service at the Universite´ de Montre´ al (IRIC), including functional annotation by Mascot (49). RESULTS AND DISCUSSION Puriﬁcation of mt-RNase P without the use of detergents To preserve the integrity of a postulated large complex that combines mt-RNase P with other mitochondrial func- tions, we have developed puriﬁcation procedures that do not make use of detergents or other highly disruptive con- ditions as previously applied [such as detergent, high salt, EDTA, heat shock etc; (16–18)]. For this we start with highly puriﬁed yeast mitochondria, gently disrupt the or- ganelles and extract a soluble matrix fraction by centrifu- gation, which represents 57% of total mitochondrial proteins. When separating the soluble extract on a glycerol gradient (10–70%; for details see ‘Materials and Methods’ section), most of the material moves into the glycerol phase, in support of the idea that most soluble proteins are organized in large complexes. In fact, tracing of the protein subunit of mt-RNase P (Rpm2p) by in vivo GFP ﬂuorescence labeling reveals that about half (54%) of the GFP-Rpm2p is in the soluble mitochondrial matrix fraction, and that most of it (81%) is associated with a high-molecular-weight complex as it migrates far into the glycerol gradient (35–40%; Figure 1). This gradient fraction also contains the bulk of RNase P activity (Supplementary Figure S1). GFP-labeled cells grow normally on non-fermentable substrates, indicating that the GFP fusion does not lead to functional disturbances. As gradient procedures have only limited resolution, we set out to ﬁnd an alternative, non-disruptive and prepara- tive puriﬁcation procedure for large protein complexes. This led us to develop a variant of BN–PAGE (50), in which all steps are performed under physiological condi- tions (e.g. pH 7), and in the absence of detergents (named non-denaturing polyacrylamide gel electrophoresis column, or NEC; for details see ‘Materials and Methods’ section). Instead of using a slab gel, preparative NEC separates in a large column with a continuous poly- acrylamide gel, and fractions are continuously recovered in a dialysis cup as they leave the column. Tracking of mt-RNase P by in vitro cleavage of a mitochondrial pre-tRNAproline precursor identiﬁes 70% of the total mt-RNase P activity in fractions 7–9 (with a total of 20 fractions; Figure 2A). To investigate the purity of frac- tions 7–9, they were separated by a second, regular slab-gel NEC that has more resolution than preparative NEC, and at a gel concentration that is optimal for sep- aration close to the complex’s size range. Several complexes were identiﬁed in all three fractions, but only one of them occurs regularly (Figure 3A), which is the putative mt-RNase P-containing complex. Therefore, for further large-scale puriﬁcation, fractions 7, 8 and 9 were pooled and processed on a second preparative NEC column at a higher polyacrylamide concentration (7.5%). Under this condition, mt-RNase P activity elutes exclusively in fraction 38 (for a total of 50 samples; Figure 2B and Supplementary Figure S2). Slab-gel NEC Nucleic Acids Research, 2012, Vol. 40, No. 4 1731 analysis conﬁrms that it contains a single complex (Figure 3A), which remains stable following treatments with DNase, RNase and to some degree ( 50%) with a mild detergent (digitonin; Figure 3B). Taken together, we argue that this complex closely represents the in vivo or- ganization of a set of soluble mitochondrial matrix proteins, combining a variety of RNA maturation and other functions as shown in the following experiments. Association of 50- and 30-tRNA processing activities The processing activity of the puriﬁed complex was sub- sequently tested in an in vitro assay, with a different pre-tRNAproline substrate that has both 50-leader and 30-trailer extensions. We ﬁnd essentially two forms of pro- cessed RNAs, the mature tRNAproline and an intermediate pre-tRNAproline with a 50-leader (Figure 4), revealing the presence of a 30-tRNA processing that is somewhat more effective than RNase P. This result is consistent with the previously observed co-fractionation of RNase P and RNase Z in early steps of yeast mt-RNase P puri- ﬁcation (18). Presence of RPM1 RNA and Rpm2p protein in the complex To further characterize the homogeneity and composition of this tRNA processing complex, we tracked the presence of the known yeast mt-RNase P core RNA and protein components (i.e. the RPM1 RNA subunit and Rpm2p). Proteomic analysis by mass spectrometry (LC–MS/MS, see ‘Materials and Methods’ section) shows that the Rpm2p is present only in this complex, together with a variety of other proteins (see below). Likewise, an extrac- tion and analysis of the endogenous RNA of the puriﬁed complex demonstrate the presence of the two (un-degraded) small and large subunit rRNAs, as well as several RNAs of 70–90 nt (Figure 5A). As expected, an RNA with the predicted size of an intact RPM1 RNA (427 nt) is absent, which is in agreement with previous in- vestigations on puriﬁed yeast mt-RNaseP [e.g. (16)], in which only fragments of RPM1 RNA are detected. In fact, consistent with these studies, we ﬁnd by RT–PCR and sequencing the same two RPM1 RNA fragments of 52, 89 and 71 nt (Figure 5B and Supplementary Figure S3). Our results thus conﬁrm that an intact RPM1 RNA subunit is not required for RNase P activity in vitro (16), and that the mt-RNase P core subunits are integrated within a large complex when isolated under non-denaturing conditions. Protein composition of the mt-RNase P (RMT) complex Proteomic analysis with liquid chromatography and tandem mass spectrometry (LC–MS/MS) reveals the presence of an unexpected high number of proteins (130) in the puriﬁed ‘native’ RNase P/RNase Z complex (con- ﬁrmed by three independent analyses; Supplementary Table S1). These proteins are implicated in numerous functions including (as expected) RNA processing, but also metabolism (e.g. TCA cycle), translation (two aminoacyl–tRNA synthetases; two translation elongation factors; an incomplete set of ribosomal proteins but