1734 Nucleic Acids Research, 2012, Vol. 40, No. 4 Figure 5. Two rRNAs subunits and fragments of RPM1 RNA are components of the RMT complex. (A) Agarose gel electrophoresis of the RNA extracted from the RMT complex. The prominent small and large S-rRNA) (L-rRNA) rRNA subunits are indicated (S-rRNA and L-rRNA). Lane 1, DNA size marker kb+ Lane 2, RNA size marker RiboRuler High Range; Lane 3, RNA extracted from RMT complex; Lane 4, RNA size marker RiboRuler Low Range. (B) RPM1 RNA of the RMT complex was reverse transcribed and PCR ampliﬁed using different primer combinations. Lane 1, DNA size marker kb+ lane 2, RNA control without RT–PCR ampliﬁcation; lane 3, negative control with primers; lanes 4 to 8 RT–PCR products of 89, 71 and 51 nt were identiﬁed. The diffuse RNAs migrating close to 35 bp in lanes 4, 6, 7 and 8 are primers and/or primer dimers. procedure, the mitochondrial ribosome contains about 70 proteins (52,53). The presence of only 35 mito-ribosomal proteins in the RMT complex falls short of about one half of the protein components, yet contains the un-degraded large and small subunit rRNAs (Figure 5A). This may be interpreted in two ways: (i) the RMT complex results from dissociation of a large membrane-attached complex that contains the intact ribosome. The dissociation may be caused by experimental manipulation (e.g. mechanical ex- traction procedure). It has been reported that the mito- chondrial ribosome is attached to the inner membrane (40), which would be consistent with our result that mt-RNase P and the RMT complex is partially membrane-attached. Alternatively, (ii) the RMT complex represents a biogenesis intermediate, with an incomplete set of ribosomal proteins that are being added to the rRNA subunits. The ﬁnding of metabolic proteins (e.g. the complete TCA cycle) and chaperons in this complex is consistent with the observed co-puriﬁcation of metabolic (TCA cycle) and ribosomal proteins in enriched human mt-RNase P fractions (2). Interestingly, the RMT complex also contains several chaperons that are involved in protein maturation, modiﬁcation, targeting and assembly steps. Lack of RMT complex in a FAS II deletion mutant A recent investigation of yeast mutants demonstrates that deﬁciency in any enzyme of the mitochondrial fatty acid type II biosynthetic pathway (FASII) leads to impaired 50-tRNA processing in yeast mitochondria and an overall pleiotropic phenotype (24). Our data reveal the presence of one regular component of the pathway (Oar1p; 3-Oxoacyl-[acyl-carrier-protein] reductase; FASII biosynthesis) in the RMT complex. Two other FAS II enzymes (Htd2p and Mct1p) were also identiﬁed, but only once in the three LC-MS/MS experiments. This is Figure 6. Biogenesis and stability of the RMT complex are affected in the yeast oar1 mutant. Soluble mitochondrial multiprotein complexes (50 mg of total protein) of the yeast wild strain and oar1 mutant were separated on linear 4–14% BN–PAGE, overnight (140 V and 9 mA; 4 C). Gels were stained with Serva Blue G250. probably due to their relatively low level of cellular ex- pression (51). Consistent with the above observations, the RMT complex is no longer discernable in an Oar1 deletion mutant, and the amount of other complexes is strongly reduced (Figure 6). Our data support the interpretation that lack of FAS II enzymes indirectly affects RNAse P assembly and its activity (24), as well as enzymes in the TCA cycle (24,54). As a consequence of lacking mature