Identification and characterization of high molecular weight complexes formed by matrix AAA proteases and prohibitins in mitochondria of Arabidopsis thaliana

Abstract

We identify and characterize two matrix (m)-AAA proteases (AtFtsH3 and AtFtsH10) present in the mitochondria of Arabidopsis thaliana. AtFtsH3 is the predominant protease in leaves of wild type plants. Both proteases assemble with prohibitins (PHBs) into high molecular weight complexes (approximately 2 MDa), similarly to their yeast counterparts. A smaller PHB complex (approximately 1 MDa), without the m-AAA proteases, was also detected. Unlike in yeast, stable prohibitin-independent high molecular weight assemblies of m-AAA proteases could not be identified in A. thaliana. AtFtsH3 and AtFtsH10 form at least two types of m-AAA-PHB complexes in wild type plants. The one type contains PHBs and AtFtsH3, and the second one is composed of PHBs and both AtFtsH3 and AtFtsH10. Complexes composed of PHBs and AtFtsH10 were found in an Arabidopsis mutant lacking AtFtsH3 (ftsh3). Thus, both AtFtsH3 and AtFtsH10 may form hetero- and homo-oligomeric complexes with prohibitins. The increased level of AtFtsH10 observed in ftsh3 suggests that functions of the homo- and hetero-oligomeric complexes containing AtFtsH3 can be at least partially substituted by AtFtsH10 homo-oligomers. The steady-state level of the AtFtsH10 transcripts did not change in ftsh3 compared with wild type plants, but we found that almost twice more of the AtFtsH10 transcripts were associated with polysomes in ftsh3. Based on this result, we assume that the AtFtsH10 protein is synthesized at a higher rate in the ftsh3 mutant. Our results provide the first data on the composition of m-AAA and PHB complexes in plant mitochondria and suggest that the abundance of m-AAA proteases is regulated not only at the transcriptional but also at the translational level.

FIGURE 1.

AtFtsH3 and AtFtsH10 restore growth of the yta10 and/or yta12 null mutants on glycerol. Yeast strain W303 or Δyta10 and/or Δyta12 mutants were transformed with recombinant plasmid pCM185 carrying AtFtsH3 (+ AtFtsH3) or AtFtsH10 (+ AtFtsH10) genes cloned into pCM185 vector or empty vector (+ vector). 10-Fold serial dilutions of cells were replica-plated on YP plates containing 2% glucose (YPD) or 4% glycerol (YPG) and incubated at 30 °C for 3 days. WT, wild type.

FIGURE 2.

Increased abundance of AtFtsH10 in ftsh3 and ftsh3-1 mutants. A, mitochondria isolated from wild type (Wt) plants and ftsh3 and ftsh3-1 mutants were separated by 10% SDS-PAGE, transferred to polyvinylidene difluoride membrane, and probed with antibodies raised against epitopes specific to AtFtsH3 and AtFtsH10 or an epitope common to AtFtsH3 and AtFtsH10 (anti-AtFtsH3/10 antibodies). AtVDAC1 (porin1) was used as a loading control. B, levels of AtFtsH3 + AtFtsH10 proteins (anti-AtFtsH3/10 antibody (Ab)) or AtFtsH10 protein (anti-AtFtsH10 antibody) in ftsh3 mutant relative to wild type (Wt) plants. C, steady-state level of the AtFtsH3 mRNAs relative to the AtFtsH10 mRNAs in leaves of wild type plants. Relative quantities of the mRNAs were counted using real time PCR and external standards (see “Experimental Procedures” for details). Data are means ± S.E. from three independent experiments. *, nonspecific band.

FIGURE 3.

Expression of AtFtsH10 gene in ftsh3 mutant. A, level of AtFtsH10 transcript in ftsh3 mutant and wild type (Wt) plants. RNA samples were isolated from whole 6-week-old plants. Data are means ± S.E. from three independent experiments. B, turnover of AtFtsH10 protein in wild type (Wt) and ftsh3 mitochondria. Mitochondria were incubated for 6 h. Steady-state levels of AtFtsH10 protein were analyzed in 1.5-h intervals by Western blotting. Left, results obtained for intact mitochondria. Right, results obtained for mitochondria treated with DDM at time 0. Data are representative of two independent experiments. C, abundance of AtFtsH10 transcript in polysomal RNA from wild type plants and ftsh3 mutant. RNA was isolated from adult leaves harvested from 10- to 11-week-old plants. The AtFtsH10 transcript was standardized relative to the AtAct2 transcript. The difference in abundance of the AtFtsH10 transcript in polysomal RNA from ftsh3 mutant and from wild type plants is statistically significant (Student's t test, p value = 0.01). The relative abundance of the AtFtsH10 transcript in total RNA was set as 1. Data presented as mean ± S.E. are representative of two biological repeats, each performed in three technical repeats.

FIGURE 4.

Characteristics of high molecular weight complexes of PHBs and m-AAA proteases. A, high molecular weight complexes of AtPHBs, AtFtsH3, and AtFtsH10 separated on two-dimensional BN/SDS-polyacrylamide gels. Wild type (Wt) mitochondria solubilized with digitonin were separated in 3–12% BN-PAGE in the first dimension and in 10% SDS-PAGE in the second dimension, transferred onto polyvinylidene difluoride membrane, and probed with anti-PHBs, anti-AtFtsH3, and anti-AtFtsH10 antibodies. For PHBs, a lane from the first dimension is also presented in order better to visualize the existence of two complexes of PHBs. The molecular weight of the m-AAA and PHB complexes was estimated in relation to the positions of respiratory chain complexes. B, co-immunoprecipitation of AtFtsH10 protein. Wild type mitochondria (100 μg) were solubilized with digitonin. Co-immunoprecipitation was carried out using anti-AtFtsH10 antibodies or preimmune antiserum (negative control). Immunoprecipitants were analyzed by SDS-PAGE and immunoblotting. Input, mitochondria used for immunoprecipitation. C, high molecular weight complex of AtFtsH10 in ftsh3 mutant. Mitochondria solubilized with digitonin were separated by 5% nongradient BN-PAGE in order better to visualize the AtFtsH10 complex. Left, Coomassie staining of respiratory chain complexes. Right, immunoblotting with anti-AtFtsH10 antibodies. D, immunodepletion of AtFtsH10. Wild type mitochondria (100 μg) were solubilized with digitonin and incubated for 7 h with protein A-Sepharose beads with cross-linked anti-AtFtsH10 antibodies or preimmune serum (negative control). After centrifugation, supernatant was taken for immunoblotting with anti-AtFtsH3 and anti-AtFtsH10 antibodies. AtVDAC1 (porin1) was used as a loading control.

FIGURE 5.

Stability of m-AAA and PHB complexes in DDM. Mitochondria solubilized with either digitonin or DDM were separated by two-dimensional BN/SDS-PAGE, transferred on polyvinylidene difluoride membrane, and immunoblotted for PHBs and AtFtsH10 (A and C) or PHBs and AtFtsH3 (B). A and B, results for wild type mitochondria. C, results for mitochondria isolated from ftsh3 mutant.