this observation is that there is a structural association between mt-RNase P and FAS II that is impaired with deletion of the FAS II subunits. In fact, this led us to the hypothesis that a super-structure may combine some or all of the above-mentioned mitochondrial functions into a large physical unit. Indications that RNase P may be associated with other functions also come from mt-RNase P puriﬁcation experiments. In early isolation steps, its activity co-fractionates with the tRNA 30-processing activity (18), and puriﬁcation to homogen- eity is achieved at an extremely low yield, despite the use of high salt and detergent. In yeast mitochondria, 30-tRNA processing is an endonucleotic cleavage (25), which is accomplished by a multifunctional RNase Z (11,26,27). In yeast, both the nuclear and mitochondrial forms of RNase Z are encoded by a nuclear gene (TRZ1) (15). Again, this enzyme is also implicated in ribosomal RNA maturation (26). Controlling RNA levels (balance between RNA synthe- sis and degradation) is vital for both the regulation and functioning of the mitochondrial system (28–30). RNA degradation is mediated principally by (arguably) small multiprotein complexes, like the exosome in the cytoplasm of eukaryotes (31) or the degradosome in bacteria (32). The activity responsible for RNA turnover in mitochondria is a 30- to 50-processive exoribonuclease (33), which is organized in an RNA degradosome complex (mtEXO), as ﬁrst described in S. cerevisiae (34). The highly puriﬁed mtEXO complex is composed of only two protein subunits, the exoribonuclease (35), and an NTP-dependent RNA helicase (related to the DExH superfamily) (36). Yet, the degradosome apparently asso- ciates with the mitochondrial ribosome, as mtEXO co-puriﬁes with ribosomal proteins that are difﬁcult to remove (34). These observations reinforce the hypothesis that the RNA degradosome is part of a large super- structure that associates with a variety of mitochondrial functions. In this study, we demonstrate that mt-RNase P and tRNA Z activities are part of a large ribo-nucleoprotein complex, which also includes the RNA degradosome, ﬁve additional RNA processing proteins, plus other mitochon- drial functions. We further show that the biogenesis of this complex is impaired in a mutant that is deﬁcient in type II fatty acid synthesis, rationalizing its known pleiotropic tRNA processing phenotype discussed above. MATERIALS AND METHODS Cell culture and mitochondrial isolation Saccharomyces cerevisiae (BY 4743) was kindly provided by Dr S. Michnick (Universite´ de Montr e´al), and yeast GFP (Green Fluorescent Protein) constructs used in this study (MATa his3 1 leu2 0 met15 0 ura3 0 RPM2-GFP::HIS3) by Dr J. Vogel (McGill). Cells were grown to an optical density of 1.5–2.5 in a medium con- taining 1% yeast extract, 2% peptone, 3% glycerol and 1% galactose, pH 5.0 (YEPGal). Mitochondrial puriﬁca- tion followed previously published procedures with slight modiﬁcations (37). Yeast cell walls were removed by Nucleic Acids Research, 2012, Vol. 40, No. 4 1729 digestion with glucanase, spheroplasts were disrupted by osmotic shock, and a crude mitochondrial fraction was isolated by differential centrifugation. Mitochondria were resuspended in ‘washing buffer’ (600 mM sucrose, 0.2 mM PMSF and 10 mM MOPS/KOH, pH 7.2) and further puriﬁed by centrifugation (60 min at 134 000g) through a discontinuous sucrose gradient (concentrations from top to bottom, 25%, 36% and 60%; 5 ml each). For a second puriﬁcation step, the mitochondrial fraction was collected and mixed with about four times its volume of 80% sucrose, layered at the bottom of a discontinuous sucrose gradients, and puriﬁed by centrifuged at 134 000g for 120 min. Intact mitochondria move upwards (‘ﬂotation’), and accumulate at the interface between the 36% and 60% sucrose layers. For enhanced purity, this two-step puriﬁcation of the mitochondria was repeated twice. Puriﬁed mitochondria were resuspended in buffer (600 mM sucrose, 0.2 mM PMSF and 10 mM Tricine/KOH, pH 7.2) and pelleted at 14 000g for 15 min. Usually, mitochondria were directly processed in further steps, but they may also be shock-frozen and kept at 80 C until use. Extraction of mitochondria soluble matrix proteins and membrane fractions For the extraction of matrix proteins, we adapted a pre- viously published procedure (37,38). Mitochondria were re-suspended at 10 mg/ml in breaking buffer (600 mM sucrose, 20 mM HEPES–KOH, pH 7.4, 10 mM EDTA) and incubated for 30 min on ice in nine volumes of 20 mM HEPES–KOH, pH 7.4, 0.5 mM EDTA and 1 mM PMSF. We used three alternative ways for rupturing mitochondria, which only differ in yield but not in banding pattern or protein composition of supercomplexes. The gentlest method is to burst mitochondria through osmotic shock, by abruptly adding 10 volumes buffer. Alternatively, the mitochon- drial suspension is adjusted to a ﬁnal sucrose concentra- tion of 0.45 M, incubated for 30 min on ice, and then homogenized in a Potter homogenizer for 90 s. The third method, for maximum yield of matrix proteins, involves mechanical disruption of mitochondria by shaking with an equal ratio of 125–212 m and 425–600 m glass beads (Sigma), in three rounds of 20 s each at 4 C. After a clar- ifying centrifugation at 12 000g for 15 min, the super- natant was subjected to centrifugation at 100 000g, for 2 h at 4 C. The pellet containing mitochondrial mem- branes was discarded, and the supernatant was subjected to a second centrifugation at 120 000g, for 30 min at 4 C. The matrix protein fraction (supernatant) was mostly used immediately, but may be stored at 80 C after shock-freezing. Protein concentrations were determined with the Bradford protein assay. A mitochondrial membrane fraction was prepared as described previously (39,40). Tracking of GFP-tagged marker proteins Soluble matrix protein complexes (1.5 mg) isolated from an Rpm2-GFP construct were separated by ultracentrifu- gation (16 h at 20 000g, Beckman SW41 rotor) on