitin-conjugating enzymes Ubc1 and Ubc4. Stabilization of Hap4 in the ubc1 ubc4 double mutant leads to increased expression of Hap2/3/4/5-target genes. Our results indicate that mitochondrial biogenesis in yeast is regulated by the functional state of mitochondria partly through ubiquitin/proteasome-dependent turnover of Hap4.

Keywords: Hap4; Saccharomyces cerevisiae; Ubc1; Ubc4; mitochondrial dysfunction; proteasome; protein turnover; transcription factor; ubiquitin-conjugating enzyme (E2 enzyme).

Figure 1

HAP4 expression is reduced in ρ⁰ cells. (A) A β-galactosidase activity assay of the expression of a HAP4-lacZ reporter gene in wild-type ρ⁺ strain (BY4741) and its ρ⁰ derivative grown in dextrose and raffinose media. β-galactosidase activities were determined as described in Materials and Methods. The data is presented as the mean ± standard deviation. A t-test was carried out and the p values indicate a significant difference between two groups of data. (B) Immunoblotting of Hap4-HA in a ρ⁺ hap4Δ mutant (BY4741 hap4) and its ρ⁰ derivative grown in dextrose and raffinose media. Equivalent amounts of protein samples based on OD₆₀₀ readings of the collected cell cultures were loaded into the lanes of the same gel. Ilv5 was included as a loading control. The result was representative of four independent sets of results. (C) Quantification of Hap4-HA/Ilv5 from Western blotting data.

Figure 2

Expression of KGD1-lacZ reporter gene correlates with Hap4-HA protein levels. (A) A β-galactosidase activity assay of KGD1-lacZ expression in ρ⁺ and ρ⁰ cells of wild-type strain (ZLY3440) and its isogenic hap4Δ mutant (DCY247). (B) (upper panel) Immunoblotting of Hap4-HA in hap4Δ cells (DCY247) carrying centromeric plasmids encoding HAP4-HA under the control of its endogenous promoter HAP4p, a heterologous promoter GPDp or TEF2p. Equivalent amounts of protein samples based on OD₆₀₀ readings of the collected cell cultures were loaded into the lanes of the same gel. Ilv5 was included as a loading control. (lower panel) Quantification of Hap4-HA/Ilv5 from two sets of Western blotting results. (C) A β-galactosidase activity assay of KGD1-lacZ expression in hap4Δ mutant cells carrying plasmids as described for panel (B).

Figure 3

Hap4-HA is less stable in ρ⁰ cells than in ρ⁺ cells. (A) Western blot analysis of Hap4-HA expression in hap4Δ mutant cells (DCY247) carrying a centromeric plasmid encoding GPDp-HAP4-HA (pDC216). Equivalent amounts of protein samples based on OD₆₀₀ readings of the collected cell cultures were loaded into the lanes of the same gel. (B) A cycloheximide chase assay of Hap4-HA stability in ρ⁺ and ρ⁰ cells of strain DCY247 grown in dextrose and raffinose media. Expression of HAP4-HA was under the control of its own promoter and the cycloheximide chase assay was conducted as described in Materials and Methods. Pgk1 was included as a loading control. (C) Quantification of Hap4-HA levels in panel (B). (D) The half-lives of Hap4-HA were determined from Western blots of Hap4-HA from ρ⁺ and ρ⁰ cells as described for panel (B) and plotted in the graph.

Figure 4

Inhibition of the activity of the 26S proteasome leads to increased stability of Hap4-HA. (A) A cycloheximide chase assay on Hap4-HA in ρ⁺ and ρ⁰ cells of an erg6Δ mutant strain (ZLY4531) with or without the treatment of the proteasome inhibitor MG132. Hap4-HA was detected using immunoblotting. Pgk1 was a loading control. (B) Increased formation of slower mobility forms of Hap4-HA in ρ⁰ cells compared to ρ⁺ cells when the proteasomal function is inhibited. ρ⁺ and ρ⁰ cells of an erg6Δ mutant strain expressing HAP4-HA from its own promoter were grown in raffinose medium with or without MG132 treatment. Protein samples were separated by SDS-PAGE and Hap4-HA was detected by Western blotting. The gel picture in the upper panel was enhanced to visualize the putative ubiquitinated forms of Hap4-HA. (C) Hap4-HA is stabilized in a pre1 pre4 mutant, which is defective in proteasome protease function. ρ⁺ and ρ⁰ cells of wild-type strain (15Daub) and its isogenic pre1 pre4 double mutant (PY555) were grown in dextrose or raffinose medium as indicated. Hap4-HA was detected by immunoblotting. Pgk1 was a loading control.

Figure 5

A ubc1Δ ubc4Δ double mutation stabilizes Hap4-HA. (A,B) A cycloheximide chase assay on Hap4-HA stability in ρ⁰ cells of wild type (BY4741 in panel A; Y0002 in panel B) and respective isogenic ubc mutant strains as indicated grown in raffinose medium. Hap4-HA was detected using Western blotting. (C) Hap4-HA is stabilized in ρ⁺ and ρ⁰ cells of a ubc1/4Δ mutant (ZLY3359) grown in dextrose or raffinose medium as indicated. (D) Quantification of Hap4-HA levels from ρ⁺ and ρ⁰ cells of wild type (WT, Y0002) and isogenic ubc1/4Δ mutant (ZLY3359) grown in dextrose and raffinose medium over the cycloheximide chase period. The means of remaining Hap4-HA levels during the chase period from two independent sets of experiments were plotted in the graphs.

Figure 6

Hap4 stabilization in ubc1Δ ubc4Δ double mutant cells results in increased expression of Hap2/3/4/5-target genes. β-galactosidase assays on the expression of KGD1-lacZ and SDH1-lacZ reporter genes in wild-type (WT, Y0002) and isogenic mutant strains (hap4Δ, AHY145; ubc1/4Δ, ZLY3359; ubc1/4Δ hap4Δ, ZLY3839) grown in dextrose (A) and raffinose media (B).